THE STORY OF THE RESEARCH FOR A NEW TREATMENT OF CANCER, DEGENERATIVE DISEASES AND IN REGENERATION OF TISSUES.

The paradigm of complexity and some aspects of theory of information, of linguistics and of semiology are fundamental in understanding the process of cancerogenesis and in determining the correct therapeutic approach to tumoral diseases. To go into these problems in more depth way I have to ask you to be patient and to follow the mental processes and the experiments that I made.

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At the beginning of 1982….

At the beginning of 1982 I studied the relationship between the agents that cause cancer, mutations and malformations and focused my attention on some of the data of literature, that showed how carcinogens administrated during pregnancy had had different effects. The administration of carcinogens before or during organogenesis causes in fact a high rate of malformations in offspring, but no tumor induction. Once organogenesis is complete the frequency of tumor induction rises with a concomitant decrease in the rate of malformations.

MD. Pier Mario Biava

The question was why these different effects take place ?

The answer was immediately clear:

During organogenesis all processes of cell differentiation take place and they could stop the action of factors which cause cancer. Some malformations of tissues and organs are possible but these tissues and organs are made up of differentiated cells. During organogenesis some substances with regulatory properties are perhaps present to prevent the indiscriminate multiplication of cells. These regulators are able to differentiate the mutated stem cells. Could these regulators control the multiplication of tumor cells? Were tumor cells similar to mutated stem cells? In order to answer to these questions some experiments were carried out.

Here we summarize now several researches conducted over the past 35 years in vitro as well as in vivo and finally clinical studies on cases of hepatocellular carcinoma at intermediate-advanced stage having administered factors extracted during stem cells differentiating processes.

Lastly, we report recent experiments that showed that stem cells differentiating factors (SCDFs) are able to prevent neurodegenerative processes in mouse hippocampus cell line and to significantly ameliorate psoriasis.

EXPERIMENTAL RESEARCHES AND CLINICAL TRIALS

The treatment of oncologic and degenerative diseases:

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The first Experiments were published on Cancer Letter in 1988

Abstract

Based on the hypothesis that the development of cancer is actively inhibited during embryonic life, the effects on tumor growth of homogenates of different tissues (embryos, uteri at ninth day of pregnancy, non-pregnant uteri and normal liver) were investigated in syngeneic C57BL/6 female mice. Primary tumor growth and spontaneous pulmonary metastasis formation were completely suppressed in the group of mice treated with pregnant uteri homogenates. Embryos, non-pregnant uteri and normal liver homogenates were ineffective.

Click here to see: Effects of treatment with embryonic and uterine tissue homogenates on Lewis lung carcinoma development.

http://www.cancerletters.info/article/0304-3835%2888%2990287-X/abstract

The most important scientific articles in which were published the results of the researches here recorded are:

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Activation of anti-oncogene p53 produced by embryonic extracts in vitro tumor cells.

ACTIVATION OF ANTI-ONCOGENE P53 PRODUCED BY EMBRYONIC EXTRACT

  • Results demonstrate that substances present in the embryo during cell differentiation are able to activate p53

Download: Tumor Marker Oncology, volume 12 Winter 1997

The following are the main images related to the different studies carried out on embryo differentiation factors:

The state of the art of research includes both in vitro works on molecular dynamics involving stem differentiation factors and in vivo work: on mice and on men. It remains necessary to implement in-vivo research, especially clinical.

Specifically, studies have been conducted on the following aspects:

  • Slower growth of tumor cell lines
  • Block of the cell cycle, activatinig trascriptional way the oncosupressor gene p53
  • regulating in post-traslational way the Retinoblastoma protein (pRb)
  • Animal studies
  • Protein Analysis of Zebrafish Embryo Extract
  • Clinical study of 200 patients to evaluate possible side effects
  • Randomized clinical study in 179 patients with intermediate or advanced hepatocarcinoma

Slowed down and/or cell cycle block action

It has been shown that in the embryonic microenvironment there are factors that regulate the expression of the p53 co-repressor, activating it, and post-translationally pRb. In fact, with the differentiation of stem cell differentiating factors of different tumor lines, a block of cell cycle was observed in G1-S phase.

Activation of anti-oncogene p53

Through cytofluorometry and immunohistochemistry, a significant increase in the concentration of p53 protein has been demonstrated in specific lines of cellular tumor lines such as glioblastoma multiforme, melanoma and hepatocarcinoma treated with stem differentiation factors. This increase is a consequence of the transcriptional regulation of the p53 oncosoppress gene.

Download the publication: ACTIVATION OF ANTI-ONCOGENE P53 PRODUCED BY EMBRYONIC EXTRACT

Before:

Before

After:

After

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CELL PROLIFERATION CURVES OF DIFFERENT HUMAN TUMOR LINES AFTER IN VITRO TREATMENT WITH ZEBRAFISH EMBRYONIC EXTRCTS

Abstract:

Five tumor cell lines of different origin (glioblastoma, melanoma, kidney adenocarcinoma, breast carcinoma and lymphoblastic leukemia) were treated in vitro with the extracts from zebrafish embryos collected at four different developmental stages. All cell lines responded with a significant slowing of the proliferation when treated with the extracts taken during the stages of cell differentiation, while no slowing effect was observed when they were treated with the extract taken from a merely multiplicative stage. These results suggest that a complex network of molecular factors during embryo differentiation may help abnormally proliferating cells to normalize their cycle, and that the administration of embryonic cell differentiation factors may be a useful tool in cancer therapy. On the other hand, it is known that the stem cells can be differentiated into different types of cells in relationship to different kinds of embryonic microenvironment. Since this network of cell differentiation factors may normalize the altered expression of genes, we suggest it as a sort of physiological gene therapy.

ABSTRACT:
Five tumor cell lines of different origin (glioblastoma, melanoma, kidney adenocarcinoma, breast carcinoma and lymphoblastic leukemia) were treated in vitro with the extracts from zebrafish embryos collected at four different developmental stages. All cell lines responded with a significant slowing of the proliferation when treated with the extracts taken during the stages of cell differentiation, while no slowing effect was observed when they were treated with the extract taken from a merely multiplicative stage. These results suggest that a complex network of molecular factors during embryo differentiation may help abnormally proliferating cells to normalize their cycle, and that the administration of embryonic cell differentiation factors may be a useful tool in cancer therapy. On the other hand, it is known that the stem cells can be differentiated into different types of cells in relationship to different kinds of embryonic microenvironment. Since this network of cell differentiation factors may normalize the altered expression of genes, we suggest it as a sort of physiological gene therapy.

Click here to open the link to the abtract:

http://zfin.org/cgi-bin/webdriver?MIval=aa-pubview2.apg&OID=ZDB-PUB-021017-40

These blocking mechanisms of the cell cycle have been observed in several tumor cell lines.
Specifically, tumor cell lines were investigated of:

  • kidney adenocarcinoma
  • Lymphoblastic Leukemia
  • Breast cancer
  • Melanoma
  • Glioblastoma

kidney-adenocarcinoma:

Lymphoblastic-Leukemia:

Breast-cancer:

Melanoma:

Glioblastoma:

Still in 2001 : Xenopus laevis embryos share antigens with zebrafish embryos and with human malignant neoplasms.

Abstract:

Previous experiments have demonstrated that antigens present in Xenopus laevis embryos are shared with human malignant neoplasms. In fact it is known that antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to different antigens present in human tumors. The aim of the the present study was to evaluate whether these antisera react also to different antigens contained in zebrafish embryos at different stages of cell differentiation. This was done with the agar-gel immunodiffusion method performed according to an already described protocol. The results that we obtained show that both antisera raised in against pellet and supernatant fractions of Xenopus laevis embryos react to antigens of zebrafish at different stages of cell differentiation.

Previous experiments have demonstrated that antigens present in Xenopus laevis embryos are shared with human malignant neoplasms. In fact it is known that antisera raised in rabbit against pellet and supernatant fraction of Xenopus laevis embryos react to different antigens present in human tumors. The aim of the present study was to evaluate whether these antisera react also to different antigens contained in zebrafish embryos at different stages of cell differentiation. This was done with the agar-gel immunodiffusion method performed according to an already described protocol.
The results that we obtained show that both antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to antigens of zebrafish at different stages of cell differentiation.
It has already been described that antibodies produced by immunizing rabbit to Xenopus laevis embryo fractions react with a variety of human malignant neoplasms. In fact antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to different antigens contained in 50 of 55 different tumors. These reactions were demonstrated histologically.1
The antigens in the supernatant fraction are probably different from those in the pellet fraction. Antisera raised against the pellet fraction react with carbohydrate antigens.2 The nature of the antigens in the supernatant is not known yet. These previous results suggested that Xenopus laevis ambryos may contain an “oncodevelopmental” carbohydrate re-expressed in human tumors and that Xenopus may be an useful source for tumor associated antigens of human malignant tumors.
It may be that the antigens of Xenopus laevis embryos are preserved during phylogeny and that they are shared with other different embryos. The aim of the present study is to evaluate if the antisera raised in rabbit to the pellet and supernatant fractions of Xenopus laevis embryos react with antigens of zebrafish embryos at different stages of cell differentiation.

Figure 1
Zebrafish: middle – blastula – gastrula
Antitsera R 750-I (supernatant) A R 755-I (pellet) B

Figure 2
Zebrafish: 5 somites
Antisera R 750-I (supernatant) A R 755-I (pellet) B

Figure3
Zebrafish: 20 somites
Antisera R 750-I (supernatant) A R 755-I (pellet) B

Materials and Methods
The embryos of zebrafish at the stages of middle-blastula-gastrula, 5 somites, 20 somites were washed in distilled water and placed in a solution of pure glycerol and 30% of ethanol at a 4:1 ratio. The embryos were sonicated with 2 cycles of 10 seconds each and further treated with a turboemulsifier. 35 microliters of these solutions were used in an agar-gel immunodiffusion test. The methods of preparation of antisera raised in rabbit to pellet and supernatant fractions of Xenopus laevis embryos have already been described.1,2 A solution of 1 gram of agar-gel diluted in 40 ml. of distilled water and in 10 ml. of TBE (Tris Boric acid EDTA) was put in a plastic support. The lyophilized antisera were resuspended in 500 microliters of distilled water and 35 microliters of this solution were distributed in different wells of agar gel. 35 microliters of extracts of zebrafih at different stages of cell differentiation were put in different wells, in front of the antisera at a distance of 1 cm. The agar-gel with antisera and embryonic extracts were incubated for 24 hours at 22 degrees centigrade. The agar gel was stained with Coomassie Blue for 15 minutes, then washed with distilled water and finally destained in a solution composed by 10% acetic, 45% ethanol, 45,% distilled water.

Results
The results that we obtained show that both antisera raised in rabbit to pellet and supernatant fraction of Xenopus laevis embryos react to antigens of zebrafish at the three stage of cell differentiation. In fact the Fig. 1, Fig.2 and Fig.3 show that the antigens of zebrafish embryos at the stages of 50% of epiboly, 5 somites, 20 somites react with antisera R750-1 raised in rabbit to supernatant (part A of the figure) and with antisera R755-1 raised in rabbit to pellet (part B).

Conclusions
These results suggest that Xenopus laevis embryos share antigens with zebrafish embryos. These antigens are conserved during phylogeny. They are expressed in the zebrafish embryo since the beginning of cell differentiation and are present until last organogenesis. These antigens are more expressed at the end than at the beginning of organogenesis, as the results illustrated in Fig. 3 show in comparison with the results of the Fig. 1 and 2. In any case, these antigens shared with different species of embryos are re-expressed in different human tumors as already described. Tumor cells re-express several others “oncodevelopmental” antigens in addition to those described from us. For this reason, tumor cells can be considered as mutated embryonic cells, in which “gene configurations” are similar to those present in embryo during the steps of multiplication comprised between two stages of cell differentiation. This hypothesis has been already put forward in previous reports.3,4,5,6,7,8 On the other hand, tumor cell genome is normally affected by a dramatically high number of altered genes, most of which playing an important role in normal embryo development. In fact, during tumorigenesis some embryonic genes are activated or mutated, leading the cell to an uncontrolled multiplication program.
Many substances present in embryo during organogenesis are able to reduce tumor growth in vitro or in vivo, because they regulate some important genes that control cell differentiation and multiplication.5,7 In fact, our previous works demonstrated that substances present in embryo during cell differentiation are able to reduce tumor growth in vivo4 and to activate the tumor suppressor p53 in different tumor cell lines in vitro.5 Other works led to the same hypothesis: in fact the transplantation of teratocarcinoma cells into the mouse blastocyst origins normal chimeric mice, since the teratocarcinoma cells are led to differentiate in various kinds of tissues9 Otherwise, the transplantation of embryonic stem cells in mice origins teratocarcinomas.10 Our results suggest that Xenopus laevis and zebrafish embryos may be an useful source for tumor associated antigens of human malignant tumors.

P. M. Biava,
Ospedale Civile SSG Milano,
Italia

A. Monguzzi,
Ospedale Civile SSG Milano,
Italia

D. Bonsignorio,
Ospedale Civile SSG Milano,
Italia

A. Frosi,
Ospedale Civile SSG Milano,
Italia

S. Sell,
Albany Medical College,
Albany, NY,USA;

J. V. Klavins,
Albert Einstein College Of Medicine,
New York, NY, US;

1. Klavins J. V., Sell S., Fuchs A.
J.Tumor Marker Oncol. 11/2: 36 (1996).
2. Zhang S., Sell S., Livingston P.O., Klavins J. V.
J. Tumor Marker Oncol. 12(2):52(1997).
3. Biava P. M., Fiorito A., Negro C.,
Mariani M. Cancer Lett. 41: 265-270 (1988).
4. Biava P. M., Carluccio A.
Biol. Medizin. 5:247-249(1995).
5. Biava P.M., Carluccio A.
J. Tumor Marker One. 12,4: 9-15 (1997).
6. Biava P. M.
Complessita e cancro. Leadership Medica – Anno XV. 1- (1999).
7. Biava P. M. Bonsignorio D.
J. Tumor Marker Onc. 17-47-54 (2002)
8. Biava P. M., et al
J. Tumor Marker Onc. 17-59-64 (2002). Stewart T. A., Mintz B.
9. Proc. Natl. Acad. Sci
USA, 78 :6314-6318.(1981)
10. Reubinoff B. E. et al.
Nat. Biotechnol. 18(4) : 399-404 (2000).

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Cancer and cell differentiation: a model to explain malignancy:

Cell Proliferation is affected by factors that are found in embryos and in pregnant uteri. This factors are organized in a network wohse complexity should be unscattered to retain its full efficacy. This is particulary true for embryos whose complex of molecular factors represents a closed microenviroment that can normalize the behavior of abnormously growing cell populations via a regulatory process involving key-role proteins of cell cycle homeostasis.

  • To read more, please download: Cancer and cell differentiation: a model to explain malignancy.
Download: Cancer and cell differentiation: a model to explain malignancy.pdf

Still in 2002: Mother-embryo cross-talk: the anti-cancer substances produced by mother and embryo during cell differentiation. A review of experimental data: J. Tumor Marker Oncology

Introduction
During pregnancy, a close cross-talk between mother and developing embryo is formed, made of a complex network of circulating molecular factors. This cross-talk is necessary for the prevention of pregnancy-threatening events, including the establishment of abnormally proliferating cell clones which may damage the integrity of this cross-talk.

Dwnload: Mother-embryo cross-talk: the anti-cancer substances produced by mother and embryo during cell differentiation.

Animal Studies:

The effects of stem differentiation factors on inhibition of tumor growth were in vivo tested on females of singular C57BL / 6 mice from the weight of 18-20 grams to which a subcutaneous Lewis primary carcinoma injection was performed. Therefore, both the size of the primary tumor, and the survival time of the mice, have been evaluated. In terms of development of the primary tumor, an extremely significant difference (P <0.001) was observed between treated and control mice (Figure 1) and thus also with regard to the survival ratio, always in favor of the treated mouse.

Biava, P.M.; Bonsignorio, D.; Hoxha, M.; Impagliazzo, M.; Frosi,
A.; Larese, F.; Negro, C. (2002) Mother-embryo cross-talk: the anti-cancer substances produced by mother and embryo during cell differentiation. A review of experimental data. J. Tumor Marker Oncol., 2002, 17, 55-58

Still in 2002: Post-traslational modification of the retinoblastoma protein (pRb) induced by in vitro administration of Zebrafish embryonic extracts on human kidney adenocarcinoma cell line

Tumor cells share several key-role features with embryonic cells. Tumor development is reduced or even suppressed in embryos during early differentiation processes suggest that factors present in the developing embryo may effect tumor growth. Experiments carried out in our lab showed that treatment of several tumor cell lines with embryonic and/or pregnant uteri homogenates inhibits tumor growth both in vitro and in vivo.

Download: Post-traslational modification of the retinoblastoma protein (pRb) induced by in vitro administration of Zebrafish embryonic extracts on human kidney adenocarcinoma cell line, from page 59.pdf

Still in 2002: Embryonic differentiation factors with anticancer properties:preliminary clinical results in the therapy for advanced tumors

It is possible to activate the p53 onco-suppressor after treatment with embryonic extracts on different tumor cells. The evidence that embryonic factors of cell differentiation can be used as a physiological gene therapy of cancer constituted the objective basis to prepare a therapy to test in human cancer. As a result, different products containing specific embryonic differentiation factors were prepared.

Download: Embryonic differentiation factors with anticancer properties:preliminary clinical results in the therapy for advanced tumors
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Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: an open randomized clinical trial

Abstract

There is no standard treatment for patients with advanced hepatocellular carcinoma (HCC). We developed a product containing stem cells differentiation stage factors (SCDSF) that inhibits tumor growth in vivo and in vitro. The aim of this open randomized study was to assess its efficacy in patients with HCC not suitable for resection, transplantation, ablation therapy, or arterial chemoembolization. A total of 179 consecutive patients were enrolled. We randomly assigned the patients to receive either SCDSF or only conservative treatment. Primary end points were tumor response and survival. Secondary end points were performance status and patient tolerance. Randomization was stopped at the second interim analysis (6 months) of the first 32 patients recruited when the inspection detected a significant difference in favor of treatment (p = 0.037). The responses to the therapy obtained in 154 additional patients confirmed previous results. Evaluation of survival showed a significant difference between the group of patients who responded to treatment versus the group with progression of disease (p < 0.001). Of the 23 treated patients with a performance status (PS) of 1, 19 changed to 0. The study indicated the efficacy of SCDSF treatment of the patients with intermediate-advanced HCC.

Download: Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: an open randomized clinical trial
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Zebrafish embryo proteins induce apoptosis in human colon cancer cells

Abstract

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G(2)/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.

Download from :

Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2).

Abstract

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G(2)/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.

 

SYNERGISTIC EFFECT BETWEEN CHEMOTHERAPY AND DIFFERENTIATION FACTORS

IMAGES OF TUMOR CELLS CaCo2

In order to verify the synergistic effect between chemotherapy and differentiation factors, cancer cell lines of colon were treated with:

  • 5-FLUORURACIL
  • DIFFERENTIATION FACTORS
  • 5-FLUOROURACIL + DIFFERENTIATION FACTORS

As can be seen from the table emerges a powerful synergistic activity in the association of Differentiation factors with Fluorouracil.

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Reprogramming of normal and cancer stem cells. Curr. Phar. Biotechnol

Abstract:

Over the last decade there has been an exponential rise in our understanding of the biochemical mechanisms controlling cell proliferation and differentiation. While the four transcription factors Oct4, Sox2, Klf4 and cMyc have shown to be sufficient to induce pluripotency in fibroblasts, there has in addition been much research into the mechanisms and pathways of cell differentiation and the specific properties of stem cells, namely their plasticity and capacity for trans-differentiation.

Download from: http://www.ingentaconnect.com/content/ben/cpb/2011/00000012/00000002/art00001

Abstract:

Over the last decade there has been an exponential rise in our understanding of the biochemical mechanisms controlling cell proliferation and differentiation. While the four transcription factors Oct4, Sox2, Klf4 and cMyc have shown to be sufficient to induce pluripotency in fibroblasts, there has in addition been much research into the mechanisms and pathways of cell differentiation and the specific properties of stem cells, namely their plasticity and capacity for trans-differentiation. These studies have allowed progress at very fundamental level, with the prospect of further progress – until recent years quite unimaginable – in the field of reparative, regenerative and transplant medicine. In fact, from the present time, the genetic engineering production of regulatory factors identified through such research, has allowed the production of new tissues and a new category of cell therapy products, in which the main biological action is carried out by cells or tissues, albeit in the presence of organic or inorganic matrices or coatings. Examples of this type of product are anti-tumor vaccines, in vitro cultivated skin, products made of structural and cellular elements for the reconstruction of bones, cartilages, teeth, etc. From the best, most analytical and detailed characterization of stem cells, then, it has become clear that some tumor cell behaviors – that have a crucial role in determining their malignity – can be attributed to the presence of cells with characteristics similar to those of stem cells. The field of cancer research is consequently also witnessing a surge in studies designed to identify the metabolic pathways common to tumor and stem cells. This will in turn cast light on which micro-environment factors can direct these pathways towards differentiation and induce cancerous cells to behave less aggressively. From this point of view, over recent years there has been a lively return to studies that were very significant in the 70’s and 80’s, on the role of the embryonic micro-environment in conditioning tumor cell behavior towards normal phenotypes. This research is now underway, and will in all probability lead to important results over the next few years. Against this background, this special issue on “Reprogramming of normal and cancer stem cells” focuses on research in terms of conditioning the fate of normal and tumor stem cells with a view to new prospects for therapies. The issue therefore begins with articles covering the possibility of reprogramming normal stem cells, including through use of biomaterials, and goes on to consider what characteristics of tumor stem cells can allow them to be identified and studied. This is followed by a series of further articles illustrating the role of the micro-environment in conditioning the fate of a tumor cell. A number of metabolic pathways characterizing and common to both stem and tumor cells are examined, in order to gain a better understanding of the possibilities of conditioning the fate of both cell types; in addition, the role played by infectious and inflammatory diseases in the genesis of tumor diseases is also considered. Today, in fact, we know that inflammatory processes can support rather than hinder tumor growth, and also that pro-inflammatory cytokines can promote tumor proliferation, inhibiting the cell pathways that are able to block the neoplastic growth. The special issue goes on with a series of articles taking a close look at the specific role played by the micro-environment in conditioning the destiny of the tumor stem cells present in some tumors, for example breast and retinoblastoma tumor, and the role played by the use of normal stem cells in treating disorders such as hematological diseases. One article also considers the risks run by some reprogramming techniques: for example, creating embryo cells via parthenogenesis can give rise to tumors. The issue continues with various articles illustrating in close detail the role of the embryonic micro-environment in conditioning the destiny of tumor cells. In this context, one review takes a look at general aspects, while others consider aspects that can help clarify the mechanisms underlying the capacity of factors of this type of microenvironment to reprogram a tumor cell. One mathematical model sets out from a description of the state of cell differentiation, making use of existing data from studies of tumor growth slowing, linked to the use of such factors, with the goal of shedding light on aspects such as fitness, dosage, and administration time for the differentiation factors on improvement in tumor inhibitory response. Other articles illustrate a number of clinical cases of full regression of hepatocellular carcinomas in intermediate-advanced stages observed following administration of stem cell differentiation factors, and describe the molecular mechanisms that might explain these inhibitory responses on the tumor growth. It should be noted that the randomized and controlled clinical studies launched to date using stem cell differentiation factors are limited to patients with intermediate-advanced stage of hepatocellular carcinomas where other therapies were no longer possible. These factors are at present used only for hepatocellular carcinomas, since it has been demonstrated that substances capable of slowing one tumor’s growth may be inefficacious for another type. Finally, it is important to note that research into the possibility of reprogramming normal and tumor stem cells requires a complex approach to the issue. In fact, the problem requires the study of networks of substances and genes involved in the reprogramming phase, demanding skills in a variety of different areas of research, not simply of medical/biological, but also mathematical/ computational and modeling, in view of the complexity and non-linearity of the processes being studied. A paradigm shift is underway, and the future will witness our engagement in increasing numbers of scientific studies requiring cross-disciplinary skills.

The new paradigm and the new ideas were well understood many years ago by Professor John Klavins, who has been for a long time President of the International Academy of Tumor Marker Oncology. Professor Klavins has always sustained my studies on reprogramming cancer cells, though the possibility of controlling tumor growth by using reprogramming factors was not considered realistic at the time I began studying it.

I wish to dedicate this work to my friend John Klavins.

ACKNOWLEDGEMENTS

Many thanks to Roberta Zorovini and Silvia Cefalo from Smile Tech srl, via Valdirivo 19 Trieste, Italy ([email protected]), for their accurate and thorough work as organizing and editing secretariat.

Document Type: Research Article

DOI: http://dx.doi.org/10.2174/138920111794295873

Publication date: 1 febbraio 2011

Still in 2011 : Cancer cell reprogramming: stem cell differentiation stage factors and an agent based model to optimize cancer treatment. Curr. Phar. Biotechnol.

Abstract

The recent tumor research has lead scientists to recognize the central role played by cancer stem cells in sustaining malignancy and chemo-resistance. A model of cancer presented by one of us describes the mechanisms that give rise to the different kinds of cancer stem-like cells and the role of these cells in cancer diseases. The model implies a shift in the conceptualization of the disease from reductionism to complexity theory. By exploiting the link between the agent-based simulation technique and the theory of complexity, the medical view is here translated into a corresponding computational model.

From: https://www.ncbi.nlm.nih.gov/pubmed/21044002

Abstract

The recent tumor research has lead scientists to recognize the central role played by cancer stem cells in sustaining malignancy and chemo-resistance. A model of cancer presented by one of us describes the mechanisms that give rise to the different kinds of cancer stem-like cells and the role of these cells in cancer diseases. The model implies a shift in the conceptualization of the disease from reductionism to complexity theory. By exploiting the link between the agent-based simulation technique and the theory of complexity, the medical view is here translated into a corresponding computational model. Two main categories of agents characterize the model, 1) cancer stem-like cells and 2) stem cell differentiation stage factors. Cancer cells agents are then distinguished based on the differentiation stage associated with the malignancy. Differentiation factors interact with cancer cells and then, with varying degrees of fitness, induce differentiation or cause apoptosis. The model inputs are then fitted to experimental data and numerical simulations carried out. By performing virtual experiments on the model’s choice variables a decision-maker (physician) can obtains insights on the progression of the disease and on the effects of a choice of administration frequency and or dose. The model also paves the way to future research, whose perspectives are discussed.

Download: Cancer Cell Reprogramming

Still in 2011 : Embryonic morphogenetic field induces phenotypic reversion in cancer cells. Curr. Phar. Biotechnol.

Abstract

Cancer cells introduced into developing embryos can be committed to a complete reversion of their malignant phenotype. It is unlikely that such effects could be ascribed to only few molecular components interacting according to a simple linear-dynamics model, and they claim against the somatic mutation theory of cancer. Some 50 years ago, Needham and Waddington speculated that cancer represents an escape from morphogenetic field like those which guide embryonic development. Indeed, disruption of the morphogenetic field of a tissue can promote the onset as well as the progression of cancer. On the other hand, placing tumor cells into a “normal” morphogenetic field – like that of an embryonic tissue – one can reverse malignant phenotype, “reprogramming” tumor into normal cells.

From: https://www.ncbi.nlm.nih.gov/pubmed/21044001

Abstract

Cancer cells introduced into developing embryos can be committed to a complete reversion of their malignant phenotype. It is unlikely that such effects could be ascribed to only few molecular components interacting according to a simple linear-dynamics model, and they claim against the somatic mutation theory of cancer. Some 50 years ago, Needham and Waddington speculated that cancer represents an escape from morphogenetic field like those which guide embryonic development. Indeed, disruption of the morphogenetic field of a tissue can promote the onset as well as the progression of cancer. On the other hand, placing tumor cells into a “normal” morphogenetic field – like that of an embryonic tissue – one can reverse malignant phenotype, “reprogramming” tumor into normal cells. According to the theoretical framework provided by the thermodynamics of dissipative systems, morphogenetic fields could be considered as distinct attractors, to which cell behaviors are converging. Cancer-attractors are likely positioned somewhat close to embryonic-attractors. Indeed, tumors share several morphological and ultra-structural features with embryonic cells. The recovering of an “embryonic-like” cell shape might enable the gene regulatory network to reactivate embryonic programs, and consequently to express antigenic and biochemical embryonic characters. This condition confers to cancer an unusual sensitivity to embryonic regulatory cues. Thus, it is not surprising that cancer cells exposed to specific embryonic morphogenetic fields undergoes significant modifications, eventually leading to a complete phenotypic reversion.

Still in 2011 :

Zebrafish stem cell differentiation stage factors suppress Bcl-xL release and enhance 5-Fu-mediated apoptosis in colon cancer cells.

Abstract

Stem cell differentiation stage factors (SCDSF), taken from Zebrafish embryos during the stage in which totipotent stem cells are differentiating into pluripotent stem cells, have been shown to inhibit proliferation and induce apoptosis in colon tumors. In order to ascertain if these embryonic factors could synergistically/additively interact with 5-Fluorouracil (5-Fu), whole cell-count, flow-cytometry analysis and apoptotic parameters were recorded in human colon cancer cells (Caco2) treated with Zebrafish stem cell differentiation stage factors (SCDSF 3 µg/ml) in association or not with 5-Fu in the sub-pharmacological therapeutic range (0.01 mg/ml). Cell proliferation was significantly reduced by SCDSF, meanwhile SCDSF+5-Fu leads to an almost complete growth-inhibition. SCDSF produces a significant apoptotic effect, meanwhile the association with 5-FU leads to an enhanced additive apoptotic rate at both 24 and 72 hrs.

From: https://www.ncbi.nlm.nih.gov/pubmed/21043999

Abstract

Stem cell differentiation stage factors (SCDSF), taken from Zebrafish embryos during the stage in which totipotent stem cells are differentiating into pluripotent stem cells, have been shown to inhibit proliferation and induce apoptosis in colon tumors. In order to ascertain if these embryonic factors could synergistically/additively interact with 5-Fluorouracil (5-Fu), whole cell-count, flow-cytometry analysis and apoptotic parameters were recorded in human colon cancer cells (Caco2) treated with Zebrafish stem cell differentiation stage factors (SCDSF 3 µg/ml) in association or not with 5-Fu in the sub-pharmacological therapeutic range (0.01 mg/ml). Cell proliferation was significantly reduced by SCDSF, meanwhile SCDSF+5-Fu leads to an almost complete growth-inhibition. SCDSF produces a significant apoptotic effect, meanwhile the association with 5-FU leads to an enhanced additive apoptotic rate at both 24 and 72 hrs. SCDSF alone and in association with 5-Fu trigger both the extrinsic and the intrinsic apoptotic pathways, activating caspase-8, -3 and -7. SCDSF and 5-Fu alone exerted opposite effects on Bax and Bcl-xL proteins, meanwhile SCDSF+5-Fu induced an almost complete suppression of Bcl-xL release and a dramatic increase in the Bax/Bcl-xL ratio. These data suggest that zebrafish embryo factors could improve chemotherapy efficacy by reducing anti-apoptotic proteins involved in drug-resistance processes.

0
The zebrafish embryo derivative affects cell viability of epidermal cells: a possible role in the treatment of psoriasis.

Abstract

In patients affected by psoriasis, use of a topical formula containing a derivative of zebrafish embryos was associated with reduced skin inflammation and dermal turnover, as well as a generally better outcome. In an attempt to understand the molecular mechanisms lying beyond these findings, we investigated the anti-proliferative effects of the zebrafish embryos derivative by addressing the mitochondrial function (MTT assay) and cell nuclei distribution (Hoestch staining). In cell cultures stimulated with fetal calf serum (FCS) or epidermal growth factor (EGF), the zebrafish derivative significantly inhibited cell proliferation induced by either approach, although the effect was stronger in cells stimulated with FCS. These results suggest that the zebrafish embryos derivative may dampen increased cell proliferation; this observation may be relevant to cutaneous pathologies related to altered proliferative mechanisms, including psoriasis.

From: https://www.ncbi.nlm.nih.gov/pubmed/24005140

Abstract

In patients affected by psoriasis, use of a topical formula containing a derivative of zebrafish embryos was associated with reduced skin inflammation and dermal turnover, as well as a generally better outcome. In an attempt to understand the molecular mechanisms lying beyond these findings, we investigated the anti-proliferative effects of the zebrafish embryos derivative by addressing the mitochondrial function (MTT assay) and cell nuclei distribution (Hoestch staining). In cell cultures stimulated with fetal calf serum (FCS) or epidermal growth factor (EGF), the zebrafish derivative significantly inhibited cell proliferation induced by either approach, although the effect was stronger in cells stimulated with FCS. These results suggest that the zebrafish embryos derivative may dampen increased cell proliferation; this observation may be relevant to cutaneous pathologies related to altered proliferative mechanisms, including psoriasis.

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Still in 2013:

Rediscovering Meaning

Abstract

The present crisis of the Western countries involves not just the world of politics, finance, and the economy, but also the ecosystem balance, the world of the values on which the model of social organization and economic development is based. The crisis is therefore systemic, epoch-making, and overcoming it will require radical changes, above all in the way we think, in our scale of values, and therefore our culture. On the other hand, for decades the world—in particular the West—has been experiencing a cultural sea-change that is challenging many of the pillars of society that have stood for centuries. The young people of today and their use of technological social networks to develop non-profit initiatives for social change may create a more positive future while maintaining our human values.

From: World Futures

The Journal of New Paradigm Research

https://www.tandfonline.com/doi/full/10.1080/02604027.2012.762196

Abstract

The present crisis of the Western countries involves not just the world of politics, finance, and the economy, but also the ecosystem balance, the world of the values on which the model of social organization and economic development is based. The crisis is therefore systemic, epoch-making, and overcoming it will require radical changes, above all in the way we think, in our scale of values, and therefore our culture. On the other hand, for decades the world—in particular the West—has been experiencing a cultural sea-change that is challenging many of the pillars of society that have stood for centuries. The young people of today and their use of technological social networks to develop non-profit initiatives for social change may create a more positive future while maintaining our human values.

0
A systemic approach to cancer treatment: tumor cell reprogramming focused on endocrine-related cancers.

Abstract

The term “cancer cell reprogramming” is used to define any kind of intervention aimed at transforming cancer cells into terminally differentiated cells. Using this approach, new technologies have been applied with different methods for a more systemic approach to cancer treatment. This review reports on advances of these technologies, including our personal contributions, mainly carried out on endocrine-related cancers. Some of the interventions, aimed at reverting cancer cells into a normal phenotype, are based on the evidence that tumor development is suppressed by the embryonic microenvironment. On the basis of this rationale, experiments have been conducted using stem cell differentiation stage factors (SCDSFs) taken at different stages of development of Zebrafish embryos, oocyte extracts, or naïve human umbilical cord matrix derived stem cells (UMDSCs).

From: https://www.ncbi.nlm.nih.gov/pubmed/24304275

Abstract

The term “cancer cell reprogramming” is used to define any kind of intervention aimed at transforming cancer cells into terminally differentiated cells. Using this approach, new technologies have been applied with different methods for a more systemic approach to cancer treatment. This review reports on advances of these technologies, including our personal contributions, mainly carried out on endocrine-related cancers. Some of the interventions, aimed at reverting cancer cells into a normal phenotype, are based on the evidence that tumor development is suppressed by the embryonic microenvironment. On the basis of this rationale, experiments have been conducted using stem cell differentiation stage factors (SCDSFs) taken at different stages of development of Zebrafish embryos, oocyte extracts, or naïve human umbilical cord matrix derived stem cells (UMDSCs). SCDSFs induce significant growth inhibition on different tumor cell lines in vitro, likely because of increases in cell cycle regulatory molecules, such as p53 and pRb. Treatment with these factors activates apoptosis and differentiation related to caspase-3. This is achieved via p73 apoptotic-dependent pathway activation with a concurrent normalization of the E-cadherin and beta-catenin ratio. Extracts from prophase amphibian oocytes could reprogram relevant epigenetic alterations in MCF-7 and HCC1954 breast cancer cell lines, while un-engineered (naïve) human UMDSCs attenuated growth of MDA-231 human breast carcinoma cells. A product prepared for human treatments, containing SCDSFs at very low doses, yielded favorable results in breast cancer and in intermediate-advanced hepatocellular carcinoma. Other reprogramming interventions used in the models of breast, prostate and ovarian cancer cell lines are described. Finally, current and future perspectives of this novel technology are discussed and a new hallmark of cancer is suggested: the loss of differentiation of cancer cells.

Still in 2014:

Changing the endocrine dependence of breast cancer: data and hypotheses.

Abstract

Among the most common human cancers, often only breast and prostate cancers have advantage of hormone dependence. For a long time, this advantage permitted breast cancer to be efficaciously managed in the adjuvant and metastatic settings with low side effects by endocrine therapy. Unfortunately, soon or afterward hormone dependence is lost in most patients. In breast cancer, de novo or acquired hormone resistance is an hot issue and the focus of endless debate. Although a lack of oestrogen receptors (ERs) is considered to be the main reason for de novo hormone resistance, many studies have been conducted and many different mechanisms have been hypothesised to account for acquired hormone resistance.

From: https://www.ncbi.nlm.nih.gov/pubmed/24304277

Abstract

Among the most common human cancers, often only breast and prostate cancers have advantage of hormone dependence. For a long time, this advantage permitted breast cancer to be efficaciously managed in the adjuvant and metastatic settings with low side effects by endocrine therapy. Unfortunately, soon or afterward hormone dependence is lost in most patients. In breast cancer, de novo or acquired hormone resistance is an hot issue and the focus of endless debate. Although a lack of oestrogen receptors (ERs) is considered to be the main reason for de novo hormone resistance, many studies have been conducted and many different mechanisms have been hypothesised to account for acquired hormone resistance. Thus far, hormone resistance appears to be occasionally delayed or avoided in “in vivo” experiments. However, this finding did not have a significant benefit in current clinical practice. The principal aim of this review article is to sum up and update the issue of changing the endocrine dependence of breast cancer. Recent molecular insights extensively elucidating and shedding new light on this very controversial issue are considered. Moreover, based on our recent reports, a new mechanistic interpretation of and a therapeutic approach for overcome hormone resistance are proposed.

Still in 2014:

The role of neuroendocrine cells in prostate cancer: a comprehensive review of current literature and subsequent rationale to broaden and integrate current treatment modalities.

Abstract

Neuroendocrine prostate carcinoma (NE-PCa) is a heterogeneous disease. Due to a high prevalence of NE (neuroendocrine) differentiation in patients who receive prolonged androgen deprivation treatment, the real incidence of NE-PCa remains unknown. Similarly, the biological steps from prostate carcinoma (PCa) toward NE differentiation are far less than definitive and, consequently, there is a lack of evidence to support any of the treatments as the “gold standard”.

From: https://www.ncbi.nlm.nih.gov/pubmed/24304274

Abstract

Neuroendocrine prostate carcinoma (NE-PCa) is a heterogeneous disease. Due to a high prevalence of NE (neuroendocrine) differentiation in patients who receive prolonged androgen deprivation treatment, the real incidence of NE-PCa remains unknown. Similarly, the biological steps from prostate carcinoma (PCa) toward NE differentiation are far less than definitive and, consequently, there is a lack of evidence to support any of the treatments as the “gold standard”.

MATERIALS AND METHODS:

A systematic literature search was conducted using the PubMed, Scopus, and Embase databases to identify original articles and review articles regarding NE-PCa . Keywords were “prostate cancer” and “neuroendocrine”. Articles published between 1995 and 2013, were reviewed and selected with the consensus of all of the authors.

RESULTS:

Fifty-one articles were selected by the authors for the purpose of this review. The principle findings were reported into some subsections: Epidemiology, Biological steps of NE differentiation (with some principle articles on animal and in vitro, since there is very little in the literature on human studies); for the treatment options, we had to expand the search on PubMed to a larger timeframe and selection since very little was specifically found in the first criteria: surgery, radiotherapy, ablative techniques, immunomodulation and epigenetic therapy were then reviewed. A multidisciplinary approach, advocated by many authors, although promising, has failed to demonstrate increased survival rates. Limitations of this review include the lack of a clear definition of NE-PCa and consequently, the lack of strong evidence provided by a large series with long-term follow-up.

CONCLUSIONS:

Supported from this extensive review, we propose it is worthwhile to investigate a new multimodal therapeutic approach to address advanced NE-PCa starting from a debulking (with radical intent) of the disease plus epigenetic therapy with stem cell differentiation stage factors (SCDSFs). In addition immunotherapy can be used to treat the cancer presenting phenotype in association with chemomodulation plus ablative therapies, in case of advanced or recurrent diseases. SCDSFs may be utilized to regulate cancer stem cells and possible new phenotypes could also be associated with ablative therapies. Hormonal deprivation, radiotherapy, chemotherapy, ex vivo vaccines and targeted therapies could also be used and reserved in case of failure.

Still in 2014:

Human Stem Cell Exposure to Developmental Stage Zebrafish Extracts: a Novel Strategy for Tuning Stemness and Senescence Patterning

Abstract

BACKGROUND: Zebrafish exhibits extraordinary ability for tissue regeneration. Despite growing investigations dissecting the molecular underpinning of such regenerative potential, little is known about the possibility to use the chemical inventory of the zebrafish embryo to modulate human stem cell dynamics.

RESULTS: Late developmental stage extracts decreased cell viability and elicited caspase-3 mediated apoptosis. This effect did not involve Bax or Bcl-2 transcription. Conversely, early developmental stage ZF1 did not affect cell viability or apoptosis, albeit increasing Bax/Bcl-2 mRNA ratio. ZF1 enhanced transcription of the stemness/pluripotency genes Oct-4, Sox-2 and c-Myc. ZF1 also induced the transcription of TERT, encoding the catalytic subunit of telomerase, as well as the gene expression of Bmi-1, a chromatin remodeler acting as a major telomerase-independent repressor of senescence. These transcriptional responses were restricted to the action of early stage factors, since they were not elicited by late developmental stage ZF5.

From: http://www.cellr4.org/article/1226

Abstract

BACKGROUND: Zebrafish exhibits extraordinary ability for tissue regeneration. Despite growing investigations dissecting the molecular underpinning of such regenerative potential, little is known about the possibility to use the chemical inventory of the zebrafish embryo to modulate human stem cell dynamics.

METHODS: Extracts from zebrafish embryo were collected at different developmental stages, referred to as ZF1, ZF2, ZF3 (early stages), and ZF4, ZF5 (late stages). Human adipose-derived stem cells (hASCs), isolated from microfractured fat tissue obtained with a novel non-enzymatic method (Lipogems), were cultured in absence or presence of each developmental stage extract. Cell viability was assessed by MTT assay. Nuclear morphology was investigated by cell-permeable dye 4’,6-DAPI. Caspase-3 activity was assessed by ELISA. Gene transcription was monitored by real-time PCR.

RESULTS: Late developmental stage extracts decreased cell viability and elicited caspase-3 mediated apoptosis. This effect did not involve Bax or Bcl-2 transcription. Conversely, early developmental stage ZF1 did not affect cell viability or apoptosis, albeit increasing Bax/Bcl-2 mRNA ratio. ZF1 enhanced transcription of the stemness/pluripotency genes Oct-4, Sox-2 and c-Myc. ZF1 also induced the transcription of TERT, encoding the catalytic subunit of telomerase, as well as the gene expression of Bmi-1, a chromatin remodeler acting as a major telomerase-independent repressor of senescence. These transcriptional responses were restricted to the action of early stage factors, since they were not elicited by late developmental stage ZF5.

CONCLUSIONS: Exposure to early developmental stage zebrafish embryo extracts may enhance stem cell expression of multipotency and activate both telomerase-dependent and -independent antagonists of cell senescence. These outcomes may prove rewarding during prolonged expansion in culture, as it occurs in most cell therapy protocols.

Margherita Maioli, Federica Facchin, and Eva Bianconi equally contributed to the study

INTRODUCTION

Zebrafish maintains a remarkably higher ability than mammals to repair complex tissues after injury, including the heart and the central nervous system. For this reason, zebrafish embryos and their stem cells have been increasingly studied to unravel the molecular mechanisms underlying such regenerative potential, or to dissect evolutionary conserved pathways that may account for the regenerative action afforded by stem cells across different species.

Transplantation of human cord blood-derived CD34+ (hCD34+) cells into pregastrulation zebrafish embryos revealed that these human cells cosegregated with presumptive zebrafish hemangioblasts, being involved in early development of the embryonic vasculature of the recipient 1. Conversely, postgastrulation transplant resulted in the recruitment of hCD34+ cells into developing vessels, where their biology was mainly shifted to a paracrine action 2. These human cells were also found to accelerate vascular repair in adult zebrafish, after transplantation in a model of vascular regeneration induced by caudal fin amputation 3. These observations indicate unexpected developmental skills in human stem/progenitor cells and show that the possibility to modulate their differentiating and/or paracrine repertoire within the zebrafish embryo is tightly dependent from the microenvironmental context in a developmental stage-dependent fashion.

There is also growing evidence that embryonic development and tumorigenesis are closely correlated, as it can be inferred from the fact that they share several molecular pathways and regulatory molecules 4 , 5 , 6 , 7 , 8 , 9 , 10, and from the high tumorigenic risk associated with the acquirement of a pluripotent embryonic-like state, as it occurs during the preparation of human induced pluripotent stem cells 11 , 12. To this end, it has been shown that cell proliferation curves of different human cancer cell lines could be slowed down following exposure to zebrafish embryo extracts harvested during the stages of cell differentiation, with no significant antiproliferative effect in the presence of extracts taken from a merely duplicative stage 13 , 14 , 15 , 16 , 17. These observations also provide intriguing cues on the possibility to consider the tumorigenic process as a developmental deviation susceptible to control by regulators of cell differentiation, tracing a glimpse for future strategies of cancer (stem) cell reprogramming in the presence of differentiation stage factors produced by normal stem cells.

Despite the growing body of knowledge on the biology of the zebrafish embryo and stem cells, including the chance to use their secretome to impact on cancer cell dynamics, comparatively little is known about the possibility to exploit the chemical milieu provided at different developmental stages by the zebrafish embryo to modulate the homeostasis of human stem cells.

In the current study, we explored this novel perspective by exposing to zebrafish extracts, yielded at different developmental stages, human adipose-derived stem cells (hASCs), isolated from a microfractured fat tissue obtained with a novel non-enzymatic method and device (Lipogems) 18. We found that only the treatment with early developmental stage extracts was able to modulate the stem cell expression of multipotency, and elicited the transcriptional activation of two major mechanisms capable of counteracting stem cell senescence, including the gene expression of TERT, the catalytic subunit of telomerase, and the gene transcription of Bmi-1, a member of the Polycomb and Trithorax families group of repressors, acting as an essential factor for the self-renewal of adult stem cells, and as a key telomerase independent repressor of cell aging 19 , 20 , 21 , 22. Late, but not early developmental stage extracts induced a significant decrease in stem cell proliferation and the activation of pro-apoptotic signatures.

METHODS

Fat tissue processing, hASC harvesting and culture

According to the policies approved by the Institutional Review Boards for Human Studies local ethical committees, all tissue samples were obtained after informed consent. Human subcutaneous adipose tissue samples were obtained from lipoaspiration procedures and processed by using the Lipogems device, as previously described 23.

A volume of 1.5 ml of Lipogems product has been seeded in a T75 flask precoated with human fibronectin (0.55 μg/cm2) (Sigma-Aldrich Co., St. Louis, MO, USA) and human collagen I-III (0.50 μg/cm2) (ABCell-Bio), cultured in α-MEM medium supplemented with 20% heat-inactivated FBS, antibiotics (200 units/ml penicillin, 100 μg/ml streptomycin), L-Glutamine (1%), and incubated at 37°C in a humidified atmosphere with 5% CO2. Medium has been changed every 4 days, but the Lipogems product was maintained in culture for two weeks, then it was eliminated. At confluence, released cells were detached by treatment with trypsin-EDTA (Sigma-Aldrich Co., St. Louis, MO, USA), and subcultured. Experiments were performed at passage 3-5. All cell cultures were maintained 24 hours in standard conditions before treatments.

Zebrafish embryo extracts

Zebrafish embryos were harvested and processed as previously described 24 at 5 different developmental stages: 50% epiboly [5 hours post fertilization (hpf)], tail bud (10 hpf), 5 somites (12 hpf), 20 somites (19 hpf) and pharyngula (24 hpf), referred to as ZF1, ZF2, ZF3, ZF4 and ZF5, respectively. The embryos were washed in distilled water for 60 sec at the density of 800 eggs/ml. Extracts were prepared in a glycero-alcoholic solution (60% glycerol, 5% ethanol, 0.12% potassium sorbate and 0.08% sodium benzoate) and stored at 4°C until use. A 0.5% dilution of glycero-alcoholic solution in α-MEM with complements was used as a control in all experiments.

BCA protein assay

Protein content of each zebrafish embryo extract was determined with BCA protein assay kit, following the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA). A serial dilution of bovine serum albumin was used as standard, and NanoDrop (Nanodrop ND 1000 v.3.8.1, Wilmington, DE, USA) was used to determine the protein content of the extracts.

Cell viability

hASCs at the density of 5,000 cells/cm2 were cultured in a 24-well plate and incubated for 24 hours prior to starting the treatments. Cells were treated with ZF1, ZF2, ZF3, ZF4, or ZF5, each at four different concentrations: 0.01, 0.1, 1, or 10 μg/ml. As controls, hASCs were treated with a 0.5% glycero-alcoholic solution. The number of viable cells was determined after 24 and 72 hours of treatment, using the 3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyl-tetrazolium bromide (MTT, Sigma-Aldrich Co., St. Louis, MO, USA) assay. Briefly, at defined time points, cells were incubated for 3 hours with MTT in standard conditions (previously described) and subsequently lysed with a lysis buffer (90% iso-propanol, 10% TritonX-100 and 0.008% HCl). Absorbance of formazan salt was measured at 595 nm using a microplate reader (Opsys MR Microplate Reader; Dynex Technologies) and data were analyzed in GraphPad by one-way ANOVA followed by Dunnett’s post-hoc test. Each treatment was performed in triplicate, and the whole experiment was repeated in cells derived from at least three independent subjects.

Caspase-3 activity

To determine caspase-3 activity in hASCs treated in the absence or presence of ZF extracts, 10,000 cells/well were seeded in a 96-well black clear-bottom plate and cultured for 24 hours in standard conditions. Cells were treated with ZF4, or ZF5 at 0.1, 1, or 10 μg/ml for 72 hours. hASCs treated with a 0.5% glycero-alcoholic solution were used as control cells. Caspase-3 activity was determined using the Caspase-3 Colorimetric In-Cell ELISA Kit (Pierce Biotechnology, Rockford, IL, USA), following the manufacturer’s instructions, including staining with Janus Green as whole cell stain. Each treatment was performed in duplicate, and the whole experiment was repeated in cells derived from three independent subjects.

Assessment of nuclear morphology

To further assess cell apoptosis, hASCs were seeded at a density of 7,000 cells/cm2 onto coverslips placed in 24-well plates and maintained in standard conditions for 24 hours before treatments. Cells were treated with ZF4, or ZF5 at 10 μg/ml or with 0.5% glycero-alcoholic solution in α-MEM with complements as controls. After 72 hours, cells were fixed for 20 min using 4% formaldehyde, stained with UltraCruz Mounting medium for fluorescence with DAPI (cell-permeable dye 4’,6-DAPI) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and examined using a fluorescence microscope (Leitz Orthoplan; Leica DFC300 FX Digital Color Camera) to visualize the chromatin condensation and/or fragmentation typical for apoptotic cells. Each treatment was performed in duplicate, and the whole experiment was repeated in cells derived from three independent subjects. At least 200 cells, from different areas of the coverslips, were subjected to visual score for each sample.

RNA extraction and RT-PCR

hASCs were cultured in a 6-well plate at the density of 7,000 cells/cm2 and incubated for 24 hours before treatment. Cells were treated for 72 hours with the indicated ZF at 10 μg/ml or with 0.5% glycero-alcoholic solution in α-MEM with complements as a control. Each treatment was performed in duplicate, and the whole experiment was repeated in cells derived from three independent subjects.

Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. RNA was subjected to reverse transcription (RT) using the following conditions: 2 µg total RNA, Moloney murine leukaemia virus reverse-transcriptase (Promega, Madison, WI; used with companion buffer) 400 U, oligo dT-15 2.5 µM, Random Hexamers 2 µM, dNTPs 500 µM each. RT reaction was performed in a final volume of 50 µl for 60 min at 37°C. In order to verify that the RT reaction was successful, amplification of the human Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was performed, using specific primers (Table 1). GAPDH amplification was performed in a final volume of 25 µl, using the following conditions; 1 µl cDNA, 0.2 µM each primer, 12.5 µl BioMix Red (Bioline, Taunton, MA); initial denaturation for 2 min at 94°C; 25 cycles of 30 s at 94°C, 30 s at 61°C (annealing temperature of GAPDH primers), 30 s at 72°C followed by a final extension for 7 min at 72°C. Amplicon detection was performed by gel electrophoresis in 1.5% agarose gel in TAE 1X (Merck) stained with 0.5 μg/ml Etidium Bromide (SIGMA) and visualized with UV-light.

Real-time PCR

Quantitative real-time PCR was performed using an iCycler Thermal Cycler (Bio-Rad). Two μl cDNA were amplified in 50-μl reactions using Platinum Supermix UDG (Invitrogen), 200 nM each primer, 10 nM fluorescein (Bio-Rad), and Sybr Green. After an initial denaturation step at 94°C for 10 min, temperature cycling was initiated. Each cycle consisted of 94°C for 15 s, 55-59°C for 30 s and 60°C for 30 s, the fluorescence being read at the end of this step. The primers used were specific and spanning exons. Their list is reported in Table 1.

To evaluate the quality of product of real-time PCR assays, melting curve analysis was performed after each assay. Relative expression was determined using the “delta-CT method”25 with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as a reference gene.

Data analysis

Data are presented as mean ± standard deviation. As indicated in the legend of each figure, the statistical analysis was determined by one-way ANOVA followed by Dunnett’s post-hoc test, or by two-tailed unpaired Student’s t test. A p value less than 0.05 was assumed as the limit of significance.

RESULTS

Late- but not early-developmental stage extracts from zebrafish embryo reduce hASC viability

We first investigated whether hASC exposure to embryo extracts collected at different developmental stages may differentially affect cell viability. Cell culture in the presence of ZF4 or ZF5 only produced a slight decrease in the yield of viable cells after 24 hours (Figure 1, A), but resulted in a significant, dose-dependent reduction in the number of viable elements after 72 hours of exposure (Figure 1, B). No effect on cell viability was detected after treatment for 24 or 72 hours with early developmental stage embryo extracts, including ZF1, ZF2 or ZF3 (Figure 1, A and B).

We next investigated whether the reduced cell viability observed after treatment with late developmental stage extracts may be due to an increase in cell apoptosis. As shown in Figure 2, caspase-3 activity was significantly increased in cells treated with ZF4 or ZF5 at 10 μg/ml for 72 hours, as compared with the control cells. Caspase-3 activity was not affected by ZF1, ZF2 or ZF3 (not shown).

The induction of an apoptotic trait following exposure to late developmental stage extracts was further inferred by the analysis of nuclear morphology showing evident chromatin condensation and increased percentage of apoptotic cells in the presence of 10 μg/ml ZF4 or ZF5, as compared to unexposed hASCs (Figure 3).

Gene expression analysis revealed that hASC treatment with ZF1 for 72 hours enhanced Bax transcription (Figure 4, A) along with a downregulation in the expression of Bcl-2 mRNA (Figure 4, B), encoding a major prosurvival player 26. On the other hand, exposure to the late stage ZF5 did not affect significantly both Bax and Bcl-2 transcription (Figure 4, A and B).

Early-, but not late-developmental stage embryo extracts enhance the expression of stemness related genes and activate the transcription of antisenescence orchestrators

Real-time PCR shown that hASC treatment with the early developmental stage ZF1 for 72 hours was able to remarkably increase the transcription of Oct-4 and Sox-2 (Figure 5, A and B). The transcription of both genes was not affected by the late developmental stage ZF5 (Figure 5, A and B). Similar to Oct-4 and Sox-2 expression, the transcription of c-Myc was upregulated by ZF1, while being unaffected by ZF5 (Figure 5, C).

Exposure of hASCs in the presence of ZF1 led to a consistent overexpression of the TERT gene, encoding the catalytic subunit of telomerase (Figure 6, A). The gene expression of Bmi-1, a transcriptional regulator involved in chromatin remodeling and acting as a telomerase-independent repressor of senescence, was also increased by ZF1 (Figure 6, B). Stem cell treatment with ZF5 did not modify TERT transcription, but significantly reduced Bmi-1 gene expression (Figure 6, A and B).

DISCUSSION

In the last few years, a growing body of studies has been designed to exploit the rescuing potential of human mesenchymal stem cells (hMSCs) for the treatment of injured tissues. Although hMSCs have been isolated from many different tissues, their number is exiguous in all tissue sources. Meta-analysis of currently available cell therapy protocols shows that hMSCs are transplanted at high doses, between 10 and 400 million hMSCs per treatment (www.clinicaltrials.gov). The needs for expansion is particularly envisionable in protocols based on intravascular delivery of stem cells, that cannot be transplanted in form of tissue extracts (i.e. processed fat tissue). To fulfill these requirements, hMSCs undergo multiple passages and prolonged time in culture, usually 8-12 weeks. This approach has been shown to be both a risk of, and a well-established model for cell aging in vitro 27 , 28. Moreover, prolonged expansion impairs stem cell expression of pluripotency/multipotency, leading to a consistent decline in the multilineage repertoire and in the yield of differentiated cells.

hASCs are now being used as an easy-to-harvest tool for cell therapy, and exhibit phenotypic and transcriptional profiles similar to hMSCs, as well as robust multilineage potential in vitro. Despite these attractive features, hASCs also undergo significant senescence and decline in multipotency expression after multiple passages in culture 29 , 30 , 31. These findings raise cautionary notes whenever long passaged hASCs are used in a clinical setting, and prompt the needs for novel approaches that may oppose senescence and optimize the expression of multipotency in such a promising tool for cell therapy.

Here, we have exposed hASCs in the presence of zebrafish extracts harvested at different developmental stages, showing that only the late developmental stage extracts (ZF4 and 5) significantly decreased cell proliferation and viability. The ZF4/ZF5 effect involved the activation of a proapoptotic program, as shown by the derangement in nuclear morphology and the chromatin condensation, and by the activation of caspase-3. The finding that ZF5 failed to affect the transcription of Bax and Bcl-2, two major players in the modulation of apoptosis, suggests that apoptosis mediated by late stage developmental factors of zebrafish embryo is Bax-independent. This phenomenon has long been observed, as shown in the case of Bax-independent, caspase-3-related apoptosis induced by HGF in rat liver epithelial cells 32, and recently confirmed in both malignant and normal cells 33 , 34.

Unlike ZF5, ZF1-3 did not induce hASC apoptosis, nor they decreased cell viability. This different behaviour may result from a fine equilibrium between ZF1-induced transcription of Oct-4, Sox-2, TERT, Bmi-1 and c-Myc, which have all been found to inhibit apoptotic pathways 35 , 36 , 37 , 38 , 39 40, and the increase in Bax/Bcl-2 mRNA ratio observed in ZF1-exposed hASCs. This hypothesis is consonant with the emerging view that crucial modulators of apoptotic pathways do not behave as on-off transcriptional specifiers, but they rather act as non-linear boosters of the expression of active genes, based upon the adjustments in the settings of critical thresholds resulting from the inventory of regulatory players in given cell populations 41. To this end, c-Myc has also been found to act as a positive regulator of apoptosis in human embryonic stem cells 42 , 43. Further studies are needed to trace a proteomic profiling of early and late developmental stage zebrafish extracts and possibly screen factors that could specifically induce caspase activation and the release of cytocrome-c in a Bax-dependent or -independent fashion.

During zebrafish embryogenesis the expression of the pluripotency genes Pou5f1/Oct-4 and Sox-2 is timely regulated by defined factors that are mainly restricted to the early developmental pattern (during the first hpf) 44 , 45 , 46. The current data show that hASCs are able to selectively respond to factors restricted to the very early, but not late, developmental stages of zebrafish embryo with a transcriptional increase in the same two stemness-related genes necessary for the expression of family members of transcription factors that contribute to the maintenance of human stem cell pluripotency and self-renewal. We have previously shown that hASCs overexpressing Oct-4 and Sox-2 following exposure to an asymmetrically conveyed radioelectric field exhibited enhanced commitment towards cardiac, vascular, skeletal muscle, and neuronal lineages, with higher differentiating yields from Lipogems-derived hASCs than cells obtained from enzymatic digestion of the same lipoaspirate 47. Whether the currently observed increase of Oct-4 and Sox-2 in response to targeted zebrafish embryo extracts may also lead to enhanced multilineage commitment in vitro and rescuing potential in vivo remains to be established, and it is the subject of ongoing investigations.

We show that the overexpression of stemness genes elicited by ZF1 was paralleled by an increase in the transcription of both Bmi-1 and TERT. The relevance of this observation is highlighted by the fact that both genes exert a major role in counteracting aging processes in vivo and cell senescence in vitro. Bmi-1 is emerging as a major aging repressor and is transcriptionally down-regulated when cells undergo replicative senescence 48 , 49 , 50. TERT opposes cell senescence by counteracting telomere shortening. Studies on brain development in mice have correlated a decrease in TERT expression and activity with decreased neuroblast proliferation, and differentiation 51. Moreover, it has been demonstrated that MSCs or bone marrow stromal stem cells lacking telomerase activity undergo premature cellular senescence, with a progressive decline in the expression of early mesenchymal stem cell markers 52. The maintenance of stemness gene expression is also important in the prevention of cell senescence. The singular loss of the Bright/Arid3A transcription factor, the founding member of the ARID family of transcriptions factors 53 , 54, which binds directly to the promoter/enhancer regions of Oct-4 and Sox-2 contributing to their repression in both mouse embryonic fibroblasts (MEFs) and mouse embryonic stem cells (ESCs), was found to bypass the cell senescene barrier, leading to MEF reprogramming 55.

CONCLUSIONS

We show for the first time that human stem cell exposure to early developmental stage zebrafish embryo extracts may represent a useful tool to enhance stem cell expression of multipotency and activate both telomerase-dependent and -independent antagonists of cell senescence. This strategy did not require cumbersome gene manipulation through viral vector mediated gene transfer, or expensive synthetic chemistry. Further studies are in progress to investigate whether developmental stage extracts from zebrafish embryo may be used to revert cell senescence in hMSCs subjected to expansion for multiple passages in vitro, resuming their ability to differentiate along multiple lineages.

Acknowledgments

This research was supported by Wartsila Italia Spa, Trieste, Italy; Ettore Sansavini Health Science Foundation, Italy; Ministero della Salute, Italy, Ricerca Finalizzata-Progetti Cellule Staminali 2008, Italy.

The Foundation BLANCEFLOR Boncompagni Ludovisi, née Bildt; The C.M. Lerici Foundation.

Conflict of Interests:The Authors declare that they have no conflict of interests.

References

  1. Pozzoli O, Vella P, Iaffaldano G, Parente V, Devanna P, Lacovich M, Lamia CL, Fascio U, Longoni D, Cotelli F, Capogrossi MC, Pesce M. Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish. Arterioscler Thromb Vasc Biol 2011; 31: 1589-1597. (back)
  2. Pozzoli O, Vella P, Iaffaldano G, Parente V, Devanna P, Lacovich M, Lamia CL, Fascio U, Longoni D, Cotelli F, Capogrossi MC, Pesce M. Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish. Arterioscler Thromb Vasc Biol 2011; 31: 1589-1597. (back)
  3. Pozzoli O, Vella P, Iaffaldano G, Parente V, Devanna P, Lacovich M, Lamia CL, Fascio U, Longoni D, Cotelli F, Capogrossi MC, Pesce M. Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish. Arterioscler Thromb Vasc Biol 2011; 31: 1589-1597. (back)
  4. Biava PM, Bonsignorio D. Cancer and cell differentiation: a model to explain malignancy. J Tumor Marker Oncol 2002; 17: 47-54. (back)
  5. Biava PM, Nicolini A, Ferrari P, Carpi A, Sell S. A systemic approach to cancer treatment: tumor cell reprogramming focused on endocrine-related cancers. Curr Med Chem 2014; 21: 1072-1081. (back)
  6. Minz B, Illmensee K. Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc Natl Acad Sci USA1975; 72: 3585-3589. (back)
  7. Papaioannou VE, McBumey MV, Gardner RL, Evans RL. Fate of teratocarcinoma cells injected into early mouse embryos. Nature 1975; 258: 70-73. (back)
  8. Pierce GB. The cancer cell and its control by the embryo. Am J Pathol 1983; 113: 116-124. (back)
  9. Hendrix MJ, Seftor EA, Seftor REB, Kaisermeier-Kulesa J, Kulesa PM, Postovit LM. Reprogramming metastatic tumor cells with embryonic microenvironment. Nat Rev Cancer 2007; 7: 246-255. (back)
  10. Postovit LM, Maragaryan NV, Seftor EA, Kirschmann DA, Lipavski A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ. Human embryonic stem cell microenvironment suppress the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci USA 2008; 18: 105-111. (back)
  11. Ohnishi K, Semi K, Yamada Y. Epigenetic regulation leading to induced pluripotency drives cancer development in vivo. Biochem Biophys Res Commun 2014 Jul 11. pii: S0006-291X(14)01234-0. doi: 10.1016/j.bbrc.2014.07.020. [Epub ahead of print]. (back)
  12. Ohnishi K, Semi K, Yamamoto T, Shimizu M, Tanaka A, Mitsunaga K, Okita K, Osafune K, Arioka Y, Maeda T, Soejima H, Moriwaki H, Yamanaka S, Woltjen K, Yamada Y. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell 2014; 156: 663-677. (back)
  13. Biava PM, Bonsignorio D, Hoxa M. Cell proliferation curves of different human tumor lines after in vitro treatment with Zebrafish embryonic extracts. J Tumor Marker Oncol 2001; 16: 195-202. (back)
  14. Biava PM, Bonsignorio D, Hoxa M, Facco R, Ielapi T, Frati L, Bizzarri, M. Post-traslational modification of the retinoblastoma protein (pRb) induced by in vitro administration of Zebrafish embryonic extracts on human kidney adenocarcinoma cell line. J Tumor Marker Oncol 2002; 17: 59-64. (back)
  15. Cucina A, Biava PM, D’Anselmi F, Coluccia P, Conti F, Di Clemente R, Miccheli A, Frati L, Gulino A, Bizzani M. Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2). Apoptosis 2006; 9: l617-1628. (back)
  16. Biava PM, Basevi M, Biggiero L, Borgonovo A, Borgonovo E, Burigana F. Cancer cell reprogramming: stem cell differentiation stage factors and an agent based model to optimize cancer treatment. Curr Pharm Biotechnol 2011; 12: 231-242. (back)
  17. Livraghi T, Meloni F, Frosi A, Lazzaroni S, Bizzani M, Frati L, Biava PM. Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: an open randomized clinical trial. Oncol Res 2005; 15: 399-408. (back)
  18. Bianchi F, Maioli M, Leonardi E, Olivi E, Pasquinelli G, Valente S, Mendez AJ, Ricordi C, Raffaini M, Tremolada C, Ventura C. A new nonenzymatic method and device to obtain a fat tissue derivative highly enriched in pericyte-like elements by mild mechanical forces from human lipoaspirates. Cell Transplant 2013; 22: 2063-2077. (back)
  19. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003; 425: 962-967. (back)
  20. Guney I, Sedivy JM. Cellular senescence, epigenetic switches and c-Myc. Cell Cycle 2006; 5: 2319-2323. (back)
  21. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 2003; 423: 255-260. (back)
  22. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ, Clarke MF. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 2003; 423: 302-305. (back)
  23. Bianchi F, Maioli M, Leonardi E, Olivi E, Pasquinelli G, Valente S, Mendez AJ, Ricordi C, Raffaini M, Tremolada C, Ventura C. A new nonenzymatic method and device to obtain a fat tissue derivative highly enriched in pericyte-like elements by mild mechanical forces from human lipoaspirates. Cell Transplant 2013; 22: 2063-2077. (back)
  24. Livraghi T, Meloni F, Frosi A, Lazzaroni S, Bizzani M, Frati L, Biava PM. Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: an open randomized clinical trial. Oncol Res 2005; 15: 399-408. (back)
  25. Pfaffl MW. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res 2001; 29: e45. (back)
  26. Kelly PN, Strasser A. The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy. Cell Death Differ 2011; 18: 1414-1424. (back)
  27. Estrada JC, Torres Y, Benguría A, Dopazo A, Roche E, Carrera-Quintanar L, Pérez RA, Enríquez JA, Torres R, Ramírez JC, Samper E, Bernad A. Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death Dis 2013; 4: e691. (back)
  28. Izadpanah R, Kaushal D, Kriedt C, Tsien F, Patel B, Dufour J, Bunnell BA. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res 2008; 68: 4229-4238. (back)
  29. Estrada JC, Torres Y, Benguría A, Dopazo A, Roche E, Carrera-Quintanar L, Pérez RA, Enríquez JA, Torres R, Ramírez JC, Samper E, Bernad A. Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death Dis 2013; 4: e691. (back)
  30. Gruber HE, Somayaji S, Riley F, Hoelscher GL, Norton HJ, Ingram J, Hanley EN Jr. Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence. Biotech Histochem 2012; 87: 303-311. (back)
  31. Han SM, Han SH, Coh YR, Jang G, Chan Ra J, Kang SK, Lee HW, Youn HY. Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells. Exp Mol Med 2014; 46: e101. (back)
  32. Conner EA, Teramoto T, Wirth PJ, Kiss A, Garfield S, Thorgeirsson SS. HGF-mediated apoptosis via p53/bax-independent pathway activating JNK1. Carcinogenesis 1999; 20: 583-590. (back)
  33. Mullauer FB, Kessler JH, Medema JP. Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion. Apoptosis 2009; 14: 191-202. (back)
  34. Lei X, Chen Y, Du G, Yu W, Wang X, Qu H, Xia B, He H, Mao J, Zong W, Liao X, Mehrpour M, Hao X, Chen Q. Gossypol induces Bax/Bak-independent activation of apoptosis and cytochrome c release via a conformational change in Bcl-2. FASEB J 2006; 20: 2147-2149. (back)
  35. Wen K, Fu Z, Wu X, Feng J, Chen W, Qian J. Oct-4 is required for an antiapoptotic behavior of chemoresistant colorectal cancer cells enriched for cancer stem cells: effects associated with STAT3/Survivin. Cancer Lett 2013; 333: 56-65. (back)
  36. Lin Y, Yang Y, Li W, Chen Q, Li J, Pan X, Zhou L, Liu C, Chen C, He J, Cao H, Yao H, Zheng L, Xu X, Xia Z, Ren J, Xiao L, Li L, Shen B, Zhou H, Wang YJ. Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells. Mol Cell 2012; 48: 627-640. (back)
  37. Thiel G. How Sox2 maintains neural stem cell identity. Biochem J 2013; 450: e1-2. (back)
  38. Feng R, Zhou S, Liu Y, Song D, Luan Z, Dai X, Li Y, Tang N, Wen J, Li L. Sox2 protects neural stem cells from apoptosis via up-regulating survivin expression. Biochem J 2013; 450: 459-468. (back)
  39. Madonna R, Taylor DA, Geng YJ, De Caterina R, Shelat H, Perin EC, Willerson JT. Transplantation of mesenchymal cells rejuvenated by the overexpression of telomerase and myocardin promotes revascularization and tissue repair in a murine model of hindlimb ischemia. Circ Res 2013; 113: 902-914. (back)
  40. Lüscher B, Vervoorts J. Regulation of gene transcription by the oncoprotein MYC. Gene 2012; 494: 145-160. (back)
  41. Nie Z, Hu G, Wei G, Cui K, Yamane A, Resch W, Wang R, Green DR, Tessarollo L, Casellas R, Zhao K, Levens D. c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell 2012; 151: 68-79. (back)
  42. Nie Z, Hu G, Wei G, Cui K, Yamane A, Resch W, Wang R, Green DR, Tessarollo L, Casellas R, Zhao K, Levens D. c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell 2012; 151: 68-79. (back)
  43. Sumi T, Tsuneyoshi N, Nakatsuji N, Suemori H. Apoptosis and differentiation of human embryonic stem cells induced by sustained activation of c-Myc. Oncogene 2007; 26: 5564-5576. (back)
  44. Lunde K, Belting HG, Driever W. Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade. Curr Biol 2004; 14: 48-55. (back)
  45. Onichtchouk D, Geier F, Polok B, Messerschmidt DM, Mössner R, Wendik B, Song S, Taylor V, Timmer J, Driever W. Zebrafish Pou5f1-dependent transcriptional networks in temporal control of early development. Mol Syst Biol 2010; 6: 354. (back)
  46. Kotkamp K, Mössner R, Allen A, Onichtchouk D, Driever W. A Pou5f1/Oct4 dependent Klf2a, Klf2b, and Klf17 regulatory sub-network contributes to EVL and ectoderm development during zebrafish embryogenesis. Dev Biol 2014; 385: 433-447. (back)
  47. Maioli M, Rinaldi S, Santaniello S, Castagna A, Pigliaru G, Delitala A, Bianchi F, Tremolada C, Fontani V, Ventura C. Radio electric asymmetric conveyed fields and human adipose-derived stem cells obtained with a non-enzymatic method and device: a novel approach to multipotency. Cell Transplant 2013 Aug 30. doi: 10.3727/096368913X672037. [Epub ahead of print]. (back)
  48. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003; 425: 962-967. (back)
  49. Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001; 19: 180-192. (back)
  50. Rando TA. Stem cells, ageing and the quest for immortality. Nature 2006; 441: 1080-1086. (back)
  51. Itahana K, Zou Y, Itahana Y, Martinez JL, Beausejour C, Jacobs JJ, Van Lohuizen M, Band V, Campisi J, Dimri GP. Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1. Mol Cell Biol 2003; 23: 389-401. (back)
  52. Klapper W, Parwaresch R, Krupp G. Telomere biology in human aging and aging syndromes. Mech Ageing Dev 2001; 122: 695-712. (back)
  53. Herrscher RF, Kaplan MH, Lelsz DL, Das C, Scheuermann R, Tucker PW. The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: a B cell-specific trans-activator that describes a new DNA-binding protein family. Genes Dev 1995; 9: 3067-3082. (back)
  54. Wilsker D, Patsialou A, Dallas PB, Moran E. ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development. Cell Growth Differ 2002; 13: 95-106. (back)
  55. Popowski M, Templeton TD, Lee BK, Rhee C, Li H, Miner C, Dekker JD, Orlanski S, Bergman Y, Iyer VR, Webb CF, Tucker H. Bright/Arid3A Acts as a Barrier to Somatic Cell Reprogramming through Direct Regulation of Oct4, Sox2, and Nanog. Stem Cell Reports 2014; 2: 26-35. (back)
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Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells

As the modern society is troubled by multi-factorial diseases, research has been conducted on complex realities including chronic inflammation, cancer, obesity, HIV infection, metabolic syndrome and its detrimental cardiovascular complications as well as depression and other brain disorders. Deterioration of crucial homeostatic mechanisms in such diseases invariably results in activation of inflammatory mediators, chronic inflammation, loss in immunological function, increased susceptibility to diseases, alteration of metabolism, decrease of energy production and neuro-cognitive decline. Regulation of genes expression by epigenetic code is the dominant mechanism for the transduction of environmental inputs, such as stress and inflammation to lasting physiological changes. Acute and chronic stress determines DNA methylation and histone modifications in brain regions which may contribute to neuro-degenerative disorders. Nuclear glucocorticoids receptor interacts with the epigenoma resulting in a cortisol resistance status associated with a deterioration of the metabolic and immune functions. Gonadal steroids receptors have a similar capacity to produce epigenomic reorganization of chromatine structure. Epigenomic-induced reduction in immune cells telomeres length has been observed in many degenerative diseases, including all types of cancer. The final result of these epigenetic alterations is a serious damage to the neuro-endocrine-immune-metabolic adaptive systems. In this study, we propose a treatment with stem cells differentiation stage factors taken from zebrafish embryos which are able to regulate the genes expression of normal and pathological stem cells in a different specific way.

Download: Complex Therapeutical Approaches to Complex Diseases
Download: Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells

Still in 2015:

Getting an Insight into the Complexity of Major Chronic Inflammatory and Degenerative Diseases: A Potential New Systemic Approach to Their Treatment.

From: https://www.ncbi.nlm.nih.gov/pubmed/26201608

As the modern society is troubled by multi-factorial diseases, research has been conducted on complex realities including chronic inflammation, cancer, obesity, HIV infection, metabolic syndrome and its detrimental cardiovascular complications as well as depression and other brain disorders. Deterioration of crucial homeostatic mechanisms in such diseases invariably results in activation of inflammatory mediators, chronic inflammation, loss in immunological function, increased susceptibility to diseases, alteration of metabolism, decrease of energy production and neuro-cognitive decline. Regulation of genes expression by epigenetic code is the dominant mechanism for the transduction of environmental inputs, such as stress and inflammation to lasting physiological changes. Acute and chronic stress determines DNA methylation and histone modifications in brain regions which may contribute to neuro-degenerative disorders. Nuclear glucocorticoids receptor interacts with the epigenoma resulting in a cortisol resistance status associated with a deterioration of the metabolic and immune functions. Gonadal steroids receptors have a similar capacity to produce epigenomic reorganization of chromatine structure. Epigenomic-induced reduction in immune cells telomeres length has been observed in many degenerative diseases, including all types of cancer. The final result of these epigenetic alterations is a serious damage to the neuro-endocrine-immune-metabolic adaptive systems. In this study, we propose a treatment with stem cells differentiation stage factors taken from zebrafish embryos which are able to regulate the genes expression of normal and pathological stem cells in a different specific way.

Still in 2015:

The PI3K-AKt-mTOR Pathway and New Tools to Prevent Acquired Hormone Resistance in Breast Cancer

Abstract

Acquired hormone resistance is an old hurdle and still represents to be a constant challenge in oncology for the medical community. Most recently, mainly following the results of BOLERO-2 study, the activation of the PI3K-AKT-mTOR pathway is considered clinically relevant for tumor escape from hormone dependence in breast cancer. In the BOLERO-2 trial, a combination of everolimus, mTOR inhibitor, and exemestane significantly prolonged the median progression free survival (PFS) compared to exemestane alone in advanced breast cancer patients with acquired endocrine resistance. Therefore, the inhibitors of the PI3K-AKT-mTOR pathway are a new class of drugs in great expansion joined with great expectation. This review article focuses on this special issue and briefly reports on the results of clinical trials using PI3K-AKT-mTOR inhibitors. However, the emergence of resistance to this new class of drugs, evidenced by the basic research and the relatively less benefit shown in the clinical trials, has been emerging as a new undesirable complication. Therefore, the principal elucidated mechanisms of the resistance to the inhibitors of the PI3K-AKT-mTOR pathway and the related potential therapeutic strategies are described. A more general immunological approach to delay acquired hormone resistance has also been considered and commented upon.

From: https://www.ncbi.nlm.nih.gov/pubmed/26201609

Abstract

Acquired hormone resistance is an old hurdle and still represents to be a constant challenge in oncology for the medical community. Most recently, mainly following the results of BOLERO-2 study, the activation of the PI3K-AKT-mTOR pathway is considered clinically relevant for tumor escape from hormone dependence in breast cancer. In the BOLERO-2 trial, a combination of everolimus, mTOR inhibitor, and exemestane significantly prolonged the median progression free survival (PFS) compared to exemestane alone in advanced breast cancer patients with acquired endocrine resistance. Therefore, the inhibitors of the PI3K-AKT-mTOR pathway are a new class of drugs in great expansion joined with great expectation. This review article focuses on this special issue and briefly reports on the results of clinical trials using PI3K-AKT-mTOR inhibitors. However, the emergence of resistance to this new class of drugs, evidenced by the basic research and the relatively less benefit shown in the clinical trials, has been emerging as a new undesirable complication. Therefore, the principal elucidated mechanisms of the resistance to the inhibitors of the PI3K-AKT-mTOR pathway and the related potential therapeutic strategies are described. A more general immunological approach to delay acquired hormone resistance has also been considered and commented upon.

0
New Views in the Integrative Treatment of Oncologic Disease: Stem Cell Differentiation Stage Factors and Their Role in Tumor Cell Reprogramming

On the basis of the evidence that tumor development is suppressed by the embryonic microenvironment, some experiments using the factors taken from Zebrafish embryo at precise stages of cell differentiation were made. These experiments demonstrated a significant growth inhibition on different tumor cell lines in vitro. The observed mechanism of tumor growth inhibition is connected with the key-role cell cycle regulation molecules, such as p53 and pRb, which are modified by transcriptional or post-translational processes. Research on apoptosis and differentiation revealed that treatment with these factors induces caspase-3 with a p73 apoptotic-dependent pathway activation and a concurrent significant normalization of e-cadherin and beta-catenin ratio. Other experiments found a synergistic effect on the colon cancer proliferation curve after the concurrent treatment with these factors and 5-fluorouracil. Finally, a product prepared for human therapy demonstrated 19.8% regression and 16% stable disease in an open randomized clinical trial on intermediate-advanced hepatocellular carcinoma. The aims of this article is to recall in a synthetic way all the aforementioned researches to explain deeply the rationale of this approach of reprogramming cancer cells.

From: World Futures

The Journal of New Paradigm Research

https://www.tandfonline.com/doi/full/10.1080/02604027.2016.1143290?scroll=top&needAccess=true

On the basis of the evidence that tumor development is suppressed by the embryonic microenvironment, some experiments using the factors taken from Zebrafish embryo at precise stages of cell differentiation were made. These experiments demonstrated a significant growth inhibition on different tumor cell lines in vitro. The observed mechanism of tumor growth inhibition is connected with the key-role cell cycle regulation molecules, such as p53 and pRb, which are modified by transcriptional or post-translational processes. Research on apoptosis and differentiation revealed that treatment with these factors induces caspase-3 with a p73 apoptotic-dependent pathway activation and a concurrent significant normalization of e-cadherin and beta-catenin ratio. Other experiments found a synergistic effect on the colon cancer proliferation curve after the concurrent treatment with these factors and 5-fluorouracil. Finally, a product prepared for human therapy demonstrated 19.8% regression and 16% stable disease in an open randomized clinical trial on intermediate-advanced hepatocellular carcinoma. The aims of this article is to recall in a synthetic way all the aforementioned researches to explain deeply the rationale of this approach of reprogramming cancer cells.

Still in 2016:

Cancer: A Problem of Developmental Biology; Scientific Evidence for Reprogramming and Differentiation Therapy

Abstract:

Current medical literature acknowledges that embryonic micro-environment is able to suppress tumor development. Administering carcinogenic substances during organogenesis in fact leads to embryonic malformations, but not to offspring tumor growth. Once organogenesis has ended, administration of carcinogenic substances causes a rise in offspring tumor development. These data indicate that cancer can be considered a deviation in normal development, which can be regulated by factors of the embryonic microenvironment. Furthermore, it has been demonstrated that teratoma differentiates into normal tissues once it is implanted in the embryo. Recently, it has been shown that implanting a melanoma in Zebrafish embryo did not result in a tumor development; however, it did in the adult specimen. This demonstrates that cancer cells can differentiate into normal tissues when implanted in the embryo. In addition, it was demonstrated that other tumors can revert into a normal phenotype and/or differentiate into normal tissue when implanted in the embryo. These studies led some authors to define cancer as a problem of developmental biology and to predict the present concept of “cancer stem cells theory”. In this review, we record the most important researches about the reprogramming and differentiation treatments of cancer cells to better clarify how the substances taken from developing embryo or other biological substances can induce differentiation of malignant cells. Lastly, a model of cancer has been proposed here, conceived by one of us, which is consistent with the reality, as demonstrated by a great number of researches. This model integrates the theory of the “maturation arrest” of cancer cells as conceived by B. Pierce with the theory which describes cancer as a process of deterministic chaos determined by genetic and/or epigenetic alterations in differentiated cells, which leads a normal cell to become cancerous. All the researches here described demonstrated that cancer can be considered a problem of developmental biology and that one of the most important hallmarks of cancer is the loss of differentiation as already described by us in other articles.

From: https://www.ncbi.nlm.nih.gov/pubmed/26343109

Abstract

Current medical literature acknowledges that embryonic micro-environment is able to suppress tumor development. Administering carcinogenic substances during organogenesis in fact leads to embryonic malformations, but not to offspring tumor growth. Once organogenesis has ended, administration of carcinogenic substances causes a rise in offspring tumor development. These data indicate that cancer can be considered a deviation in normal development, which can be regulated by factors of the embryonic microenvironment. Furthermore, it has been demonstrated that teratoma differentiates into normal tissues once it is implanted in the embryo. Recently, it has been shown that implanting a melanoma in Zebrafish embryo did not result in a tumor development; however, it did in the adult specimen. This demonstrates that cancer cells can differentiate into normal tissues when implanted in the embryo. In addition, it was demonstrated that other tumors can revert into a normal phenotype and/or differentiate into normal tissue when implanted in the embryo. These studies led some authors to define cancer as a problem of developmental biology and to predict the present concept of “cancer stem cells theory”. In this review, we record the most important researches about the reprogramming and differentiation treatments of cancer cells to better clarify how the substances taken from developing embryo or other biological substances can induce differentiation of malignant cells. Lastly, a model of cancer has been proposed here, conceived by one of us, which is consistent with the reality, as demonstrated by a great number of researches. This model integrates the theory of the “maturation arrest” of cancer cells as conceived by B. Pierce with the theory which describes cancer as a process of deterministic chaos determined by genetic and/or epigenetic alterations in differentiated cells, which leads a normal cell to become cancerous. All the researches here described demonstrated that cancer can be considered a problem of developmental biology and that one of the most important hallmarks of cancer is the loss of differentiation as already described by us in other articles.

Still in 2016:

Immunotherapy and Hormone-therapy in Metastatic Breast Cancer: A Review and an Update

Abstract

Historically, antiestrogen is the first targeted therapy used in breast cancer treatment. In fact, its rationale lies in the molecular pathways elucidated by basic research. In estrogen receptor (ER)-alpha positive metastatic breast cancer patients, hormone-therapy remains the first option of treatment. While tamoxifen concomitant with suppression of ovarian function with luteinizing hormone releasing hormone (LHRH) agonists is the standard first line treatment in premenopausal, third generation aromatase inhibitors (AIs) are the first line standard hormone therapy in postmenopausal. However, the development of acquired resistance during antiestrogen therapy continues to be a central clinical problem. This review provides an update on the antiestrogen action and report on immunological treatment of the advanced disease by some cytokines. Interleukin-2, interleukin-12 and interferons used alone or in combination demonstrated an anti-tumor action directly and/or through synergism with antiestrogens. A rationale for the addition of interferon-beta and interleukin-2 to antiestrogens is described. Furthermore, we summarize and interpret the clinical and laboratory data of a recent long-term hormone- immunotherapy study in metastatic endocrine dependent breast cancer patients. Prospective randomized trials are necessary to confirm some recent promising results based on an immunological approach in addition to antiestrogens to overcome or delay acquired hormone resistance.

From: https://www.ncbi.nlm.nih.gov/pubmed/26844558

Abstract

Historically, antiestrogen is the first targeted therapy used in breast cancer treatment. In fact, its rationale lies in the molecular pathways elucidated by basic research. In estrogen receptor (ER)-alpha positive metastatic breast cancer patients, hormone-therapy remains the first option of treatment. While tamoxifen concomitant with suppression of ovarian function with luteinizing hormone releasing hormone (LHRH) agonists is the standard first line treatment in premenopausal, third generation aromatase inhibitors (AIs) are the first line standard hormone therapy in postmenopausal. However, the development of acquired resistance during antiestrogen therapy continues to be a central clinical problem. This review provides an update on the antiestrogen action and report on immunological treatment of the advanced disease by some cytokines. Interleukin-2, interleukin-12 and interferons used alone or in combination demonstrated an anti-tumor action directly and/or through synergism with antiestrogens. A rationale for the addition of interferon-beta and interleukin-2 to antiestrogens is described. Furthermore, we summarize and interpret the clinical and laboratory data of a recent long-term hormone- immunotherapy study in metastatic endocrine dependent breast cancer patients. Prospective randomized trials are necessary to confirm some recent promising results based on an immunological approach in addition to antiestrogens to overcome or delay acquired hormone resistance.

0
Tissue Regeneration without Stem Cell Transplantation: Self-Healing Potential from Ancestral Chemistry and Physical Energies

Abstract

The human body constantly regenerates after damage due to the self-renewing and differentiating properties of its resident stem cells. To recover the damaged tissues and regenerate functional organs, scientific research in the field of regenerative medicine is firmly trying to understand the molecular mechanisms through which the regenerative potential of stem cells may be unfolded into a clinical application. The finding that some organisms are capable of regenerative processes and the study of conserved evolutionary patterns in tissue regeneration may lead to the identification of natural molecules of ancestral species capable to extend their regenerative potential to human tissues. Such a possibility has also been strongly suggested as a result of the use of physical energies, such as electromagnetic fields and mechanical vibrations in human adult stem cells. Results from scientific studies on stem cell modulation confirm the possibility to afford a chemical manipulation of stem cell fate in vitro and pave the way to the use of natural molecules, as well as electromagnetic fields and mechanical vibrations to target human stem cells in their niche inside the body, enhancing human natural ability for self-healing.

From: https://www.hindawi.com/journals/sci/2018/7412035/

Abstract

The human body constantly regenerates after damage due to the self-renewing and differentiating properties of its resident stem cells. To recover the damaged tissues and regenerate functional organs, scientific research in the field of regenerative medicine is firmly trying to understand the molecular mechanisms through which the regenerative potential of stem cells may be unfolded into a clinical application. The finding that some organisms are capable of regenerative processes and the study of conserved evolutionary patterns in tissue regeneration may lead to the identification of natural molecules of ancestral species capable to extend their regenerative potential to human tissues. Such a possibility has also been strongly suggested as a result of the use of physical energies, such as electromagnetic fields and mechanical vibrations in human adult stem cells. Results from scientific studies on stem cell modulation confirm the possibility to afford a chemical manipulation of stem cell fate in vitro and pave the way to the use of natural molecules, as well as electromagnetic fields and mechanical vibrations to target human stem cells in their niche inside the body, enhancing human natural ability for self-healing.

1. Introduction

The human body continuously regenerates due to the peculiar properties of its resident stem cells.

These cells possess the unique ability to self-renew and differentiate, and the balance between these two processes defines the stem cell fate and their primary role in tissue regeneration [1].

Regeneration is the recovery of the organ structure and function after injury and it is at the basis of our self-healing potential and therefore of the preservation of human health. Such a process exhibits remarkable grading in the way it is fashioned in living organisms, since, within the same species, the regenerative potential is different among the various organs [2].

To rescue damaged tissues and restore functional organ mass, huge efforts have been made in the growing field of regenerative medicine, engaging scientific research in the understanding of the molecular mechanisms through which the regenerative potential of stem cells (as human mesenchymal stem cells – hMSCs) may be unfolded into a clinical application [3]. Stem cells in fact have the capability to differentiate into a wide range of adult cells and the discovery and isolation of them paved the way to new hopes in the regenerative field.

On the other hand, many aspects of the cell-based therapy prevent the use of stem cells to regenerate organs and tissues: among them, a large amount of stem cells is required and the senescence process occurs during primary cell expansion. Moreover, it is not easy to isolate stem cells and to commit all of them toward a specific phenotype, since they can differentiate in all types of mature cells, including cancer cells. Therefore, a proper set up of in vitro MSC expansion, cryopreservation, and banking should be necessary to establish safety and efficacy in transplanted patients.

In addition, most of the applications of stem cells directed on patients are still under the phase of experimental trials, except for some procedures actually used in clinical practice, as the bone marrow transplantation in hematology [4].

Even tissue engineering, one of the branches of the regenerative medicine based upon tissue regeneration from cells with the aid of biomaterials and growth factors, still is facing several problems. In fact, the regenerated tissues usable by patients are still very limited, as skin, bone, cartilage, capillary, and periodontal tissues [5]. Moreover, the engineered artificial tissue still exhibits some limitation correlated to the dimensions of the construct that cannot be used for the recovery of serious defects. Actually, the only amenable engineered tissues with a tridimensional structure are vases, cave structures like the trachea [6], or tissues which are not physiologically scattered, since the viability of cells seeded on a scaffold gradually decreases with thickness. Even the use of growth factors alone or in association with 3D constructs is still considered as being not completely safe since the resulting influence on recipient’s environment remains in part to be established. Finally, other hurdles remain, such as finding the best scaffold, the most suitable bioreactor, and the optimal solution for seeding different cell populations in order to have a relevant mature material implantable on patients.

All these issues need to be addressed before cells or engineered constructs can be used routinely in the clinical setting. Therefore, multiple studies have long been running to address the modulation of relevant physiological traits known to be involved in tissue homeostasis and in the activation of the stem cell niches. To this end, besides investigating the effects of synthetic molecules, many researchers have also focused their attention on the effects elicited by natural molecules and physical energies. Their findings are reported below.
2. Natural Molecules

The ability to regrow body parts is common to a lot of animal species, although the regenerative potential varies among taxa [7]. Some phyla are able to rebuild every part of the body, while others cannot regenerate internal organs [8].

Danio rerio (zebrafish) is among the organisms capable of amazing regenerative processes, prompting the needs for uncovering the underlying regeneration strategies. Zebrafish is since recently commonly used as an animal model of organogenesis and regeneration, owing to its ability to regenerate complex organs, like the heart, the central nervous system, and the limbs, at an extraordinarily higher efficacy than humans [2, 3, 9–16]. Another species exhibiting astonishing regenerative potential is the Mexican axolotl (Ambystoma mexicanum), which can make self-copies regenerating a missing limb, tail, or parts of the brain, heart, and lower jaw. Other creatures in the spotlight for their regenerative capabilities are the salamanders, as well as several frogs [17], or the tunicates [18]. Despite their evolutionary distance, as in the case of zebrafish which is separated by about 450 million years from humans, our stem cells can still sense ancestral microenvironmental cues from these species, as shown by the finding that human cord blood CD34+ cells are recruited into early vasculogenesis upon transplantation in pre-gastrulation, but not post-gastrulation zebrafish embryos [19]. Akin to this view is the finding that conserved transcriptional responses have been discovered among the differentiation of hMSCs, Xenopus embryogenesis, and axolotl regeneration, identifying common networks across model species that are associated with depolarization (changes in cellular resting potential) [20].

On the whole, these findings and the deployment of comparative biology into the analysis of conserved evolutionary patterns in tissue regeneration may lead to the identification of natural molecules capable to extend their regenerative potential from ancestral species to human tissues through the manipulation of common/similar mechanisms in their resident stem cells.

Investigation of the role of natural molecules in stem cell biology is becoming a growing area of inquiry. Psoralidin, for example, a natural phenolic compound found in the seeds of Psoralea corylifolia, has been seen to inhibit NOTCH1 in breast cancer stem cells and in breast cancer cells, leading to a growth arrest and inhibition of epithelial to mesenchymal transition (EMT) [21]. Moreover, two herbal extracts (Tithonia diversifolia leaf extract and Momordica foetida extract) led to a decrease of the adipogenesis and accumulation of lipid droplets in human adipose-derived stem cells (hADSCs) [22, 23]. Two natural compounds, honokiol (a low-molecular-weight polyphenol isolated from the genus Magnolia) and hyperoside (a flavonoid compound extracted from Hypericum perforatum), were shown to potentially induce the differentiation into neurons in the murine embryonic carcinoma cell line P19 [24]. Synthetic compounds created by the assembly of natural molecules have also been proven effective in the modulation of stem cell biology in vitro and in vivo. To this end, mixed esters of naturally occurring molecules, such as hyaluronan mixed esters with butyric and retinoic acids (HBR), have been shown to remarkably increase cardiogenesis and vasculogenesis in mouse embryonic stem cells [25] and hMSCs [26], enhancing the ability of term placenta hMSCs of promoting the regeneration of infarcted myocardium in vivo in both small (rat) and large (pig) animal models with post infarct heart failure [27, 28]. Intriguingly, in the myocardium of infarcted rats, HBR itself acted through the intracellular release of its natural grafted molecules to afford significant decrease in infarct size, and apoptotic myocytes, leading to reverse myocardial remodeling, normalization in myocardial contractility, and increase in vital myocardial mass and metabolism, through the enhancement/recruitment of the number of endogenous stro-1 (a mesenchymal stem cell marker)-positive stem cells, the increase in the number of local elements with pericyte identity and important revascularization processes [29]. This finding shows the feasibility of chemical targeting damaged organs to afford tissue survival and repair without stem cell transplantation. Consonant with these results, a simple cocktail of hyaluronic, butyric, and retinoic acids was able to improve islet graft revascularization and function by adipose tissue-derived hMSCs in diabetic rats [30].

The addition of melatonin to this mixture of natural molecules was able to shift the commitment of hMSCs towards an osteogenic fate, indicating the feasibility of creating a multicomponent, multitarget ensemble of natural agents to chemically redirect the multilineage repertoire of hMSCs [31].

A major breakthrough in the effort of using natural arrays of molecules to drive cellular fates under normal and pathological conditions came by the discovery that extracts from zebrafish embryos obtained at different developmental stages were able to counteract the proliferation rate of several cancer cell lines [32–35]. Extracts from the beginning, intermediate and final embryonic development stages led to an evident increase in p53 expression in association with the growth reduction [33]. In some cancer cell lines, such as kidney adenocarcinoma, the proliferation decrease was associated with changes in pRb phosphorylation, a cell cycle modulator [34]. Moreover, in colon adenocarcinoma cells, an activation of the p73-dependent apoptotic pathway was observed [35]. A mixture of early, middle, and late developmental stage zebrafish extracts was also able to enhance cell survival to toxic stimuli, as shown by the reduction in mortality observed in cells from mouse hippocampal slices (CA1 area) that had been subjected to serum deprivation or NMDA (N-methyl-D-aspartate) treatment [36]. These findings and previous observations showing that embryonic microenvironment is able to suppress tumor development during cell differentiating processes [37, 38] led us to further investigate whether zebrafish embryonic factors may also be exploited in a developmental stage manner to control essential features in stem cell dynamics. To this end, we successfully used early-stage developmental zebrafish extracts (obtained from 5.15 hours post fertilization embryos) on early-passage hADSCs to enhance the stem cell expression of multipotency, and the transcription of TERT, encoding the catalytic subunit of telomerase, as well as the gene expression of BMI1, a chromatin remodeler acting as a major telomerase-independent repressor of senescence [39].

On the whole, the above mentioned studies, showing the possibility to afford a chemical manipulation of stem cell fate in vitro, may pave the way to the use of natural or synthetic chemistry to target human stem cells where they are already resident in all body tissues. This would lead to the development of a regenerative medicine executed without the needs for (stem) cell or tissue transplantation.
3. Physical Energies

The possibility of using physical energies to boost regenerative processes has been strongly suggested by the ability of electromagnetic fields and mechanical vibrations to drive efficient in situ reprogramming of the differentiating and regenerative potential of our endogenous stem cells.

We are in fact embedded in a wide variety of physical stimuli, including electromagnetic fields, light radiation, and mechanical oscillatory patterns. In this sense, our life which contains a seeming infinity of rhythmic oscillations, including calcium and pH intracellular oscillations [40–42], as well as the rhythmic expression of genes and proteins [43, 44], can be considered as a part of the vibratory nature of the universe.

It is now evident that our cells perceive and generate energies like magnetic fields and mechanical oscillations [45–47]. Cells contain a network of microtubules that, due to their electrical polarity and intrinsic vibration modes, is able to generate high-frequency electric fields with radiation features [48]. Applying scanning tunneling microscopy (STM) to microtubules growing onto a nanoelectrode array, within an artificial cell replica designed to pump electromagnetic frequencies, has shown the existence of resonance patterns between the tubulin dimers, or the whole microtubules, and the applied frequencies [49]. STM also provided evidence that such resonance patterns could be imaged as specific “tunneling current profiles” corresponding to the pumped electromagnetic frequencies [49]. The frequency region selectivity for engaging particular types of conformational modifications establishes that pure mechanical changes can be remotely managed in an atomically way by using electromagnetic fields.

The importance of the microtubule network as an information-transporting-system is also deduced by the finding of multilevel memory-switching properties in a single brain microtubule [50]. Even DNA, despite its role of storage and expression of genetic information, when considered as an electrically charged vibrational entity, may contribute to cell polarity, also by virtue of its constant assembly into different loops and domains that are an essential component of the nanomechanics and nanotopography imparted to this macromolecule by transcription factors and molecular motors. Accordingly, electromagnetic resonance frequency spectra have been revealed for DNA, which was found to exhibit electromagnetic resonances in the wide frequency range from KHz, MHz, GHz, to THz [51].

Recently, regenerative medicine has been focused on the use of biophysical stimuli to modulate cellular dynamics [52]. Physical factors in the cellular microenvironment, including matrix mechanics, cell geometry and shape, mechanical forces, and nanotopographical aspects of the extracellular matrix, can modulate the stem cell fate [53, 54]. There is evidence that this type of regulation is highly affected by coexisting insoluble, adhesive, mechanical, and topological cues contained and dynamically regulated within the stem cell niche [55, 56]. Biophysical stimuli can be sensed and transduced into intracellular biochemical and functional responses by stem cells, a process known as mechano-transduction [55]. The stem cell sensory machinery can at the same time perceive and integrate several signals from the niche and turn them into coherent responses affording downstream modulation of gene expression and stem cell fate [55, 57–59].

For years, scientists tried to drive stem cell fate by the aid of chemistry, increasing cell proliferation with growth factors or fabricating 3D constructs derived from the combination of stem cells or mature adult cells, with natural or artificial polymers. Only in the last years, efforts have been made to interact with cells in vivo, directly on patients or on animal models, and in vitro on cell cultures. Recently, some research groups have shown the possibility to use physical stimuli directly on patients, tissues, and cells [60].

The idea behind the use of physical stimuli on tissues and body was already proposed in 1974 by Richard Nuccitelli who gained evidence on endogenous ionic current and interaction with electric field in multicellular animal tissues [61]. Nowadays, it is possible to explain changes in cellular behavior, following electromagnetic stimulation, considering an effect on cell polarity [62] and on the stem cell niche in the body [63].

The use of physical energies for therapeutic purpose is now well known, being approved by the Food and Drug Administration (FDA) and used on patients. Several devices based on different physical mechanisms have been designed, and the beneficial effects have been observed directly on patients. Ultrasounds have been used for medical purposes since the 1950 in some pathological situations, such as tendinitis or bursitis [64].

Even the use of extremely low-frequency electromagnetic fields (ELF-EMFs) with frequencies lower than 100 Hz, and magnetic field intensity spanning from 0.1 to 20 mT, became a useful therapy for soft tissue regeneration, fracture repair, and osteoporosis treatment [65]. The mechanisms of action of ELF-EMFs are not clear yet. However, it has been shown that electric currents can accelerate cell activation [66] and influence epigenetic remodeling. In particular, the use of 50 Hz ELF-EMF on GC-2 cells decreased genome-wide methylation and the expression of DNA methyltransferases [67] in neural stem cells (NSCs) isolated from the hippocampus of newborn mice. Moreover, the ELF-EMF irradiation at 1 mT, and 50 Hz, for 12 days enhanced NSC proliferation and neuronal cell fate specification through Cav1 channel-dependent regulation and histone modification [68]. These results show the feasibility of using physical stimuli to affect cell fate.

Within this context, we have first demonstrated the possibility to use ELF-EMFs to modulate the gene transcription of essential growth regulatory peptides in adult myocardial cells [60] and to enhance cardiogenesis and terminal differentiation into spontaneously beating myocardial cells in mouse embryonic stem (ES) cells [69]. Then, by the aid of a radio electric asymmetric conveyer (REAC), we found that properly conveyed radioelectric fields of 2.4 GHz could produce important biological effects in mouse ES cells and human adult stem cells. In both cell types, we showed that REAC-conveyed radioelectric fields elicited an increase of the expression of stemness-related genes, followed by the commitment towards neuronal, myocardial, and skeletal muscle lineages [70, 71]. The same differentiating outcomes were induced by REAC exposure in human skin fibroblasts [72]: for the first time, human non-stem somatic adult cells were committed to lineages in which they would never otherwise appear. This effect was mediated by a biphasic change in pluripotency gene expression, a temporary overexpression followed by a down regulation, and did not require the use of viral vector-mediated gene transfer technologies or cumbersome synthetic chemistry.

Noteworthy, REAC exposure of hADSCs was able to turn stem cell senescence, occurring after prolonged (up to 30 passage) in vitro expansion, into a reversible phenomenon, associated with a decrease in the expression of senescence-associated β-galactosidase and an increase in TERT gene expression and telomere length. The REAC action also enhanced the gene transcription of BMI1 and that of stemness-related genes, establishing a telomerase-independent arm for senescence reversal [73]. These findings may have important biomedical implications, since senescent stem cells decrease their self-renewal and differentiation potential, reducing their ability for tissue regeneration in vivo and the possibility of a prolonged expansion in vitro prior to transplantation.

Compounding the wide-ranging biological effects of REAC stimulation is the observation that this technology was able to promote neurological and morphofunctional differentiation in PC12 cells [74], a rat adrenal pheochromocytoma cell line displaying metabolic features of Parkinson’s disease. Cell response to the electromagnetic field was mediated by the transcriptional activation of neurogenic genes, as neurogenin-1, β3-tubulin, and nerve growth factor (NGF), and was associated with a consistent increase in the number of cells expressing both β3-tubulin and tyrosine hydroxylase [74]. These findings open the new perspective of using physical energies in the treatment of neurodegenerative diseases and in the reprogramming of cancer (stem) cells into normal regenerative elements. More recently, we found that the REAC action could be significantly counteracted by stem cell treatment with 4-methylumbelliferone (4-MU), a potent repressor of type-2 hyaluronan (HA) synthase and endogenous HA synthesis [75]. This observation suggests that REAC-mediated responses may have occurred through an essential pleiotropic role of this glycosaminoglycan in regulating (stem) cell polarity.

Extracorporeal shock waves (ESW) represent another type of biophysical stimuli that is increasingly being applied in the field of regenerative medicine and that could be classified as “mechanotherapy” (i.e., extracorporeal shock wave therapy, ESWT). In fact, ESW are “mechanical” waves, characterized by an initial positive very rapid phase, of high amplitude, followed by negative pressure, producing a “microexplosion” that can be directed on a target zone (body, tissue, or cells) in order to stimulate or modify the cells in their behavior. Shock waves are generated by an electrohydraulic device that produces underwater high-voltage condenser spark discharge, conveyed by an elliptical reflector on tissues or cells.

In the 1980s, shock waves were used in urology (lithotripsy) to disintegrate renal stones [76]. Then, ESW application has been extended to other fields, showing promising hopes for promoting tissue healing and the recovery from pathological disorders. One of the first applications was in the orthopedic field, in order to induce neovascularization and improve blood supply and tissue regeneration. Investigations on the use of this technology spread progressively, and leading to its application in the treatment of musculoskeletal disorders [77], tendon pathologies [78], bone healing disturbances, and vascular bone diseases [79]. The use of ESW has also been extended to the field of dermatology for the wound healing disturbances and ulcers. However, to date, the exact mechanism through which cells convert mechanical signals into biochemical responses is not well understood yet. Emphasis has been placed so far into mechanisms mediated by ATP release and P2 receptor activation that may foster cell proliferation and tissue remodeling via Erk1/2 activation [80, 81], as well as PI-3K/AKT and NF-κB signaling pathways, and the implication of TLR3 signaling and subsequent TLR4. Several studies performed in vitro proved the effect of ESW on cell modulation through “mechano-transduction”. Recently, ESW were found to activate ADSCs through MAPK, PI-3K/AKT, and NF-κB signaling pathways [82, 83] and to induce in HUVEC cells an overexpression of angiogenic factors and of caveolin-1, a constitutive protein of caveolae, implicated in the regulation of cell growth, lipid trafficking, endocytosis, and cell migration [84].

In addition, the ESWT effect on cell behavior proved to be a dose-dependent phenomenon. In a study published by Zhang and coworkers, cells exposed to low-energy ESW (0.04 e 0.13 mJ/mm2) improved the expression of some angiogenic factors, such as eNOS, Ang-1, and Ang-2. On the other hand, at higher energy, ESW induced a reduction in angiogenic factor expression and an increase in apoptosis [85]. These findings suggest that the biological effects of shock waves strongly correlated with the intensity of applied energy and thus with the related mechanical forces.

Recently, the effects of shock waves have been characterized on the expression of IL-6, IL-8, MCP-1, and TNF-α in human periodontal ligament fibroblasts [86]. Following an early inhibition on the expression of pro-inflammatory mediators, shock waves elicited a dose-related increase in IL-6 and IL-8, while down-regulating TNF-α expression [86]. Most of the literature showed an anti-inflammatory effect of ESWT in vivo [59, 78, 79, 87, 88]. Nevertheless, the pro-inflammatory effect of ESWT partially observed on cells in vitro may suggest a pro-activator event mediated by cytokine and chemokine expression. It was supposed that the shock wave impulses on cells were able to create a pro-inflammatory milieu, mediated by mechano-transduction [80]. However, this mechanism may involve a more complex action on the whole niche architecture, with the embedded (stem) cells behaving as sensors and activators of the regenerative response.

In actual fact, mechanical vibration may represent a relevant modality to affect stem cell reprogramming in vivo without having to resort to transplantation procedures. In this regard, we have shown and patented for the first time the cell ability to exhibit “vibrational” (nanomechanical) signatures of their health and their multilineage repertoire [89]. Wide-ranging vital processes are fashioned around the nanomechanical features of subcellular structures, like the microtubular networks, imparting feature characteristic of connectedness and synchronization that can be transferred and recorded from the cell surface. Atomic force microscopy (AFM) can be used to gain insights on cellular nanomechanical properties [89, 90], providing the chance to identify vibrational signatures that can be used to drive lineage-specific commitments in different stem cell populations in vitro or even in vivo to promote endogenous rescue in diseased organs.
4. Conclusion

The emerging view of a (stem) cell biology governed by physical forces and influenced by ancestral natural molecules may lead us to reinterpret the way we envision the field of regenerative medicine for a near future.

In fact, due to the diffusive nature of electromagnetic fields and mechanical vibrations, the chance is emerging to target and reprogram the stem cells where they are, enhancing our natural ability for self-healing without the needs for (stem) cell transplantation which still shows remarkable limitations.
Conflicts of Interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.
Authors’ Contributions

Federica Facchin and Eva Bianconi contributed equally as co-first authors to this study.
Acknowledgments

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: funded by Eldor Lab, Milan, Italy, and AMeC (Associazione Medicina e Complessità), Via Valdirivo 19, 34100 Trieste, Italy.
References

C. Chen, J. M. Fingerhut, and Y. M. Yamashita, “The ins(ide) and outs(ide) of asymmetric stem cell division,” Current Opinion in Cell Biology, vol. 43, pp. 1–6, 2016. View at Publisher · View at Google Scholar · View at Scopus
T. L. Tal, J. A. Franzosa, and R. L. Tanguay, “Molecular signaling networks that choreograph epimorphic fin regeneration in zebrafish – a mini-review,” Gerontology, vol. 56, no. 2, pp. 231–240, 2010. View at Publisher · View at Google Scholar · View at Scopus
W. Shi, Z. Fang, L. Li, and L. Luo, “Using zebrafish as the model organism to understand organ regeneration,” Science China Life Sciences, vol. 58, no. 4, pp. 343–351, 2015. View at Publisher · View at Google Scholar · View at Scopus
T. Reinfjell, M. Tremolada, and L. K. Zeltzer, “A review of demographic, medical, and treatment variables associated with health-related quality of life (HRQOL) in survivors of hematopoietic stem cell (HSCT) and bone marrow transplantation (BMT) during childhood,” Frontiers in Psychology, vol. 8, pp. 8–253, 2017. View at Publisher · View at Google Scholar · View at Scopus
S. Caddeo, M. Boffito, and S. Sartori, “Tissue engineering approaches in the design of healthy and pathological in vitro tissue models,” Frontiers in Bioengineering and Biotechnology, vol. 5, pp. 5–40, 2017. View at Publisher · View at Google Scholar
J. X. Law, L. L. Liau, B. S. Aminuddin, and B. H. I. Ruszymah, “Tissue-engineered trachea: a review,” International Journal of Pediatric Otorhinolaryngology, vol. 91, pp. 55–63, 2016. View at Publisher · View at Google Scholar · View at Scopus
E. M. Tanaka and P. W. Reddien, “The cellular basis for animal regeneration,” Developmental Cell, vol. 21, no. 1, pp. 172–185, 2011. View at Publisher · View at Google Scholar · View at Scopus
A. E. Bely, “Evolutionary loss of animal regeneration: pattern and process,” Integrative and Comparative Biology, vol. 50, no. 4, pp. 515–527, 2010. View at Publisher · View at Google Scholar · View at Scopus
W. Goessling and T. E. North, “Repairing quite swimmingly: advances in regenerative medicine using zebrafish,” Disease Models & Mechanisms, vol. 7, no. 7, pp. 769–776, 2014. View at Publisher · View at Google Scholar · View at Scopus
S. E. Brockerhoff and J. M. Fadool, “Genetics of photoreceptor degeneration and regeneration in zebrafish,” Cellular and Molecular Life Sciences, vol. 68, no. 4, pp. 651–659, 2011. View at Publisher · View at Google Scholar · View at Scopus
T. Becker and C. G. Becker, “Axonal regeneration in zebrafish,” Current Opinion in Neurobiology, vol. 27, pp. 186–191, 2014. View at Publisher · View at Google Scholar · View at Scopus
K. D. Poss, L. G. Wilson, and M. T. Keating, “Heart regeneration in zebrafish,” Science, vol. 298, no. 5601, pp. 2188–2190, 2002. View at Publisher · View at Google Scholar · View at Scopus
K. D. Poss, M. T. Keating, and A. Nechiporuk, “Tales of regeneration in zebrafish,” Developmental Dynamics, vol. 226, no. 2, pp. 202–210, 2003. View at Publisher · View at Google Scholar · View at Scopus
K. K. McCampbell and R. A. Wingert, “New tides: using zebrafish to study renal regeneration,” Translational Research, vol. 163, no. 2, pp. 109–122, 2014. View at Publisher · View at Google Scholar · View at Scopus
C. Kizil, J. Kaslin, V. Kroehne, and M. Brand, “Adult neurogenesis and brain regeneration in zebrafish,” Developmental Neurobiology, vol. 72, no. 3, pp. 429–461, 2012. View at Publisher · View at Google Scholar · View at Scopus
K. Kikuchi, “Advances in understanding the mechanism of zebrafish heart regeneration,” Stem Cell Research, vol. 13, no. 3, pp. 542–555, 2014. View at Publisher · View at Google Scholar · View at Scopus
J. A. Coffman, S. Rieger, A. N. Rogers, D. L. Updike, and V. P. Yin, “Comparative biology of tissue repair, regeneration and aging,” NPJ Regenerative Medicine, vol. 1, no. 1, article 16003, 2016. View at Publisher · View at Google Scholar
M. Hamada, S. Goricki, M. S. Byerly, N. Satoh, and W. R. Jeffery, “Evolution of the chordate regeneration blastema: differential gene expression and conserved role of notch signaling during siphon regeneration in the ascidian Ciona,” Developmental Biology, vol. 405, no. 2, pp. 304–315, 2015. View at Publisher · View at Google Scholar · View at Scopus
O. Pozzoli, P. Vella, G. Iaffaldano et al., “Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 31, no. 7, pp. 1589–1597, 2011. View at Publisher · View at Google Scholar · View at Scopus
V. P. Pai, C. J. Martyniuk, K. Echeverri, S. Sundelacruz, D. L. Kaplan, and M. Levin, “Genome–wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation,” Regeneration, vol. 3, no. 1, pp. 3–25, 2016. View at Publisher · View at Google Scholar
S. Suman, T. P. Das, and C. Damodaran, “Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells,” British Journal of Cancer, vol. 109, no. 10, pp. 2587–2596, 2013. View at Publisher · View at Google Scholar · View at Scopus
C. Di Giacomo, L. Vanella, V. Sorrenti et al., “Effects of Tithonia diversifolia (Hemsl.) A. Gray extract on adipocyte differentiation of human mesenchymal stem cells,” PLoS One, vol. 10, no. 4, article e0122320, 2015. View at Publisher · View at Google Scholar · View at Scopus
R. Acquaviva, C. Di Giacomo, L. Vanella et al., “Antioxidant activity of extracts of Momordica Foetida Schumach. et Thonn,” Molecules, vol. 18, no. 3, pp. 3241–3249, 2013. View at Publisher · View at Google Scholar · View at Scopus
T. Chen, J. Wang, M. Liu, L. Y. Zhang, and H. Liao, “Screening of natural compounds with neuronal differentiation promoting effects in a cell–based model,” Chinese Journal of Natural Medicines, vol. 13, no. 8, pp. 602–608, 2015. View at Publisher · View at Google Scholar · View at Scopus
C. Ventura, M. Maioli, Y. Asara et al., “Butyric and retinoic mixed ester of hyaluronan. A novel differentiating glycoconjugate affording a high throughput of cardiogenesis in embryonic stem cells,” The Journal of Biological Chemistry, vol. 279, no. 22, pp. 23574–23579, 2004. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, S. Santaniello, A. Montella et al., “Hyaluronan esters drive Smad gene expression and signaling enhancing cardiogenesis in mouse embryonic and human mesenchymal stem cells,” PLoS One, vol. 5, no. 11, article e15151, 2010. View at Publisher · View at Google Scholar · View at Scopus
C. Ventura, S. Cantoni, F. Bianchi et al., “Hyaluronan mixed esters of butyric and retinoic acid drive cardiac and endothelial fate in term placenta human mesenchymal stem cells and enhance cardiac repair in infarcted rat hearts,” The Journal of Biological Chemistry, vol. 282, no. 19, pp. 14243–14252, 2007. View at Publisher · View at Google Scholar · View at Scopus
A. Simioniuc, M. Campan, V. Lionetti et al., “Placental stem cells pre–treated with a hyaluronan mixed ester of butyric and retinoic acid to cure infarcted pig hearts: a multimodal study,” Cardiovascular Research, vol. 90, no. 3, pp. 546–556, 2011. View at Publisher · View at Google Scholar · View at Scopus
V. Lionetti, S. Cantoni, C. Cavallini et al., “Hyaluronan mixed esters of butyric and retinoic acid affording myocardial survival and repair without stem cell transplantation,” The Journal of Biological Chemistry, vol. 285, no. 13, pp. 9949–9961, 2010. View at Publisher · View at Google Scholar · View at Scopus
G. Cavallari, E. Olivi, F. Bianchi et al., “Mesenchymal stem cells and islet cotransplantation in diabetic rats: improved islet graft revascularization and function by human adipose tissue–derived stem cells preconditioned with natural molecules,” Cell Transplantation, vol. 21, no. 12, pp. 2771–2781, 2012. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, V. Basoli, S. Santaniello et al., “Osteogenesis from dental pulp derived stem cells: a novel conditioned medium including melatonin within a mixture of hyaluronic, butyric, and retinoic acids,” Stem Cells International, vol. 2016, Article ID 2056416, 8 pages, 2016. View at Publisher · View at Google Scholar · View at Scopus
P. M. Biava, D. Bonsignorio, and M. Hoxa, “Cell proliferation curves of different human tumor lines after in vitro treatment with zebrafish embryonic extracts,” Journal of Tumor Marker Oncology, vol. 16, pp. 195–202, 2001. View at Google Scholar
P. M. Biava and A. Carluccio, “Activation of anti–oncogene p53 produced by embryonic extracts in vitro tumor cells,” Journal of Tumor Marker Oncology, vol. 12, pp. 9–15, 1977. View at Google Scholar
P. M. Biava, D. Bonsignorio, M. Hoxa et al., “Post–translational modification of the retinoblastoma protein (pRb) induced by in vitro administration of zebrafish embryonic extracts on human kidney adenocarcinoma cell line,” Journal of Tumor Marker Oncology, vol. 17, pp. 59–64, 2002. View at Google Scholar
A. Cucina, P. M. Biava, F. D’Anselmi et al., “Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2),” Apoptosis, vol. 11, no. 9, pp. 1617–1628, 2006. View at Publisher · View at Google Scholar · View at Scopus
P. M. Biava, S. Canaider, F. Facchin et al., “Stem cell differentiation stage factors from zebrafish embryo: a novel strategy to modulate the fate of normal and pathological human (stem) cells,” Current Pharmaceutical Biotechnology, vol. 16, no. 9, pp. 782–792, 2015. View at Publisher · View at Google Scholar · View at Scopus
L. Einhorn, “Are there factors preventing cancer development during embryonic life?” Oncodevelopmental Biology and Medicine, vol. 4, no. 3, pp. 219–229, 1983. View at Google Scholar
M. S. Lakshmi and G. V. Sherbet, Embryonic and Tumour Cell Interactions, G. V. Sherbet, Ed., Karger Basel, New York, NY, USA, 1974.
S. Canaider, M. Maioli, F. Facchin et al., “Human stem cell exposure to developmental stage zebrafish extracts: a novel strategy for tuning stemness and senescence patterning,” CellR4, vol. 2, article e1226, 2014. View at Google Scholar
C. Ventura, M. C. Capogrossi, H. A. Spurgeon, and E. G. Lakatta, “Kappa–opioid peptide receptor stimulation increases cytosolic pH and myofilament responsiveness to Ca2+ in cardiac myocytes,” American Journal of Physiology-Heart and Circulatory Physiology, vol. 261, 5 Part 2, pp. H1671–H1674, 1991. View at Publisher · View at Google Scholar
C. Ventura, H. Spurgeon, E. G. Lakatta, C. Guarnieri, and M. C. Capogrossi, “Kappa and delta opioid receptor stimulation affects cardiac myocyte function and Ca2+ release from an intracellular pool in myocytes and neurons,” Circulation Research, vol. 70, no. 1, pp. 66–81, 1992. View at Publisher · View at Google Scholar
M. Nivala, C. Y. Ko, M. Nivala, J. N. Weiss, and Z. Qu, “The emergence of subcellular pacemaker sites for calcium waves and oscillations,” The Journal of Physiology, vol. 591, no. 21, pp. 5305–5320, 2013. View at Publisher · View at Google Scholar · View at Scopus
R. Kageyama, T. Ohtsuka, and T. Kobayashi, “The Hes gene family: repressors and oscillators that orchestrate embryogenesis,” Development, vol. 134, no. 7, pp. 1243–1251, 2007. View at Publisher · View at Google Scholar · View at Scopus
Y. Masamizu, T. Ohtsuka, Y. Takashima et al., “Real–time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 5, pp. 1313–1318, 2006. View at Publisher · View at Google Scholar · View at Scopus
G. Albrecht-Buehler, “Rudimentary form of cellular ‘vision’,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 17, pp. 8288–8292, 1992. View at Publisher · View at Google Scholar · View at Scopus
G. Albrecht-Buehler, “A long-range attraction between aggregating 3T3 cells mediated by near-infrared light scattering,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 14, pp. 5050–5055, 2005. View at Publisher · View at Google Scholar · View at Scopus
G. Uzer, W. R. Thompson, B. Sen et al., “Cell mechanosensitivity to extremely low–magnitude signals is enabled by a LINCed nucleus,” Stem Cells, vol. 33, no. 6, pp. 2063–2076, 2015. View at Publisher · View at Google Scholar · View at Scopus
D. Havelka, M. Cifra, O. Kucera, J. Pokorny, and J. Vrba, “High–frequency electric field and radiation characteristics of cellular microtubule network,” Journal of Theoretical Biology, vol. 286, no. 1, pp. 31–40, 2011. View at Publisher · View at Google Scholar · View at Scopus
S. Sahu, S. Ghosh, D. Fujita, and A. Bandyopadhyay, “Live visualizations of single isolated tubulin protein self–assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule,” Scientific Reports, vol. 4, no. 1, p. 7303, 2014. View at Publisher · View at Google Scholar · View at Scopus
S. Sahu, S. Ghosh, K. Hirata, D. Fujita, and A. Bandyopadhyay, “Multi–level memory–switching properties of a single brain microtubule,” Applied Physics Letters, vol. 102, no. 12, article 123701, 2013. View at Publisher · View at Google Scholar · View at Scopus
I. Cosic, D. Cosic, and K. Lazar, “Is it possible to predict electromagnetic resonances in proteins, DNA and RNA?” EPJ Nonlinear Biomedical Physics, vol. 3, no. 1, p. 5, 2015. View at Publisher · View at Google Scholar
C. Ventura, F. Bianchi, C. Cavallini, E. Olivi, and R. Tassinari, “The use of physical energy for tissue healing,” European Heart Journal Supplements: Journal of the European Society of Cardiology, vol. 17, pp. A69–A73, 2015. View at Publisher · View at Google Scholar · View at Scopus
F. M. Watt and B. L. Hogan, “Out of Eden: stem cells and their niches,” Science, vol. 287, no. 5457, pp. 1427–1430, 2000. View at Publisher · View at Google Scholar · View at Scopus
Y. Sun, C. S. Chen, and J. Fu, “Forcing stem cells to behave: a biophysical perspective of the cellular microenvironment,” Annual Review of Biophysics, vol. 41, no. 1, pp. 519–542, 2012. View at Publisher · View at Google Scholar · View at Scopus
D. Dado, M. Sagi, S. Levenberg, and A. Zemel, “Mechanical control of stem cell differentiation,” Regenerative Medicine, vol. 7, no. 1, pp. 101–116, 2012. View at Publisher · View at Google Scholar · View at Scopus
D. E. Discher, D. J. Mooney, and P. W. Zandstra, “Growth factors, matrices, and forces combine and control stem cells,” Science, vol. 324, no. 5935, pp. 1673–1677, 2009. View at Publisher · View at Google Scholar · View at Scopus
C. Ventura and L. Tavazzi, “Biophysical signalling from and to the (stem) cells: a novel path to regenerative medicine,” European Journal of Heart Failure, vol. 18, no. 12, pp. 1405–1407, 2016. View at Publisher · View at Google Scholar · View at Scopus
L. Tavazzi and C. Ventura, “Observational medicine: registries and electronic health recording for science and health systems governance,” European Journal of Heart Failure, vol. 18, no. 9, pp. 1093–1095, 2016. View at Publisher · View at Google Scholar · View at Scopus
C. Speed, “A systematic review of shockwave therapies in soft tissue conditions: focusing on the evidence,” British Journal of Sports Medicine, vol. 48, no. 21, pp. 1538–1542, 2014. View at Publisher · View at Google Scholar · View at Scopus
C. Ventura, M. Maioli, G. Pintus, G. Gottardi, and F. Bersani, “Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells,” Cardiovascular Research, vol. 45, no. 4, pp. 1054–1064, 2000. View at Publisher · View at Google Scholar · View at Scopus
R. Nuccitelli, “Endogenous ionic currents and DC electric fields in multicellular animal tissues,” Bioelectromagnetics, vol. 13, pp. 147–157, 1992. View at Publisher · View at Google Scholar · View at Scopus
I. G. Macara and S. Mili, “Polarity and differential inheritance-universal attributes of life?” Cell, vol. 135, no. 5, pp. 801–812, 2008. View at Publisher · View at Google Scholar · View at Scopus
S. W. Lane, D. A. Williams, and F. M. Watt, “Modulating the stem cell niche for tissue regeneration,” Nature Biotechnology, vol. 32, no. 8, pp. 795–803, 2014. View at Publisher · View at Google Scholar · View at Scopus
D. L. Miller, N. B. Smith, M. R. Bailey et al., “Overview of therapeutic ultrasound applications and safety considerations,” Journal of Ultrasound in Medicine, vol. 31, no. 4, pp. 623–634, 2012. View at Publisher · View at Google Scholar · View at Scopus
N. M. Shupak, “Therapeutic uses of pulsed magnetic-field exposure: a review,” URSI Radio Science Bulletin, vol. 307, pp. 9–32, 2003. View at Publisher · View at Google Scholar
M. S. Markov, “Magnetic field therapy: a review,” Electromagnetic Biology and Medicine, vol. 26, no. 1, pp. 1–23, 2007. View at Publisher · View at Google Scholar · View at Scopus
Y. Liu, W. B. Liu, K. J. Liu et al., “Effect of 50 Hz extremely low-frequency electromagnetic fields on the DNA methylation and DNA methyltransferases in mouse spermatocyte-derived cell line GC-2,” BioMed Research International, vol. 2015, Article ID 237183, 10 pages, 2015. View at Publisher · View at Google Scholar · View at Scopus
L. Leone, S. Fusco, A. Mastrodonato et al., “Epigenetic modulation of adult hippocampal neurogenesis by extremely low-frequency electromagnetic fields,” Molecular Neurobiology, vol. 49, no. 3, pp. 1472–1486, 2014. View at Publisher · View at Google Scholar · View at Scopus
C. Ventura, M. Maioli, Y. Asara et al., “Turning on stem cell cardiogenesis with extremely low frequency magnetic fields,” The FASEB Journal, vol. 19, no. 1, pp. 155–157, 2005. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, S. Rinaldi, S. Santaniello et al., “Radiofrequency energy loop primes cardiac, neuronal, and skeletal muscle differentiation in mouse embryonic stem cells: a new tool for improving tissue regeneration,” Cell Transplantation, vol. 21, no. 6, pp. 1225–1233, 2012. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, S. Rinaldi, S. Santaniello et al., “Radioelectric asymmetric conveyed fields and human adipose-derived stem cells obtained with a nonenzymatic method and device: a novel approach to multipotency,” Cell Transplantation, vol. 23, no. 12, pp. 1489–1500, 2014. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, S. Rinaldi, S. Santaniello et al., “Radio electric conveyed fields directly reprogram human dermal–skin fibroblasts toward cardiac–, neuronal–, and skeletal muscle–like lineages,” Cell Transplantation, vol. 22, no. 7, pp. 1227–1235, 2013. View at Publisher · View at Google Scholar · View at Scopus
S. Rinaldi, M. Maioli, G. Pigliaru et al., “Stem cell senescence. Effects of REAC technology on telomerase independent and telomerase–dependent pathways,” Scientific Reports, vol. 4, no. 1, p. 6373, 2014. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, S. Rinaldi, R. Migheli et al., “Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson’s disease,” Scientific Reports, vol. 5, no. 1, article 10439, 2015. View at Publisher · View at Google Scholar · View at Scopus
M. Maioli, S. Rinaldi, G. Pigliaru et al., “REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence,” Scientific Reports, vol. 6, no. 1, article 28682, 2016. View at Publisher · View at Google Scholar · View at Scopus
J. M. Kelley, “Extracorporeal shock wave lithotripsy of urinary calculi. Theory, efficacy, and adverse effects,” West Journal of Medicine, vol. 153, pp. 65–69, 1990. View at Google Scholar
C. J. Wang, “An overview of shock wave therapy in musculoskeletal disorders,” Chang Gung Medical Journal, vol. 26, no. 4, pp. 220–232, 2003. View at Google Scholar
V. Pavone, L. Cannavo, A. Di Stefano, G. Testa, L. Costarella, and G. Sessa, “Low-energy extracorporeal shock-wave therapy in the treatment of chronic insertional Achilles tendinopathy: a case series,” BioMed Research International, vol. 2016, Article ID 7123769, 4 pages, 2016. View at Publisher · View at Google Scholar · View at Scopus
A. Notarnicola and B. Moretti, “The biological effects of extracorporeal shock wave therapy (eswt) on tendon tissue,” Muscles, Ligaments and Tendons Journal, vol. 2, no. 1, pp. 33–37, 2012. View at Google Scholar
D. E. Ingber, “Cellular mechanotransduction: putting all the pieces together again,” The FASEB Journal, vol. 20, no. 7, pp. 811–827, 2006. View at Publisher · View at Google Scholar · View at Scopus
Y. J. Chen, Y. R. Kuo, K. D. Yang et al., “Activation of extracellular signal-regulated kinase (ERK) and p38 kinase in shock wave-promoted bone formation of segmental defect in rats,” Bone, vol. 34, no. 3, pp. 466–477, 2004. View at Publisher · View at Google Scholar · View at Scopus
L. Xu, Y. Zhao, M. Wang et al., “Defocused low-energy shock wave activates adipose tissue-derived stem cells in vitro via multiple signaling pathways,” Cytotherapy, vol. 18, no. 12, pp. 1503–1514, 2016. View at Publisher · View at Google Scholar · View at Scopus
L. Rinella, F. Marano, L. Berta et al., “Extracorporeal shock waves modulate myofibroblast differentiation of adipose-derived stem cells,” Wound Repair and Regeneration, vol. 24, no. 2, pp. 275–286, 2016. View at Publisher · View at Google Scholar · View at Scopus
K. Hatanaka, K. Ito, T. Shindo et al., “Molecular mechanisms of the angiogenic effects of low-energy shock wave therapy: roles of mechanotransduction,” American Journal of Physiology. Cell Physiology, vol. 311, no. 3, pp. C378–C385, 2016. View at Publisher · View at Google Scholar · View at Scopus
X. Zhang, X. Yan, C. Wang, T. Tang, and Y. Chai, “The dose-effect relationship in extracorporeal shock wave therapy: the optimal parameter for extracorporeal shock wave therapy,” The Journal of Surgical Research, vol. 186, no. 1, pp. 484–492, 2014. View at Publisher · View at Google Scholar · View at Scopus
Z. Cai, F. Falkensammer, O. Andrukhov, J. Chen, R. Mittermayr, and X. Rausch-Fan, “Effects of shock waves on expression of IL-6, IL-8, MCP-1, and TNF-α expression by human periodontal ligament fibroblasts: an in vitro study,” Medical Science Monitor, vol. 22, pp. 914–921, 2016. View at Publisher · View at Google Scholar · View at Scopus
P. Romeo, V. Lavanga, D. Pagani, and V. Sansone, “Extracorporeal shock wave therapy in musculoskeletal disorders: a review,” Medical Principles and Practice, vol. 23, no. 1, pp. 7–13, 2014. View at Publisher · View at Google Scholar · View at Scopus
R. Mittermayr, V. Antonic, J. Hartinger et al., “Extracorporeal shock wave therapy (ESWT) for wound healing: technology, mechanisms, and clinical efficacy,” Wound Repair and Regeneration, vol. 20, no. 4, pp. 456–465, 2012. View at Publisher · View at Google Scholar · View at Scopus
J. K. Gimzewski, A. Pelling, and C. Ventura, “Nanomechanical characterization of cellular activity,” Tech. Rep., International Patent WO 2008/105919 A2, 2008. View at Google Scholar
A. E. Pelling, S. Sehati, E. B. Gralla, J. S. Valentine, and J. K. Gimzewski, “Local nanomechanical motion of the cell wall of Saccharomyces cerevisiae,” Science, vol. 305, no. 5687, pp. 1147–1150, 2004. View at Publisher · View at Google Scholar · View at Scopus

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Novel Diagnostic Biomarkers of Prostate Cancer: An Update

Abstract

OBJECTIVE:

In recent years, several biomarkers alternative to standard prostate specific antigen (PSA) for prostate cancer (PCa) diagnosis have become available. The aim of this systematic review is to assess the current knowledge about alternative serum and urinary biomarkers for the diagnosis of PCa.

MATERIAL AND METHODS:

A research was conducted in Medline, restricted to English language articles published between December 2014 and June 2018 with the aim to update previously published series on PCa biomarkers. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) criteria were used for selecting studies with the lowest risk of bias.

RESULTS:

Emerging role and actual controversies on serum and urine alternative biomarkers to standard PSA for PCa diagnosis, staging and prognosis assessment, such as prostate health index (PHI), PCA3, ConfirmMDx, Aberrant PSA glycosylation, MiPS, miRNAs are critically presented in the current review.

CONCLUSION:

Although the use of several biomarkers has been recommended or questioned by different international guidelines, larger prospective randomized studies are still necessary to validate their efficacy in PCa detection, discrimination, prognosis and treatment effectiveness. To date, only PHI and 4Kscore have shown clinical relevance for discriminating more aggressive PCa. Furthermore, a new grading classification based on molecular features relevant for PCa risk-stratification and tailoring treatment is still needed.

KEYWORDS:

Biomarkers; diagnosis; prognosis; prostate cancer; serum; urine; Biava PM.

From: https://www.ncbi.nlm.nih.gov/pubmed/30215331

Abstract

OBJECTIVE:

In recent years, several biomarkers alternative to standard prostate specific antigen (PSA) for prostate cancer (PCa) diagnosis have become available. The aim of this systematic review is to assess the current knowledge about alternative serum and urinary biomarkers for the diagnosis of PCa.

MATERIAL AND METHODS:

A research was conducted in Medline, restricted to English language articles published between December 2014 and June 2018 with the aim to update previously published series on PCa biomarkers. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) criteria were used for selecting studies with the lowest risk of bias.

RESULTS:

Emerging role and actual controversies on serum and urine alternative biomarkers to standard PSA for PCa diagnosis, staging and prognosis assessment, such as prostate health index (PHI), PCA3, ConfirmMDx, Aberrant PSA glycosylation, MiPS, miRNAs are critically presented in the current review.

CONCLUSION:

Although the use of several biomarkers has been recommended or questioned by different international guidelines, larger prospective randomized studies are still necessary to validate their efficacy in PCa detection, discrimination, prognosis and treatment effectiveness. To date, only PHI and 4Kscore have shown clinical relevance for discriminating more aggressive PCa. Furthermore, a new grading classification based on molecular features relevant for PCa risk-stratification and tailoring treatment is still needed.

KEYWORDS:

Biomarkers; diagnosis; prognosis; prostate cancer; serum; urine; Biava PM.

Still in 2019:

Zebrafish embryo extract counteracts human stem cell senescence

Abstract

Human adult stem cells hold promise for regenerative medicine. They are usually expanded for multiple passages in vitro to increase cell yield prior to transplantation. Unfortunately, prolonged culture leads to cell senescence, a major drawback from successful outcomes in cell therapy approaches. Here, we show that an extract from early Zebrafish embryo (ZF1) counteracted senescence progression in human adipose-derived stem cells (hASCs) along multiple culture passages (from the 5th to the 20th). Exposure to ZF1 strongly reduced the expression of senescence marker beta-galactosidase. Both stemness (NANOG, OCT4, and MYC) and anti-senescence (BMI1, and telomerase reverse transcriptase – TERT) related genes were overexpressed at specific experimental points, without recruitment of the cyclin-dependent kinase Inhibitor 2A (CDKN2A, ali-as p16). Increased telomerase activity was associatt-ed with TERT overexpression. Both osteogenic and adipogenic abilities were enhanced. In conclusion, hASCs exposure to ZF1 is a feasible tool to counteract and reverse human stem cell senescence in long-term culturing conditions.

From: https://www.ncbi.nlm.nih.gov/pubmed/30844738

Abstract

Human adult stem cells hold promise for regenerative medicine. They are usually expanded for multiple passages in vitro to increase cell yield prior to transplantation. Unfortunately, prolonged culture leads to cell senescence, a major drawback from successful outcomes in cell therapy approaches. Here, we show that an extract from early Zebrafish embryo (ZF1) counteracted senescence progression in human adipose-derived stem cells (hASCs) along multiple culture passages (from the 5th to the 20th). Exposure to ZF1 strongly reduced the expression of senescence marker beta-galactosidase. Both stemness (NANOG, OCT4, and MYC) and anti-senescence (BMI1, and telomerase reverse transcriptase – TERT) related genes were overexpressed at specific experimental points, without recruitment of the cyclin-dependent kinase Inhibitor 2A (CDKN2A, ali-as p16). Increased telomerase activity was associatt-ed with TERT overexpression. Both osteogenic and adipogenic abilities were enhanced. In conclusion, hASCs exposure to ZF1 is a feasible tool to counteract and reverse human stem cell senescence in long-term culturing conditions.

Still in 2019:

Active Fraction from Embryo Fish Extracts Induces Reversion of the Malignant Invasive Phenotype in Breast Cancer through Down-regulation of TCTP and Modulation of E-cadherin/β-catenin Pathway

Abstract

From: https://www.ncbi.nlm.nih.gov/pubmed/31052313

Some yet unidentified factors released by both oocyte and embryonic microenvironments demonstrated to be non-permissive for tumor development and display the remarkable ability to foster cell/tissue reprogramming, thus ultimately reversing the malignant phenotype. In the present study we observed how molecular factors extracted from Zebrafish embryos during specific developmental phases (20 somites) significantly antagonize proliferation of breast cancer cells, while reversing a number of prominent aspects of malignancy. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Counteracting the invasive phenotype is a relevant issue in controlling tumor spreading and metastasis. Moreover, such effect is not limited to cancerous cells as embryo extracts were also effective in inhibiting migration and invasiveness displayed by normal breast cells undergoing epithelial-mesenchymal transition upon TGF-β1 stimulation. The reversion program involves the modulation of E-cadherin/β-catenin pathway, cytoskeleton remodeling with dramatic reduction in vinculin, as well as downregulation of TCTP and the concomitant increase in p53 levels. Our findings highlight that-contrary to the prevailing current “dogma”, which posits that neoplastic cells are irreversibly “committed”-the malignant phenotype can ultimately be “reversed”, at least partially, in response to environmental morphogenetic influences.

From: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539734/

Abstract

Some yet unidentified factors released by both oocyte and embryonic microenvironments demonstrated to be non-permissive for tumor development and display the remarkable ability to foster cell/tissue reprogramming, thus ultimately reversing the malignant phenotype. In the present study we observed how molecular factors extracted from Zebrafish embryos during specific developmental phases (20 somites) significantly antagonize proliferation of breast cancer cells, while reversing a number of prominent aspects of malignancy. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Counteracting the invasive phenotype is a relevant issue in controlling tumor spreading and metastasis. Moreover, such effect is not limited to cancerous cells as embryo extracts were also effective in inhibiting migration and invasiveness displayed by normal breast cells undergoing epithelial–mesenchymal transition upon TGF-β1 stimulation. The reversion program involves the modulation of E-cadherin/β-catenin pathway, cytoskeleton remodeling with dramatic reduction in vinculin, as well as downregulation of TCTP and the concomitant increase in p53 levels. Our findings highlight that—contrary to the prevailing current “dogma”, which posits that neoplastic cells are irreversibly “committed”—the malignant phenotype can ultimately be “reversed”, at least partially, in response to environmental morphogenetic influences.

Keywords: tumor reversion, TCTP, embryo fish extract, cytoskeleton, E-cadherin/β-catenin, p53, Biava PM

1. Introduction

Some yet unidentified factors released by both oocyte and embryonic microenvironments demonstrated to be non-permissive for tumor development and display the remarkable ability to foster cell/tissue reprogramming, thus ultimately reversing the malignant phenotype [,].

Pioneer studies from the sixties showed that carcinoma cells are “reprogrammed” when injected into a mouse blastocyst, ultimately resulting in normal tissue originating from cancer cells []. Since then, several cancer types have been shown to undergo partial or complete reversion when exposed to embryonic environments or treated with soluble factors extracted from oocytes or embryonic cells (reviewed in []). Microenvironments derived from mouse, human embryonic stem cells, zebrafish (Danio rerio), chick, and amphibian embryo/eggs extracts were used to give an insight into the molecular reprogramming of cancer cells in response to the embryonic environment [].

This process may entail proliferation and apoptosis rate [], as well as partial or complete reversion of the malignant phenotype, including DNA demethylation, removal of repressive histone marks at the promoters of tumor suppressor genes, and expression of silenced genes [,,,].

These studies documented that the reversion program is a complex attempt, running through discrete steps in which differentiation that lead a stem cell toward a complete phenotypic commitment is recapitulated according to an ”inverse process” []. This remark substantiates the concept for which carcinoma is a caricature of the normal process of tissue renewal, or a “development gone awry” [].

The fact that the observed reversal of malignancy in cancer cells frequently involves only some features of the malignant phenotype, evidences how limited is our knowledge of the molecular and biophysical mechanisms that orchestrated the reversion.

Namely, only few factors extracted from embryo kept during a well-defined period of development share the capability to trigger tumor reversion []. For instance, when embryonal carcinoma cells were injected into 8- to 15-day mouse embryos, it was found that the ability to abolish malignancy was inversely proportional to the age of the embryo at the moment of carcinoma cell transplantation [].

Indeed, the release of morphogenetic factors significantly differs according to the different stages of embryonic development [] and we can truthfully postulate that cancer cells can only be sensitive to only some signaling molecules depending on the differentiated stage of the tumor itself. Moreover, the participation of non-molecular factors, i.e., biophysical constraints shaping the overall architecture of cell and tumor tissue, cannot be discarded and should instead be considered mandatory to ensure a full reversion of the malignant phenotype [], given that reprogramming can also be achieved only through manipulation of the biophysical properties of the microenvironment [].

We have already shown that stage developmental factors extracted from embryos of Zebrafish can efficiently arrest cancer proliferation and induce apoptosis, both in vitro [] and in vivo []. Moreover, sublingual administration of Zebrafish extracts showed to improve response to conventional chemotherapy while reducing the incidence of drug-resistance in a pilot study on human colon cancer patients [].

Mature oocytes and undifferentiated embryonic stem (ES) cells contain reprogramming factors (proteins, RNAs, lipids, small molecules) that enable these cells to reprogram a somatic nucleus to pluripotency []. However, compelling evidence is still lacking. Nevertheless, it can be inferred from in vivo studies that embryo/oocyte factors are likely low-weight soluble components, easily absorbable through the mucosa. Co-culture of breast cancer cells with embryonic mesenchyme from early stage mammary glands decreases tumor cell proliferation while stimulating acinus differentiation, as reported by a few scientific studies. Namely, both soluble and insoluble components of embryo microenvironment have demonstrated the ability to reverse neoplastic progression and “reboot” breast cancer, resulting in at least partial normalization of tumor cells [,].

Over the last decades, Zebrafish has proven to be a powerful model in cancer research. Indeed, Zebrafish displays more than 80% of all human disease-related genes, indicating that many human diseases can, in fact, be modeled in Zebrafish. Namely, an impressive body of studies demonstrated that Zebrafish can serve as a useful model to investigate tumor driving as well as anti-tumor mechanisms []. Moreover, cancer research in Zebrafish particularly benefits from the many genetic tools and transgenic strains established by the Zebrafish community over the years.

Herewith we seek to determine the stages of Zebrafish embryo development that display the most significant activity in inducing the reversal of prominent malignant features, like proliferation, migrating capacity, and invasiveness.

2. Results

2.1. Recognizing the Most Effective Embryo Fish Extract Fraction

We performed a preliminary screening to identify the most effective embryo fraction (EF). Biological activity was ascertained by considering as prominent parameter the reduction in cell viability, assessed with the Sulforhodamine B (SRB) colorimetric assay. Two concentrations (0.3 and 3.0 μg/mL) of each EF were tested in breast cancer cell lines—MCF7 and MDA-MB-231—at different time points (24, 48, and 72 h). This preliminary survey identified F6 (corresponding to the 20-somite stage) to be the most active fraction, and significant effects were also recorded in F4 and F5, for both cell lines. No significant differences were found between 0.3 and 3 μg/mL concentrations (Supplementary Materials Figures S1 and S2; Tables S1 and S2). Hereafter, only the F6 at 0.3 μg/mL was utilized in the succeeding experimental phases.

2.2. Embryo Extract Stimulates Apoptosis

Besides the reduction in cell viability as assessed with the SRB test, we investigated if EFs could foster the apoptosis rate. As expected, fractions from F1 to F5 did not induce any significant increase in apoptosis either in MCF7 or MDA-MB-231 cancer cells, at any of the time points we considered (data not shown). Instead, F6 at both 0.3 and 3 μg/mL significantly raise the apoptosis rate in MDA-MB-231 cells, while in MCF7 cells only a trend toward increased apoptosis was observed, albeit not significant (Figure 1). Given that at 72 h 5FU kills almost all cells, the possible additive effect of F6 cannot be ascertained when F6 was associated with the chemotherapic drug. Overall, data obtained with both the SRB and the MUSE test demonstrated that some embryo extracts could efficiently impair cancer cell viability.

Figure 1: Effect of Zebrafish embryo F6 fraction on apoptosis of MDA-MB-231 (a) and MCF-7 (b) cells after 72 h of treatment with F6 at 0.3 and 3 μg/mL. Histograms showing the percentage of apoptotic cells; each column represents the mean value ± SD of four independent experiments. * p < 0.05 versus ctrl by ANOVA followed by Bonferroni post-test.

2.3. Embryo Extract Reduces Cancer Cell Proliferation

Cell proliferation was investigated in MDA-MB-231 and MCF-7 cells at 24 h by comparing data recorded in cells treated with 5FU or F6 alone and in association. As shown in Figure 2, in both cell lines 5FU slightly reduces cell proliferation. F6 and 5FU+F6 significantly decreased cell growth to less than 60% of control values. Moreover, in MDA-MB-231 cells, the association 5FU+F6 further decreased cell proliferation compared to F6 alone, even if without statistical relevance. These findings evidenced that F6 significantly slows down cancer proliferation and most likely amplifies the cytostatic effect of 5FU.

Figure 2: Effect of 5FU, 5FU+F6, and F6 on proliferation of MDA-MB-231 (a) and MCF-7 (b) cells. Cell proliferation was determined after 24 h of treatment by cell count assays performed by a particle count and size analyzer. Values, expressed as fold increase of control value considered as 1, are means of three independent experiments performed in triplicate, with SD represented by vertical bars. * p < 0.05 versus ctrl by ANOVA followed by Bonferroni post-test.

2.4. Embryo Extract Antagonizes Cancer Cell Invasiveness and Migrating Capability

To ascertain to what extent F6 can significantly reverse the malignant phenotype, we plan to investigate some remarkable parameters belonging to the macroscopic–mesoscopic level, where microscopic elements are “channeled” and organized in a coherent manner in producing macroscopic features, as recorded by macroscopic parameters []. Indeed, the mesoscopic approach strives to “capture” the self-organizing process, which in turn will lead to the emergence of specific system’s properties []. Therefore, we evaluate invasiveness and migrating capability in the highly malignant cell line MDA-MB-231, given that MCF7 cells display only minimal invasive capacity. We observed that F6 dramatically reduced invasiveness below to 60% as recorded in untreated cells, while 5FU had no effect (Figure 3a,b). Both MMP2 and MMP9 have been measured to investigate their potential involvement in the observed inhibition of invasiveness. As a result, MMP9 was reduced in both F6 and 5FU+F6, while MMP2 shows a slight increase in both conditions (data not shown). Overall, such changes were of little significance and we decided to look at uPA to ascertain if invasiveness reduction in treated samples could be attributed to uPA modulation. Indeed, inhibition of invasive phenotype was further confirmed when urokinase plasminogen activator (uPA) levels were investigated in conditioned media of MDA-MB-231 cells. During tumor progression, uPA, after binding to its receptor (uPAR), activates a cascade of proteases, ultimately leading to the degradation of the basement membrane, thus fostering tumor cell invasiveness. Decreasing of uPA in breast cancer cells dramatically reduces the wound healing, migratory, invasive, and adhesive capacity of cancer cells []. In our experiments, uPA levels were significantly reduced after 24 h in 5FU- and F6-treated cells, while no additive effects were observed with the association of both (Figure 3c).However, inhibition of invasiveness in MDA-MB-231 cells can only partially be explained by downregulation of a single molecular factor, alike uPA. Indeed, in 5FU-treated cells, despite uPA reduction, invasiveness remains unchanged. Probably other factors, including cytoskeleton modifications (i.e., those involving migratory/invasive structures, like pseudopodia) play a major role. Furthermore, migration was highly hindered in both 5FU- and F6-treated groups (Figure 4a,b). Remarkably, F6 was even more efficient than 5FU in inhibiting migratory capability, while the association of both F6+5FU were shown to exert additive effects. Embryo factor demonstrated thus to be even more effective than conventional chemotherapy in reversing prominent malignant features like invasiveness and migratory behavior. To ascertain if this effect could be traced back to the epithelial–mesenchymal (EMT) features harbored by invasive cancer cells, we investigated the F6 inhibitory effects on a previously studied model of normal breast cells (MCF10A), which had been committed to EMT upon TGF-β stimulation []. Briefly, the immortalized, not transformed MCF10A breast cell line was treated with TGF-β1 for five days. Both invasiveness and motility of MCF10A cells increased to fivefold under these conditions (Figure 5a,b). Addition of 5FU only partially mitigated that increase, while F6 almost completely nullified the TGF-β increase. This finding specifically evidenced that F6 was able in interfering with the acquisition of the invasive, EMT-dependent phenotype, independently from either the malignant or the benign hallmark of cells under study.

Figure 3: Effect of 5FU, 5FU+F6, and F6 on invasion in MDA-MB-231 cells. Transwell invasion assay (a,b) and urokinase plasminogen activator (uPA) levels (c) was performed in MDA-MB-231 cells untreated (ctrl) and treated with 5FU, 5FU+F6, and F6 for 24h. Values, expressed as fold increase of control value considered as 1, are means of three independent experiments performed in duplicate, with SD represented by vertical bars. * p < 0.05; *** p < 0.001 versus ctrl; # p < 0.05 versus 5FU by ANOVA followed by Bonferroni post-test. Images were obtained by optical microscopy, with 100× magnification.

Figure 4: Effect of 5FU, 5FU+F6, and F6 on migration in MDA-MB-231 cells. Transwell migration assay (a,b) was performed in MDA-MB-231 cells untreated (ctrl) and treated with 5FU, 5FU+F6, and F6 for 24 h. Values, expressed as fold increase of control value considered as 1, are means of three independent experiments performed in duplicate, with SD represented by vertical bars. * p < 0.05; ** p < 0.01; *** p < 0.001 versus ctrl; # p < 0.05 versus 5FU by ANOVA followed by Bonferroni post-test. Images were obtained by optical microscopy, with 100× magnification.

Figure 5: Effect of 5FU, 5FU+F6, and F6 on invasion (a) and migration (b) in MCF-10A cells. Transwell assays were performed in MCF-10A cells untreated (ctrl), and pre-treated with TGF-β1 for 5 days. TGF-β1 stimulated MCF-10A cells were then treated with 5FU, 5FU+F6, and F6 for 24 h. Values, expressed as fold increase of control value considered as 1, are means of three independent experiments performed in duplicate, with SD represented by vertical bars. * p < 0.05; ** p < 0.01; *** p < 0.001 versus ctrl; # p < 0.05; ## p < 0.01; ### p < 0.001 versus TGF-β1; @ p < 0.05; @@ p < 0.01 versus 5FU by ANOVA followed by Bonferroni post-test. Images were obtained by optical microscopy, with 100× magnification.

2.5. Cytoskeleton Remodeling

Changes in the migratory/invasive phenotype are indeed mirrored by cytoskeleton rearrangement under the influence of F6. While both control and 5FU-treated MDA-MB-231 cells harbored a dense texture of stress fibers, with actin filaments distributed all along the cytosol, in F6-treated cells actin was predominantly concentrated along the membrane border (Figure 6a). Moreover, F6-treated MDA-MB-231 cells almost completely lost pseudopodia and lamellipodia, two prominent structures required by migrating/invasive cells, as evidenced in control and 5FU-treated MDA-MB-231 cells, in which polarized lamellipodia with treadmilling filaments, as well as filopodia were clearly observable. Overall, those changes enabled F6-treated cells to recover a rounded shape, with reduced spreading and smaller nucleus (Figure S3). The loss of the migratory/invasive phenotype is further confirmed when looking at the distribution of vinculin fibers and their association with actin (Figure 6b,c). Vinculin-expressing cells are able to migrate into dense three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. Indeed, vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of extra-cellular matrix []. We observed that in F6-treated cells vinculin levels are significantly reduced (Figure 7a), namely at the membrane border, where vinculin seems to be dissociated from actin filaments. Indeed, vinculin preferentially localizes inside the cytosol, thus losing contact with actin filament and impairing the migrating and invasive capabilities of cancer cells, as previously reported []. This finding should be put in correlation with ROCK1 activity. In F6-treated cells, we observed a paradoxical increase in ROCK1 levels that apparently can hardly accommodate the reduced invasiveness/motility of embryo-treated cells (Figure 7b). However, ROCK1 functions differ depending on the stiffness of the substrate upon which cells are cultivated. In stiffness conditions mimicking those observed in vivo, downregulation of ROCK1 promoted cell spreading and cell migration []; instead, high levels of activated Rho kinase and ROCK1 are required for inhibiting motility and stabilizing E-cadherin adhesions through F-actin fibers []. Namely, ROCK1 activation is considered mandatory for proper reprogramming of cells [] and for the acquisition of the correct morphological pattern in developing embryos []. On the contrary, in teratocarcinoma cells, the ROCK inhibitor Y-27632 promotes migration, accompanied by an apparent increase in focal complexes and lamellipodia and a decrease in focal adhesions and stress fibers. In this condition, reduced levels of vinculin amplify the motility inhibition triggered by ROCK1 increase []. Given that in our model we observed that ROCK1 increases and vinculin decrease, it can be hypothesized that both conditions may enhance inhibition of the migrating/invasive phenotype of MDA-MB-231 cells.

Figure 6: Distribution pattern of vinculin and F-actin in wound-healing assay performed on MDA cells cultured in control condition or exposed to 5FU, F6, and 5FU+F6. Confocal microscopy analysis of F-actin staining with rhodamine-phalloidin (red signal, (a) column) merged with anti-vinculin immunofluorescence (FITC/green signal, b column) on MDA cells subjected to wound healing assay, and cultured with or without 5FU, F6, 5FU+F6. The white arrows in the images of the left column indicate the direction of cellular movement toward the gap. In column (c) we reported higher magnification of the merging pictures shown in column (b).

Figure 7: Effect of 5FU, 5FU+F6, and F6 on expression of vinculin (a) and Rock1 (b) in MDA-MB-231 cells. Columns represent densitometric quantification of optical density (OD) of specific protein signal normalized with the OD values of GAPDH served as a loading control and they are expressed as fold increase of control value considered as 1. Each column represents the mean value ± SD of three independent experiments. * p < 0.05 versus ctrl by ANOVA followed by Bonferroni post-test. Representative western blot analysis relating to vinculin and Rock1expression in MDA-MB-231 cells untreated (ctrl) and treated with 5FU, 5FU+F6, and F6 for 24 h. GAPDH was used as loading control.

2.6. Embryo Extracts Promotes E-cadherin/β-catenin Redistribution behind Cell Membrane

MDA-MB-231 breast cancer cells showed prominent mesenchymal features, as down-regulation of E-cadherin, while β-catenin was redistributed in the cytosol and the nucleus []. Control cancer cells displayed low E-cadherin levels, while both 5FU- and F6-treated cells showed a trend, albeit not significant, toward increased release (Figure 8a); β-catenin increases significantly only in 5FU+F6-treated cells, while showing a slight, albeit not significant decrease in the other treatment conditions (Figure 8b). However, the E-cadherin/β-catenin ratio resulted significantly increased in the 5FU+F6-treated group (Figure 8c). Loss of either E-cadherin or β-catenin at the cell membrane contributes in disassembling E-cadherin/β-catenin complexes, through which cadherin sequesters β-catenin, preventing its dispersion into the cytoplasm, and its subsequent nuclear transcriptional activities [].

Figure 8: Effect of 5FU, 5FU+F6, and F6 on expression of E-cadherin (a), β-catenin (b), and E-cadherin/ β-catenin ratio (c) in MDA-MB-231 cells. Columns represent densitometric quantification of optical density (OD) of specific protein signal normalized with the OD values of GAPDH served as a loading control and they are expressed as fold increase of control value considered as 1. Each column represents the mean value ± SD of three independent experiments. * p < 0.05 versus ctrl; # p < 0.05 versus 5FU by ANOVA followed by Bonferroni post-test. Representative western blot analysis relating to E-cadherin and β-catenin expression in MDA-MB-231 cells untreated (ctrl) and treated with 5FU, 5FU+F6, and F6 for 24 h. GAPDH was used as loading control. (d) Confocal microscopy analysis of beta-catenin immunostaining (FITC/green signal) on MDA cells cultured in control condition, or with the following treatments: 5FU, F6, 5FU+F6. Distribution of β-catenin increase behind the cell membrane mostly in F6-treated cells.

In control cancer cells, overall β-catenin is lowered and mostly dispersed around in the cytoplasm, with only isolated spots at the membrane site of adhesion (Figure 8d). Instead, in F6-treated cells, immune staining unveiled a strong β-catenin signal, mostly located at the cell-to-cell junction level, thus indicating the restoring of the E-cadherin/β-catenin complexes.

2.7. Embryo Extracts Downregulate TCTP Expression in MDA-MB-231 Cells

Translationally Controlled Tumor Protein (TCTP) has emerged as a critical regulator of cell fate determination, as it regulates many different biological processes, all of which may converge to a limited set of key events that control cell fate determination and namely, tumor reversion. Aberrant expression of TCTP is frequently observed in cancer cells while silencing TCTP showed to be instrumental in promoting cancer reversion in different types of cancer cells. Namely, silencing TCTP expression in breast cancer was demonstrated to restore growth and morphological patterns reminiscent of the outgrowths generated by normal mammary epithelial cells []. Down-regulation of TCTP is usually accompanied by an increase in p53 levels, suggesting thus that up-regulation of p53 is required for enacting the reversion process. Indeed, it has been proposed that the modulation of the TCTP-p53 axis is a pre-requisite for triggering tumor reversion []. In our model, F6-treated cells showed a significant down-regulation of TCTP, while in 5FU-treated cells only a slight, not significant decrease in TCTP levels was found (Figure 9a). As expected, p53 increases in treated samples. It is worth noting that the increase specifically involves the acetylated form of p53, i.e., the active form of p53, which is resilient to MDM2-dependent degradation [] (Figure 9b).

Figure 9: Effect of 5FU, 5FU+F6, and F6 on expression of TCTP (a) and acetyl-p53 (b) in MDA-MB-231 cells. Columns represent densitometric quantification of optical density (OD) of specific protein signal normalized with the OD values of GAPDH served as a loading control and they are expressed as fold increase of control value considered as 1. Each column represents the mean value ± SD of three independent experiments. * p < 0.05; ** p < 0.01 versus ctrl; # p < 0.05 versus 5FU by ANOVA followed by Bonferroni post-test. Representative western blot analysis relating to TCTP and acetyl-p53 expression in MDA-MB-231 cells untreated (ctrl) and treated with 5FU, 5FU+F6, and F6 for 24 h. GAPDH was used as loading control.

3. Discussion

In the present study, we showed that unknown molecular factors extracted from Zebrafish embryos during specific developmental phases (20 somites) significantly antagonize proliferation of breast cancer cells, while reversing some prominent aspects of the malignant phenotype. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Counteracting the invasive phenotype is a relevant issue in controlling tumor spreading and metastasis. Moreover, such effect is not limited to cancerous cells as embryo extracts were also effective in inhibiting migration and invasiveness displayed by normal breast cells undergoing epithelial–mesenchymal transition upon TGF-β1 stimulation. The reversion program, as previously reported by several studies, involves downregulation of TCTP and the concomitant increase in p53 levels.

TCTP is a key player in the process of tumor reversion, the process in which a tumor cell is transformed into a revertant cells by losing its malignant traits—uncontrolled growth, invasiveness, and metastasis-forming capability—through the activation of a complex cascade of biochemical events, including cytoskeleton remodeling, pathways modulation, and even gene reprogramming [].

TCTP knockdown in primary mammary tumor cells from ErbB2 transgenic mice resulted in increased p53 expression and fewer stem cell-like cancer cells, while in breast cancer patients a high-TCTP status is associated with aggressive tumors and predicts a poor prognosis []. Downstream to TCTP inhibition, EMT is antagonized through cytoskeleton remodeling and rearrangement of the E-cadherin/β-catenin junctions. Those changes are considered instrumental steps in promoting the reversal of the epithelial–mesenchymal transition [], while modulation of vinculin, ROCK1, and uPA will finally antagonize the invasive/migratory proneness of breast cancer cells.

The tumor reversion we observed in our model, notwithstanding how incomplete the process can be, has the merit to highlight some essential steps that are mandatory for suppressing/achieving malignancy. Indeed, tumor reprogramming proceeds along paces that, in a reverse mode, “recapitulate” carcinogenic steps, thus allowing in ascertaining critical crossroads, which still must be investigated in depth.

The reversion process leads, sometimes, to a complete reversal but, frequently, reversion is only partially obtained. Indeed, the multistep nature of tumorigenesis is paralleled by the series of “uphill” steps required in order to achieve full reprogramming to pluripotency, and the requirement for different factors allows overcoming several barriers that are biologically designed to protect cells from the transformation, that is, to prevent cells from changing their identity. Similarly, during the reprogramming, several steps can be attained before a fully reprogrammed state could be achieved []. Moreover, depending on the internal/external interplay of constraints, a single molecular factor may eventually play opposite roles, as evidenced by the paradoxical behavior of the so-called oncogenes, recognized to act as either tumor promoters or tumor-suppressor depending on the permissive influence put forth by the context []. It is worth noting that embryo extracts promote the tumor reversion by concomitantly increasing p53 levels. In somatic cell reprogramming, it is well-recognized that the elimination of the DNA damage control checkpoint greatly boosts the efficiency of the reprogramming process []. Indeed, the elimination of the p53–p21 pathway by different means allows many of the starting cells to successfully complete the journey to full pluripotency. However, it does so at a price, which is that of genetic instability, in such a way that most of the induced pluripotent cells obtained in this manner carry genetic aberrations of different kinds []. As a result, the main potential complication in manipulating reprogrammed cells in therapeutic settings is precisely tumor generation as a result of uncontrolled growth or differentiation of the newly introduced cells into the recipient patient. Instead, in our model, down-regulation of TCTP is followed by increased activation of p53. This is a guarantee that cells cannot be further destabilized and can safely travel through the reversion pathway, until they reach a new stable, non-tumorigenic phenotype. Indeed, downstream of p53 activation, cells may be committed to apoptosis or can undergo growth arrest with subsequent differentiation, thus recovering a more physiological phenotype, and avoiding the risk to which somatic cells are exposed during reprogramming.

Treatment of breast cancer cells with 5FU is followed by a dramatic increase in apoptosis at 72 h, when cancer cells were almost all killed. However, 5FU exert only minimal, if any, effect on tumor reversion, especially when invasiveness and migrating capability are considered. Instead, addition of embryo extracts to 5FU-treated cells amplify the chemotherapy-induced cytostatic effect at early times and enhances the reversion of the invasive phenotype. This additive activity could be exploited in improving clinical response to conventional drugs, as previously reported in colon cancer patients treated with chemotherapy and embryo fish extracts [].

A major drawback of the present study is constituted by the lack of information about the identity of the “reprogramming” factors present in the pool of molecules extracted from zebrafish embryos. However, indirect evidence suggest that they could be represented by low-molecular weight components, as they are easily absorbed by oral mucosae. Studies are currently ongoing in our laboratory to ascertain the true nature of such molecular factors.

The recognition of cancer as a disease of reprogramming opens the door to therapeutic strategies directed at correcting the wrong differentiation program in an attempt to eliminate the cancerous clone from the root. Differentiation therapies are already successfully in use for some very specific cases of cancer []. Our findings provide support to this new approach, highlighting that, contrary to the prevailing current “dogma”, cancer cells do not always beget cancer cells, and malignant cells may differentiate in response to (complex) environmental influences.

Tumor cells can indeed be as amenable to reprogramming as the normal ones have shown to be. Hopefully, subsequent studies will disclose the possibility to change the natural fate of tumors and, either force them to differentiate and disappear, or convert them into cells susceptible to the newly developed targeted therapies.

4. Materials and Methods

4.1. Experimental Cell Model

The human hormone-sensitive breast adenocarcinoma cell line MCF-7 (ECACC Cat# 86012803), the human Caucasian breast adenocarcinoma MDA-MB-231 (ECACC Cat# 92020424) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The non-tumorigenic epithelial cell line MCF-10A (ATCC CRL-10317) were obtained from LGC Standards S.r.l, MI, Italy. Cells were seeded into 25 cm2 flasks (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA). MCF-7 and MDA-MB-231 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin 100 IU/mL, streptomycin 100 µg/mL, gentamycin 200 µg/mL; all from Euroclone Ltd., Cramlington, UK), MCF-10A were grown in Dulbecco’s modified Eagle’s medium/ nutrient mixture F12 Ham (Sigma-Aldrich, Merck, Darmstadt, Germany) supplemented with 10% horse serum (Euroclone Ltd., Cramlington, UK) and EGF 500 µ/5 mL (Santa Cruz Biotechnologies, Dallas, TX, USA), Hydrocortisone (50 µM), cholera toxin (0.5 mg/mL), insulin (10 mg/mL) (all from Sigma Chemical Co) and antibiotics (penicillin 100 IU/mL, streptomycin 100 µg/mL, gentamycin 200 µg/mL; all from Euroclone Ltd., Cramlington, UK). The cells were cultured at 37 °C in an atmosphere of 5% CO2 in air. The medium was changed every third day. At confluence, the cells were sub-cultured after removal with 0.05% trypsin–0.01% EDTA. In MCF-7 and MDA-MB-231 0.1 mg/mL 5-Fluorouracil (5-FU; Sigma–Aldrich), 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3µg/mL F6 were added in DMEM supplemented with 0.1% FBS. MCF-10A cells were firstly treated with 10ng/mL TGFβ1 (PeproTech catalog#100-21) for five days and on fifth day 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3 µg/mL F6 were added in F12 Ham supplemented with 0.1% horse serum.

4.2. Zebrafish Embryo Extracts

The embryos of Zebrafish (cultured under standard conditions as previously described []) were kept at different stage of development. 516 cells for each stage: blastula period (F1); 80% epiboly (F2), tailbud (F3), during the Gastrula period; 10 somites-stage (F4), 18 somites-stage (F5) and 20 somites-stage (F6), corresponding to the segmentation period, according to the developmental phases of Zebrafish embryo. Partitioning of Zebrafish embryos into the different stages of development has been assessed by three independent biologists. Measurement of total protein content for each sample and stage has been carefully recorded twice with Bradford assay. Samples were standardized (by considering protein content and number of cells) and properly stored until use. Embryos were separately collected, washed in distilled water, and dissolved with a turbo-emulsifier in cold PBS for 60 s before use.

4.3. In Vitro Toxicology Assay Kit Sulforhodamine B Based

4 × 104 cells were seeded in a 96-multiwell and stimulated with F1, F2, F3, F4, F5, F6 at concentration of 0.1, 1, 10, 0.3, 3, 30 µg/mL, respectively. After 24, 48, and 72 h the cells were fixed for 1 h at 4 °C by gently layering 1/4 volume of cold 50% (w/v) Trichloroacetic Acid (TCA Solution) on top of the growth medium, and then rinsed with water several times to remove TCA solution, serum proteins, etc. Plates were air dried and stored until use. Blank background optical density was measured in wells incubated with growth medium without cells. The 0.4% Sulforhodamine B Solution (Sigma-Aldrich Catalog Number S2902) was added in a sufficient amount to cover the culture surface area (∼50% of the culture medium volume). Cells were stained for 20–30 min and at the end of the staining period, the stain was removed, and the cells quickly rinsed with Wash Solution (1% acetic acid) until unincorporated dye was removed. The incorporated dye was then solubilized in a volume of Sulforhodamine B Assay Solubilization Solution (10 mM Tris) equal to the original volume of culture medium. Absorbance at a wavelength of 565 nm was spectrophotometrically measured.

4.4. Cell Migration Assay

The 2.5 × 104 cells non-stimulated (ctrl) and stimulated 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3µg/mL F6 respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters (Falcon, BD Biosciences, San Jose, CA, USA (upper chamber) and placed in wells of a 24-well plate (Falcon, BD Biosciences) (lower chamber), containing 0.8 mL of DMEM + 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the migratory cells on the lower surface of membranes were fixed, stained with Hemacolor® (HX54775574, Merck, Darmstadt, Germany) and examined microscopically cellular migration was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.

4.5. Cell Invasion Assay

The 2.5 × 104 cells non stimulated (ctrl) and 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6 as single agent respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10 ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters (BD Bio-CoatTM growth factor reduced MATRIGELTM invasion chamber, BD Biosciences-Discovery Labware, Two Oak Park, Bedford, MA, USA) (upper chamber) and placed in wells of a 24-well plate (Falcon, BD Biosciences) (lower chamber), containing 0.8 mL of DMEM 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the invasive cells on the lower surface of membranes were fixed, stained with Hemacolor® (HX54775574, Merck, Darmstadt, Germany) and examined microscopically. Cellular invasion was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.

4.6. Cell Proliferation

MCF-7 and MDA-MB-231 cells were seeded in 6-well culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) at a concentration of 1 × 106 cells/well in a complete medium. The following day, the cells were refed with DMEM supplemented with 0.1% FBS containing 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6. The plates were incubated for 24 h at 37 °C in an atmosphere of 5% CO2. Then, the cells were trypsinized and centrifuged, and cell pellets were resuspended in phosphate-buffered saline (PBS). Cell count was performed by a particle count and size analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Three replicate wells were used for each data point, and the experiment was performed six times.

4.7. Muse™ Annexin V & Dead Cell Kit

Cells were cultured at confluence into 25 cm2 flasks (Falcon, Becton Dickinson Labware) in a complete medium. The apoptotic assay was performed using the Muse Annexin V and Dead cell kit (Millipore Catalog No. MCH100105). Briefly, MCF-7 and MDA-MB-231 cells were stimulated with the different embryo fish factors in DMEM 0.1% FBS. On the day of the experiment they were trypsinized, centrifuged, and resuspended in DMEM, 0.1% FBS medium to have a cell suspension between 1 × 105 and 1 × 107 mL−1. 100 μL of Muse Annexin and Dead Reagent was added. Cells were incubated for 20 min in the dark, and then analyzed with the Muse TM Cell Analyzer. Each assay was performed in triplicate.

4.8. Western Blots

Control and stimulated cells were washed twice with ice-cold PBS and scraped in RIPA lysis buffer (Sigma Aldrich). A mix of protease inhibitors (Complete-Mini Protease Inhibitor Cocktail Tablets, Roche, Mannheim, Germany) and phosphatase inhibitors (PhosStop; Roche, Mannheim, Germany) was added just before use. Cellular extracts were then centrifuged at 8000× g for 10 min. The Bradford assay was used to determine protein contents. For western blot analysis, cellular extracts were separated on SDS-polyacrylamide gels and proteins were blotted onto nitrocellulose membranes (BIO-RAD, Bio-Rad Laboratories, Hercules, CA, USA). The following antibodies were analyzed: anti-vinculin (7F9): sc-73614; anti-Rock1 (H-85): sc-5560 and anti-beta-catenin sc-7963 all from Santa Cruz Biotechnology; anti-p53 (acetyl k382) ab-75754 from Abcam; anti-TPT1 (E-AB-31729) from Elabscience; anti-E-cadherin (610181) from BD Bioscience. Antigens were detected with an enhanced chemiluminescence kit (Western Bright ECL HRP Substrate, Advansta Inc., Menlo Park, CA, USA), according to the manufacturer’s instructions.

4.9. Densitometry

All Western blot images were acquired and analyzed through Imaging Fluor S densitometer (Biorad-Hercules, CA, USA). Optical density (OD) of each condition was normalized versus the signal of internal control GAPDH (anti-GAPDH #2118 from Cell Signaling Technology).

4.10. Confocal Microscopy

To evaluate the migratory phenotype of treated or non-treated cells, we perform the wound-healing assay using special double well culture inserts (Ibidi GmbH, Martinsried, Germany). Each insert was placed in 8-well μ-slides (Ibidi GmbH, Am Klopferspitz 19, D-82152 Martinsried, Germany) and 3.5 × 104 cells were placed into both wells of each insert with 70 μL of complete medium. When cells were confluent, the culture inserts were gently removed, and cells were fed with 10% FBS DMEM (CTRL), 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6 for 24 h. Then, the medium was removed and the cells were fixed with 4% paraformaldehyde for 10 min at 4 °C and washed twice for 10 min with PBS. The cells were permeabilized for 30 min using PBS, 3% BSA, 0.1% Triton X-100, followed by anti-vinculin (7F9): sc-73614, or anti anti-beta-catenin sc-7963 (all from Santa Cruz Biotechnology) staining in PBS, 3% BSA at 4 °C overnight. The cells were washed with PBS and incubated for 1 h at room temperature with appropriate secondary antibody FITC conjugated (Invitrogen Molecular Probes Eugene, OR, USA). Negative controls were processed in the same conditions besides primary antibody staining. For F-actin visualization, Rhodamine Phalloidin (Invitrogen Molecular Probes Eugene, 1: 40 dilution) was used. Cells were then washed in PBS and mounted in buffered glycerol (0.1 M, pH 9.5). Finally, analysis was conducted using a Leica confocal microscope TCS SP2 (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) equipped with Ar/ArKr and He/Ne lasers. Laser line were at 543 nm and 488 nm for TRITC and FITC excitation, respectively. The images were scanned under a 40× oil objective. To analyze the colocalization of actin and vinculin, optical spatial z series composed of about 8/10 optical section with a step size of 1 μm were performed. Color channels were merged and colocalization were analyzed with the Leica confocal software.

4.11. Urokinase-PA Zimography

To test the enzymatic activity of urokinase plasminogen activator (uPA), aliquots of conditioned media of MDA-MB-231 human breast cancer untreated control cells, 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6, and 0.3 µg/mL F6 treated cells were separated by electrophoresis in 10% polyacrylamide slab gels in the presence of SDS (SDS–polyacrylamide gels (PAGE)) under non-reducing conditions. The uPA was then visualized by placing the Triton-X100-washed gel on a casein–agar–plasminogen underlay. The lytic zones were plasminogen dependent. Molecular weights were calculated from the position of pre-stained markers subjected to electrophoresis in parallel lines. Densitometric scanning of zymographies was performed to derive a semi-quantitative estimation of protease activities. PA gelatin zimography was performed three times.

4.12. Statistical Analysis

Data were expressed as mean ± standard deviation (SD). Data were statistically analyzed with the analysis of variance (ANOVA) followed by the Bonferroni post-test. Differences were considered significant at the level of p < 0.05. Statistical analysis was performed by using GraphPad Instat software (GraphPad Software, Inc.; San Diego, CA, USA).

5. Conclusions

In the present experimental study we showed that molecular factor extracted from Zebrafish embryos isolated at the 20-somite developmental stage can reverse several malignant feature of the cancerous phenotype in a model of human breast cancer. Embryo extracts reduce cell proliferation, enhance apoptosis, and dramatically inhibit both invasiveness and migrating capabilities of cancer cells. Inhibition of migrating and invasive properties is not restricted to breast cancer cells, as embryo extracts were also effective in inhibiting the migrating phenotype adopted by normal breast cells undergoing epithelial–mesenchymal transition upon TGF-β1 stimulation. In cancerous cells embryo-induced reversion entails E-cadherin/β-catenin pathway, cytoskeleton remodeling, as well as downregulation of TCTP and the concomitant increase in p53 levels. Our findings suggest that neoplastic transformation cannot be viewed as an irreversible commitment and can be “reversed”—even partially – in response to proper morphogenetic influences.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/20/9/2151/s1.

Author Contributions

Conceptualization: M.B.; Formal analysis: A.C. (Alessandra Cucina); Funding acquisition: P.M.B., A.P.; Investigation: S.P., M.M., N.M., A.C. (Angela Catizone), G.R., E.L.; Methodology: S.P., A.C. (Alessandra Cucina), A.C. (Angela Catizone), A.H.H., S.H.A.; Resources: A.H.H., S.H.A.; Supervision: A.P., M.B.; Visualization: S.P.; Writing—original draft: M.B.; Writing—review and editing: A.C. (Alessandra Cucina), A.P., M.B.

Funding

We wish to thank Aurora Biosearch Srl for partially funding the investigations. Saleh Alwasel, Abdel Halim Harrath, and Mariano Bizzarri extend their appreciation to the International Scientific Partnership program ISPP at King Saud University for funding this research work through ISPP-122.

Conflicts of Interest

The authors declare they have not conflict of interest. The authors confirm that the funders had no influence over the study design, content of the article, or selection of this journal.

References

1. Joel M., Sandberg C.J., Boulland J.L., Vik-Mo E.O., Langmoen I.A., Glover J.C. Inhibition of tumor formation and redirected differentiation of glioblastoma cells in a xenotypic embryonic environment. Dev. Dyn. 2013;242:1078–1093. doi: 10.1002/dvdy.24001. [PubMed] [CrossRef] []
2. Hendrix M.J., Seftor E.A., Seftor R.E., Kasemeier-Kulesa J., Kulesa P.M., Postovit L.M. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat. Rev. Cancer. 2007;7:246–255. doi: 10.1038/nrc2108. [PubMed] [CrossRef] []
3. Mintz B., Illmensee K. Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. USA. 1975;72:3585–3589. doi: 10.1073/pnas.72.9.3585. [PMC free article] [PubMed] [CrossRef] []
4. Bizzarri M., Cucina A., Biava P.M., Proietti S., D’Anselmi F., Dinicola S., Pasqualato A., Lisi E. Embryonic morphogenetic field induces phenotypic reversion in cancer cells. Curr. Pharm. Biotechnol. 2011;12:243–253. doi: 10.2174/138920111794295701. [PubMed] [CrossRef] []
5. Biava P.M., Canaider S., Facchin F., Bianconi E., Ljungberg L., Rotilio D., Burigana F., Ventura C. Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells. Curr. Pharm. Biotechnol. 2015;16:782–792. doi: 10.2174/1389201016666150629102825. [PMC free article] [PubMed] [CrossRef] []
6. Giuffrida D., Rogers I.M., Nagy A., Calogero A.E., Brown T.J., Casper R.F. Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth. Cell Prolif. 2009;42:788–798. doi: 10.1111/j.1365-2184.2009.00640.x. [PMC free article] [PubMed] [CrossRef] []
7. Hansis C., Barreto G., Maltry N., Niehrs C. Nuclear reprogramming of human somatic cells by xenopus egg extract requires BRG1. Curr. Biol. 2004;14:1475–1480. doi: 10.1016/j.cub.2004.08.031. [PubMed] [CrossRef] []
8. Ferranti F., D’Anselmi F., Caruso M., Lei V., Dinicola S., Pasqualato A., Cucina A., Palombo A., Ricci G., Catizone A., et al. TCam-2 seminoma cells exposed to egg-derived microenvironment modify their shape, adhesive pattern and migratory behaviour: A molecular and morphometric analysis. PLoS ONE. 2013;8:e76192. doi: 10.1371/annotation/1c70ff15-389c-4ad7-b333-6615e98713d0. [PMC free article] [PubMed] [CrossRef] []
9. D’Anselmi F., Masiello M.G., Cucina A., Proietti S., Dinicola S., Pasqualato A., Ricci G., Dobrowolny G., Catizone A., Palombo A., et al. Microenvironment promotes tumor cell reprogramming in human breast cancer cell lines. PLoS ONE. 2013;8:e83770. doi: 10.1371/journal.pone.0083770. [PMC free article] [PubMed] [CrossRef] []
10. Allegrucci C., Rushton M.D., Dixon J.E., Sottile V., Shah M., Kumari R., Watson S., Alberio R., Johnson A.D. Epigenetic reprogramming of breast cancer cells with oocyte extracts. Mol. Cancer. 2011;10:7. doi: 10.1186/1476-4598-10-7. [PMC free article] [PubMed] [CrossRef] []
11. Abollo-Jiménez F., Jiménez R., Cobaleda C. Physiological cellular reprogramming and cancer. Semin. Cancer Biol. 2010;20:98–106. doi: 10.1016/j.semcancer.2010.02.002. [PubMed] [CrossRef] []
12. Soto A.M., Maffini M.V., Sonnenschein C. Neoplasia as development gone awry: The role of endocrine disruptors. Int. J. Androl. 2008;31:288–293. doi: 10.1111/j.1365-2605.2007.00834.x. [PMC free article] [PubMed] [CrossRef] []
13. Cucina A., Biava P., D’Anselmi F., Coluccia P., Conti F., di Clemente R., Miccheli A., Frati L., Gulino A., Bizzarri M. Zebrafish embryo proteins induce apoptosis in human colon cancer cells. Apoptosis. 2006;11:1617–1628. doi: 10.1007/s10495-006-8895-4. [PubMed] [CrossRef] []
14. Astigiano S., Damonte P., Fossati S., Boni L., Barbieri O. Fate of embryonal carcinoma cells injected into postimplantation mouse embryos. Differentiation. 2005;73:484–490. doi: 10.1111/j.1432-0436.2005.00043.x. [PubMed] [CrossRef] []
15. Tabata T., Takei Y. Morphogens, their identification and regulation. Development. 2004;131:703–712. doi: 10.1242/dev.01043. [PubMed] [CrossRef] []
16. Krause S., Maffini M.V., Soto A.M., Sonnenschein C. The microenvironment determines the breast cancer cells’ phenotype: Organization of MCF7 cells in 3D cultures. BMC Cancer. 2010;7:263. doi: 10.1186/1471-2407-10-263. [PMC free article] [PubMed] [CrossRef] []
17. Downing T.L., Soto J., Morez C., Houssin T., Fritz A., Yuan F., Chu J., Patel S., Schaffer D.V., Li S. Biophysical regulation of epigenetic state and cell reprogramming. Nat. Mater. 2013;12:1154–1162. doi: 10.1038/nmat3777. [PubMed] [CrossRef] []
18. D’Anselmi F., Cucina A., Biava P.M., Proietti S., Coluccia P., Frati L., Bizzarri M. Zebrafish stem cell differentiation stage factors suppress Bcl-xL release and enhance 5-Fu-mediated apoptosis in colon cancer cells. Curr. Pharm. Biotechnol. 2011;12:261–267. doi: 10.2174/138920111794295864. [PubMed] [CrossRef] []
19. Livraghi T., Meloni F., Frosi A., Lazzaroni S., Bizzarri M., Frati L., Biava P.M. Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: An open randomized clinical trial. Oncol. Res. 2005;15:399–408. doi: 10.3727/096504005776449716. [PubMed] [CrossRef] []
20. Proietti S., Cucina A., Giuliani A., Verna R., Palombi E., Biava P.M., Pensotti A. Fish protein extract enhances clinical response to salvage chemotherapy in colon cancer patients. Org. J. Biol. Sci. 2018;2:81–90. doi: 10.13133/2532-5876_4.8. [CrossRef] []
21. Pfeiffer M.J., Siatkowski M., Paudel Y., Balbach S.T., Baeumer N., Crosetto N., Drexler H.C., Fuellen G., Boiani M. Proteomic analysis of mouse oocytes reveals 28 candidate factors of the “reprogrammome” J. Proteome Res. 2011;10:2140–2153. doi: 10.1021/pr100706k. [PubMed] [CrossRef] []
22. Bischof A.G., Yüksel D., Mammoto T., Mammoto A., Krause S., Ingber D.E. Breast cancer normalization induced by embryonic mesenchyme is mediated by extracellular matrix biglycan. Integr. Biol. 2013;5:1045–1056. doi: 10.1039/c3ib40103k. [PubMed] [CrossRef] []
23. DeCosse J.J., Gossens C.L., Kuzma J.F., Unsworth B.R. Breast cancer: Induction of differentiation by embryonic tissue. Science. 1973;181:1057–1058. doi: 10.1126/science.181.4104.1057. [PubMed] [CrossRef] []
24. Kirchberger S., Sturtzel C., Pascoal S., Distel M. Quo natas, Danio? Recent Progress in Modeling Cancer in Zebrafish. Front. Oncol. 2017;7:186. doi: 10.3389/fonc.2017.00186. [PMC free article] [PubMed] [CrossRef] []
25. Laughlin R.B., Pines D., Schmalian J., Stojković B.P., Wolynes P. The middle way. Proc. Natl. Acad. Sci. USA. 2000;97:32–37. doi: 10.1073/pnas.97.1.32. [PMC free article] [PubMed] [CrossRef] []
26. Bertolaso M., Bizzarri M., Pensotti A., Giuliani A. Co-Emergence and Collapse: The Mesoscopic Approach for Conceptualizing and Investigating the Functional Integration of Organisms. Front. Phisiol. 2019 in press. []
27. Moirangthem A., Bondhopadhyay B., Mukherjee M., Bandyopadhyay A., Mukherjee N., Konar K., Bhattacharya S., Basu A. Simultaneous knockdown of uPA and MMP9 can reduce breast cancer progression by increasing cell-cell adhesion and modulating EMT genes. Sci. Rep. 2016;24:21903. doi: 10.1038/srep21903. [PMC free article] [PubMed] [CrossRef] []
28. Simeoni C., Dinicola S., Cucina A., Mascia C., Bizzarri M. Systems Biology Approach and Mathematical Modeling for Analyzing Phase-Space Switch During Epithelial-Mesenchymal Transition. Methods Mol. Biol. 2018;1702:95–123. doi: 10.1007/978-1-4939-7456-6_7. [PubMed] [CrossRef] []
29. Mierke C.T., Kollmannsberger P., Zitterbart D.P., Diez G., Koch T.M., Marg S., Ziegler W.H., Goldmann W.H., Fabry B. Vinculin facilitates cell invasion into three-dimensional collagen matrices. J. Biol. Chem. 2010;285:13121–13130. doi: 10.1074/jbc.M109.087171. [PMC free article] [PubMed] [CrossRef] []
30. Saunders R.M., Holt M.R., Jennings L., Sutton D.H., Barsukov I.L., Bobkov A., Liddington R.C., Adamson E.A., Dunn G.A., Critchley D.R. Role of vinculin in regulating focal adhesion turnover. Eur. J. Cell Biol. 2006;85:487–500. doi: 10.1016/j.ejcb.2006.01.014. [PubMed] [CrossRef] []
31. Breyer J., Samarin J., Rehm M., Lautscham L., Fabry B., Goppelt-Struebe M. Inhibition of Rho kinases increases directional motility of microvascular endothelial cells. Biochem. Pharmacol. 2012;83:616–626. doi: 10.1016/j.bcp.2011.12.012. [PubMed] [CrossRef] []
32. Mavria G., Vercoulen Y., Yeo M., Paterson H., Karasarides M., Marais R., Bird D., Marshall C.J. ERK-MAPK signaling opposes Rho-kinase to promote endothelial cell survival and sprouting during angiogenesis. Cancer Cell. 2006;9:33–44. doi: 10.1016/j.ccr.2005.12.021. [PubMed] [CrossRef] []
33. Sivasubramaniyan K., Pal R., Totey S., Bhat V.S., Totey S. Rho kinase inhibitor y27632 alters the balance between pluripotency and early differentiation events in human embryonic stem cells. Curr. Stem Cell Res. Ther. 2010;5:2–12. doi: 10.2174/157488810790442769. [PubMed] [CrossRef] []
34. Laeno A.M., Tamashiro D.A., Alarcon V.B. Rho-Associated Kinase Activity Is Required for Proper Morphogenesis of the Inner Cell Mass in the Mouse Blastocyst. Biol. Reprod. 2013;89:122. doi: 10.1095/biolreprod.113.109470. [PMC free article] [PubMed] [CrossRef] []
35. Mills E., LaMonica K., Hong T., Pagliaruli T., Mulrooney J., Grabel L. Roles for Rho/ROCK and vinculin in parietal endoderm migration. Cell Commun. Adhes. 2005;12:9–22. doi: 10.1080/15419060500305948. [PubMed] [CrossRef] []
36. Grille S.J., Bellacosa A., Upson J., Klein-Szanto A.J., van Roy F., Lee-Kwon W., Donowitz M., Tsichlis P.N., Larue L. The protein kinase Akt induces epithelial-mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res. 2003;63:2172–2178. [PubMed] []
37. Orsulic S., Huber O., Aberle H., Arnold S., Kemler R. E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J. Cell Sci. 1999;112:1237–1245. [PubMed] []
38. Tuynder M., Susini L., Prieur S., Besse S., Fiucci G., Amson R., Telerman A. Biological models and genes of tumor reversion: Cellular reprogramming through tpt1/TCTP and SIAH-1. Proc. Natl. Acad. Sci. USA. 2002;99:14976–14981. doi: 10.1073/pnas.222470799. [PMC free article] [PubMed] [CrossRef] []
39. Rho S.B., Lee J.H., Park M.S., Byun H.J., Kang S., Seo S.S., Kim J.Y., Park S.Y. Anti-apoptotic protein TCTP controls the stability of the tumor suppressor p53. FEBS Lett. 2011;585:29–35. doi: 10.1016/j.febslet.2010.11.014. [PubMed] [CrossRef] []
40. Ito A., Kawaguchi Y., Lai C.H., Kovacs J.J., Higashimoto Y., Appella E., Yao T.P. MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. EMBO J. 2002;21:6236–6245. doi: 10.1093/emboj/cdf616. [PMC free article] [PubMed] [CrossRef] []
41. Amson R., Pece S., Lespagnol A., Vyas R., Mazzarol G., Tosoni D., Colaluca I., Viale G., Rodrigues-Ferreira S., Wynendaele J., et al. Reciprocal repression between P53 and TCTP. Nat. Med. 2011;18:91–99. doi: 10.1038/nm.2546. [PubMed] [CrossRef] []
42. Skrypek N., Goossens S., De Smedt E., Vandamme N., Berx G. Epithelial-to-Mesenchymal Transition: Epigenetic Reprogramming Driving Cellular Plasticity. Trends Genet. 2017;33:943–959. doi: 10.1016/j.tig.2017.08.004. [PubMed] [CrossRef] []
43. Johnson D.G. The paradox of E2F1: Oncogene and tumor suppressor gene. Mol. Carcinog. 2000;27:151–157. doi: 10.1002/(SICI)1098-2744(200003)27:3<151::AID-MC1>3.0.CO;2-C. [PubMed] [CrossRef] []
44. Kawamura T., Suzuki J., Wang Y.V., Menendez S., Morera L.B., Raya A., Wahl G.M., Izpisúa Belmonte J.C. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature. 2009;460:1140–1144. doi: 10.1038/nature08311. [PMC free article] [PubMed] [CrossRef] []
45. Krizhanovsky V., Lowe S.W. Stem cells: The promises and perils of p53. Nature. 2009;460:1085–1086. doi: 10.1038/4601085a. [PMC free article] [PubMed] [CrossRef] []
46. Amson R., Karp J.E., Telerman A. Lessons from tumor reversion for cancer treatment. Curr. Opin. Oncol. 2013;25:59–65. doi: 10.1097/CCO.0b013e32835b7d21. [PubMed] [CrossRef] []
47. Biava P.M., Carluccio A. Activation of anti-oncogene p53 produced by embryonic extracts in vitro tumor cells. J. Tumor Marker Oncol. 1997;12:9–15. []

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Early Developmental Zebrafish Embryo Extract to Modulate Senescence in Multisource Human Mesenchymal Stem Cells

Abstract

From: https://www.ncbi.nlm.nih.gov/pubmed/31146388

Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton’s Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated β-galactosidase (SA β-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.

KEYWORDS:

BMI1; TERT; adipogenesis; p16; p21; p53; senescence; senescence-associated β-galactosidase activity; stem cells; zebrafish embryo extract; Biava PM

From: https://www.mdpi.com/1422-0067/20/11/2646

Abstract

Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton’s Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated β-galactosidase (SA β-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.

KEYWORDS:

BMI1; TERT; adipogenesis; p16; p21; p53; senescence; senescence-associated β-galactosidase activity; stem cells; zebrafish embryo extract, Biava PM

1. Introduction

The human body continuously relies upon its own tissue-resident stem cells to repair adult tissues and organs, and to oppose senescence-related processes [1,2,3,4,5,6]. Human mesenchymal stem cells (hMSCs) exhibit self-renewal, multilineage differentiation and can be isolated and expanded in vitro from virtually all organs, even if bone marrow and subcutaneous fat are the elected sources [7]. Furthermore, hMSCs can be isolated not only from adult tissues but also from several fetal and perinatal sources [8].
As a result, in the last decade several attempts have been made to unfold hMSC features into regenerative medicine approaches for the treatment of a variety of tissue injuries and degenerative disorders [9,10,11,12,13,14,15]. Within this context, the use of synthetic and natural molecules, as well as electromagnetic fields and mechanical vibrations, can largely contribute to the development of strategies that may counteract stem cell senescence allowing the preservation of tissue homeostasis and our innate self-healing potential [16].
In particular, in neurodegenerative diseases, strategies that prevent telomere loss or increase telomere length in MSCs, as well as the use of neuropeptides that elicit the regulation of the Wnt/β-catenin signaling pathway (involved in the shift of MSCs towards a senescent phenotype), may prevent the symptoms of neurodegenerative disorders and improve the results of MSCs-based therapy [17]. At the same time, therapeutic approaches of osteoarthritis are based on strategies able to stimulate MSCs through telomerase activators, mechanical strain, and epigenetic regulation in order to maintain their chondrogenic differentiation potential and to counteract homeostasis alterations occurring in cell in vitro cultivation and expansion [18].
Another possible and useful way to resist stem cell senescence is mimicking organisms that have developed a robust ability to regenerate tissues. Zebrafish (Danio rerio), for example, is widely used as an animal model to study regeneration and organogenesis, given its ability to regenerate organs, such as the heart or the central nervous system, at a noticeable higher efficacy than in humans [19,20,21,22,23]. Despite the evolutionary distance between humans and zebrafish, hMSCs can still perceive ancestral microenvironmental cues from this species [24] and several scientific researches in comparative biology revealed interesting conserved evolutionary patterns in tissue regeneration.
Previously, we described a putative anti-senescence action of a zebrafish extract (ZF1) which was obtained from embryos at an early stage of development (50% epiboly) in hMSCs that were isolated from the adipose tissue (human adipose tissue-derived stem cells, hASCs) and cultured from the third to the 5th passage, in order to provide a preliminary investigation of its effects on cell viability, stemness, and senescence regulatory patterning [25]. In particular, ZF1 which was given at a concentration of 10 μg/mL for 72 h did not influence cell viability or apoptosis, while it enhanced the transcription of the telomerase reverse transcriptase (TERT), BMI1 proto-oncogene, polycomb ring finger (BMI1), and the stemness POU domain class 5 homeobox 1 (POU5F1) (alias Oct-4), Sox-2, and v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) genes [25].
Recently, we investigated the role of ZF1 at the same dose and for the same exposure time on hASCs at four different, considerably more prolonged, subculture stages (5th, 10th, 15th and 20th) in order to assess additional biological responses to the treatment with ZF1. Our results showed that ZF1 is a feasible tool to modulate and reverse hASC senescence in long-term culturing conditions [26].
Since the zebrafish is considered one of the most popular vertebrate models in developmental biology and biomedical research, several transcriptomic and proteomic analyses have been increasingly used for profiling important molecules of zebrafish at different developmental stages or in specific organs/tissues or experimental conditions [27,28,29,30,31,32,33,34,35,36,37,38,39,40].
Only one study [36], among all considered, reported the transcriptome at the specific developmental stage of our interest (50% epiboly) and a second reported a list of proteins isolated from the ZF1 extract [41].
In the present study, we first explored the anti-senescence role of ZF1 in hASCs treated for 72 h with ZF1 at five different concentrations (0.01, 10, 20, 40, and 100 μg/mL), in order to evaluate the eligible ZF1 dose to use in the present work.
Then, we investigated whether the anti-senescence action of ZF1 at a defined concentration can be considered a general feature applicable to various types of hMSCs isolated from adult and perinatal tissues.
Therefore, we investigated cell proliferation, senescence-associated β-galactosidase (SA β-gal) activity, adipogenic ability, gene transcription of TERT, encoding the catalytic core of telomerase, BMI1, a transcriptional regulator acting as a major repressor of senescence, tumor protein p53 (TP53, alias p53), cyclin dependent kinase inhibitor 1A (CDKN1A, alias p21), cyclin dependent kinase inhibitor 2A (CDKN2A, alias p16), p21 protein expression in ZF1-treated hMSCs from adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and Wharton’s Jelly (hWJ-MSCs).

2. Results

2.1. hMSC Isolation and Phenotype Characterization

Cells were isolated from different sources, as described in the Material and Methods section, and were selected for their ability to adhere to plastic surfaces. After two to three weeks of in vitro culture, cell populations showed the typical fibroblast-like morphology (Figure 1) and displayed an immune phenotype consistent with multipotent mesenchymal stem cells. In fact, cell populations were positive for markers CD29, CD44, CD73, CD105, and CD166; and negative for CD14, CD34, and CD45. These results were previously described in detail [42,43].
Figure 1. Representative images of hMSCs (culture passages 5th–7th) isolated from human adipose tissue (hASCs), dental pulp (hDP-MSCs), bone marrow (hBM-MSCs), and Wharton’s Jelly (hWJ-MSCs). Cells were analyzed for morphology under optical microscopy (40× magnification and bright field illumination). Scale bar corresponds to 200 μm.

2.2. Evaluation of Different ZF1 Concentrations Cytotoxicity in hASCs

A dose-response morphological analysis of the effects elicited by ZF1 on hASCs showed that the higher concentrations (40 and 100 μg/mL) which were investigated modified cell morphology and induced high mortality. This suggested a toxicity of the treatments, and consequently, lead to their exclusion from further experiments. In contrast, treatment with a solvent or with ZF1 at 0.01, 10, and 20 μg/mL did not alter the cellular morphology, as compared with control cells from the same culture passages (5th–7th) (Figure 2).
Figure 2. Representative images of hASCs treated with ZF1 at different concentrations (0.01, 10, 20, 40, and 100 μg/mL) or with a solvent (SOLV) or untreated (CTR). Cytotoxicity was evaluated with a morphological analysis. Images were obtained with an optical microscopy (40× magnification and bright field illumination). Scale bar corresponds to 200 μm.

2.3. ZF1 Does Not Affect Proliferation in hASCs

The preliminary in vitro toxicology resazurin-based assay performed on hASCs in technical quadruplicate showed a comparable proliferation rate in all the tested experimental conditions (control, solvent, and ZF1 at 0.01, 10, and 20 μg/mL), indicating that treatments had a nontoxic effect and that ZF1 did not affect hASC proliferation (Figure 3).
Figure 3. Effects of different concentrations of ZF1 on metabolic activity in hASCs. The percentage of resazurin reduction (as an indicator of cell proliferation) in hASCs treated with ZF1 (0.01, 10, and 20 μg/mL) or a solvent (SOLV) or untreated (CTR). An analysis was conducted at 4, 24, 48, and 72 h from the beginning of the treatment and an experiment was performed in technical quadruplicate. Statistical significance was evaluated using ANOVA followed by the Tukey HSD test. Data are expressed as mean ± standard deviation (SD), n = 4.
On the basis of these results, we decided to use a solvent as a control for subsequent experiments.

2.4. ZF1 Decreases SA β-Gal Staining and Increases TERT Gene Expression in hASCs

A dose-response analysis of the effects elicited by ZF1 on SA β-gal staining was set up in hASCs to identify the most effective concentration influencing the expression of this senescence marker. Cells (culture passages 5th–7th) were treated for 72 h with ZF1 at the final concentrations of 0.01, 10, and 20 μg/mL. Although 0.01 μg/mL ZF1 was ineffective, both 10 and 20 μg/mL ZF1 significantly reduced the number of senescent hASCs positively blue stained for SA β-gal (p < 0.05) (Figure 4).
Figure 4. Effects of different concentrations of ZF1 on SA β-gal activity in hASCs. The hASCs (culture passages 5th–7th) were seeded in 6-well plates and were cultured in the presence of 0.01, 10, and 20 μg/mL ZF1, or a solvent as a control for 72 h, then processed for SA β-gal assessment. (a) Images represent hASCs after SA β-gal staining. SA β-gal positive cells are blue. The scale bar corresponds to 200 μm; (b) Positive (blue) and negative (not colored) cells were counted in at least three random fields for each technical replicate under the microscope (200× magnification and bright field illumination). Data represent the percentage of SA β-gal positive cells calculated as the number of positive cells divided by the total number of counted cells multiplied by 100 (percentage of blue cells ± SD, n = 3, statistical significance was calculated using the Student’s t-test, * p < 0.05).
Consistent with the experiments assessing the effect of ZF1 on SA β-gal activity, hASCs (isolated from one subject) and treated with ZF1 at 0.01 μg/mL concentration showed a gene expression value of the catalytic subunit of telomerase (TERT) similar to that of control cells (SOLV). In contrast, hASCs exposed to both 10 μg/mL and 20 μg/mL ZF1 resulted in a similar statistically significant increase in TERT transcription as compared with the control hASCs (SOLV) (Figure 5).

Figure 5. The effect of ZF1 treatment on TERT gene expression in hASCs. The hASCs (culture passages 5th–7th) were exposed for 72 h in the presence of 0.01, 10, and 20 μg/mL ZF1, or solvent (SOLV) as a control. The expression value of the transcripts evaluated in solvent or ZF1-treated cells was normalized to the expression levels of three reference genes, HPRT1, GAPDH and TBP. The amount of the target mRNA in the ZF1 treated cells was plotted as the fold change over the expression in control cells. The experiment was performed in technical triplicate. Statistical significance was evaluated by using the CFX Manager Software version 3.1 (Bio-Rad Laboratories) and Student’s t-test (mean ± SD, n = 3, * p < 0.05).

2.5. ZF1 Promotes Adipogenesis in hASCs

To better investigate the effect of ZF1 on hASCs, adipogenic differentiation after 0.01, 10, and 20 μg/mL treatment was evaluated and quantified via Oil Red O staining, a neutral triglycerides and lipids dye. During differentiation, the hASCs produce multiple lipid-rich vacuoles in the cytoplasm, which increased in their size and number during the two weeks of induction, and they showed an intense red color if stained with Oil Red O (Figure 6a). The red staining quantification revealed that ZF1 enhanced hASC adipogenic commitment both when cells grew in a culture medium and when cells were induced. Moreover, the statistically significant effect was dose-dependent (Figure 6b).
Figure 6. Effects of ZF1 treatment on adipogenic differentiation in hASCs at different concentrations. The hASCs (culture passages 5th–7th) were seeded in 24-well plates and were cultured in the presence of 0.01, 10, and 20 μg/mL ZF1, or solvent (SOLV) as a control for 72 h. (a) Images represent hASCs Oil red O staining after treatment with solvent (above) or ZF1 20 μg/mL (below) and adipogenic medium. Cells positive for adipogenesis showed red colored vacuoles in cytoplasm. Scale bar corresponds to 100 μm; (b) White histograms represent data derived from hASCs cultured in basal medium, while colored histograms represent those from hASCs treated with adipogenic medium. The lipid-rich vacuoles Oil Red O dye was extracted by wells and its absorbance was read at 495 nm with a spectrophotometer. Data are expressed as mean of lipid content at 495 nm absorbance ± SD. Horizontal dashed or continuous black lines represent the significance of differences between data obtained from hASCs cultured in basal and from an adipogenic medium, respectively (statistical significance was calculated using the Student’s t-test, * p < 0.05, n = 3).
Therefore, based on the above results obtained with hASCs, we decided to use ZF1 at 20 μg/mL in the following experiments performed on all the four selected hMSC types.

2.6. ZF1 and Modulation of Cell Proliferation in hMSCs Isolated from Four Different Sources

The adult stem cells, hASCs, hDP-MSCs, and hBM-MSCs, and perinatal stem cells, hWJ-MSCs, (all at culture passages 5th–7th) were treated for 72 h with 20 μg/mL ZF1 or with its solvent as a control. As shown in Figure 7a, the percentage of reduction of resazurin (as an indicator of cell proliferation) did not change in the ZF1 treated hASCs as compared with solvent treated cells at any experimental time point, in accordance with our previous assay results reported above.

Figure 7. The effects of different concentrations of ZF1 on metabolic activity in hMSCs. The hASCs (a), hBM-MSCs (b), hDP-MSCs (c) and hWJ-MSCs (d), at passages 5th–7th, were exposed for 72 h in the presence of 20 μg/mL ZF1, or solvent as a control (SOLV). An analysis of the percentage of the reduction of resazurin (as an indicator of cell proliferation) was conducted at 4, 24, 48, and 72 h from the beginning of the treatment and data are expressed as mean (n = 3) ± SD. Statistical significance was analyzed with ANOVA followed by the Tukey HSD test, * p < 0.05, ** p < 0.01.
In contrast, in hBM-MSCs, hDP-MSCs, and hWJ-MSCs cell proliferation increased after ZF1 treatment, reaching statistical significance at different experimental points: at 72 h for all three cell types (p < 0.05) and also at 24 h and 48 h for hDP-MSCs and hWJ-MSCs (Figure 7b–d).

2.7. ZF1 Reduces SA β-Gal Staining in hMSCs Isolated from Four Different Sources

All investigated hMSCs (culture passages 5th–7th) were treated for 72 h with 20 μg/mL ZF1 or with its solvent as a control and tested with the SA β-gal staining assay. As shown in Figure 8, the number of senescent stem cells expressing SA β-gal was remarkably reduced by the treatment, as compared with the control cells, in each investigated mesenchymal lineage (n = 3, p < 0.01). The different distribution of SA β-gal positive cells among ZF1-exposed and untreated hMSCs of each source is also evident in Figure 9, showing the relevant decline of senescent marker expression in ZF1 treated cells. Additionally, Figure 9 shows that the treatment with ZF1 at the investigated concentration did not alter the cellular morphology, as compared with the control cells, as previously discussed in Section 2.2.
Figure 8. ZF1 counteracts SA β-gal activity in hMSCs. The hMSCs (culture passages 5th–7th) were cultured for 72 h in the presence of 20 μg/mL ZF1, or a solvent as a control (SOLV). Then, cells were processed for SA β-gal assessment, as described in Figure 4 legend. The experiments with hASCs, hDP-MSCs, hBM-MSCs, and hWJ-MSCs were all performed with cells derived from three subjects. Data represent the percentage of SA β-gal positive cells calculated as the number of positive cells divided by the total number of counted cells multiplied by 100. All ZF1 treated cells were significantly different from the control group (Proportion in % ± SD, n = 3). Statistical significance was evaluated using the Z-test for proportional, ** p < 0.01.
Figure 9. Representative images of the effects of ZF1 treatment on SA β-gal activity in hMSCs. The hMSCs from different sources (culture passages 5th–7th) were treated with 20 μg/mL ZF1 or with a solvent (SOLV) as a control, and then analyzed for SA β-gal expression (blue color, 200× magnification and bright field illumination). Images are representative of three separate experiments (n = 3) for each hMSC type.

2.8. ZF1 and Transcriptional Modulation of Stem Cell Senescence in Multisource hMSCs

The relative quantitative real-time PCR (qPCR) analysis revealed that hASCs, hBM-MSCs, hDP-MSCs, and hWJ-MSCs (culture passages 5th–7th) all responded to a 72-h treatment with 20 μg/mL ZF1 with an evident increase in TERT gene transcription as compared with the ZF1 unexposed (control) cells (Figure 10). Next, we investigated whether ZF1 may have elicited its anti-senescence effect by acting on the transcription of other senescence modulators. In Figure 10a, we show that, in hASCs, ZF1 was also able to enhance the gene expression of BMI1, a pleiotropic transcriptional regulator acting as a major repressor of senescence [44,45,46], while the exposure to ZF1 of hBM-MSCs, hDP-MSCs or hWJ-MSCs resulted in a nonsignificant effect on BMI1 transcription (Figure 10b, 10c, and 10d). Moreover, in hASCs, the treatment with ZF1 failed to affect the transcription of TP53 (p53), as well as the gene expression of type 1A and 2A cyclin dependent kinase inhibitors, CDKN1A (p21) and CDKN2A (p16) (Figure 10a). On the contrary, in hBM-MSCs, hDP-MSCs, and hWJ-MSCs, the ZF-induced increase in TERT transcription was associated with a down-regulation in both TP53 (p53) and CDKN1A (p21) transcription, with no significant changes in CDKN2A (p16) gene expression (Figure 10b–d, respectively).
Figure 10. Effect of ZF1 on gene expression in hMSCs. The hASCs (a), hBM-MSCs (b), hDP-MSCs (c) or hWJ-MSCs (d), at passages 5th–7th, were exposed for 72 h in the presence of 20 μg/mL ZF1, or a solvent (SOLV) as a control. The expression value of the transcripts evaluated in the solvent or ZF1-treated cells, was normalized to the expression levels of three reference genes, HPRT1, GAPDH, and TBP. The amount of the target mRNA in the ZF1 treated cells was plotted as the fold change over the expression in the control cells. The experiments were all performed with cells derived from three subjects. Statistical significance was evaluated by using the CFX Manager Software version 3.1 (Bio-Rad Laboratories) and the Student’s t-test (mean ± SD, * p < 0.05, ** p < 0.01, n = 3).

2.9. ZF1 and p21 Expression in Multisource hMSCs

Consistent with the experiments assessing the effect of ZF1 on gene transcription, we decided to perform a protein investigation for p21, since it is the last protagonist of the TERT/p53/p21 pathway. Western blot analysis revealed that a 72-h treatment with 20 μg/mL ZF1 affected the expression of p21 protein in the investigated hMSCs (culture passages 5th–7th), as compared with the ZF1 unexposed (control) cells (Figure 11). In particular, we showed that in hASCs and in hBM-MSCs, the change of p21 expression was not significant, while the exposure of hDP-MSCs or hWJ-MSCs to ZF1 resulted in a statistically significant decrease of the protein, in accordance with the relative p21 gene transcription (Figure 11).
Figure 11. The Western blot analysis of p21 expression in SOLV- (as a control) and ZF1-treated hMSCs. (a) The graphs show the comparison of relative densitometric values of the bands normalized to total protein detected in stain-free acquisition in control (mean ± SD, statistical significance was evaluated using the Student’s t-test, * p < 0.05, n = 3). (b) Representative images of the Western blot for each investigated cell type.

2.10. ZF1 and Adipogenic Commitment in hMSCs

The study of the role of ZF1 in hASC adipogenesis, described above, prompted us to extend our investigation to the other three hMSC types (hDP-MSCs, hBM-MSCs, and hWJ-MSCs), performed in technical triplicate. The hMSC adipogenic commitment was evident when cells were induced by adipogenic medium, as compared with cells grown in a culture medium (data not shown). The 72-h treatment with 20 μg/mL ZF1 increased the hASC adipogenic ability (p < 0.05) as compared with the ZF1 unexposed (control) cells, as previously described in preliminary experiments. In contrast, in hDP-MSCs, hBM-MSCs, and hWJ-MSCs, the results revealed that the treatment with ZF1 did not increase adipogenesis with statistical significance (Figure 12).
Figure 12. Effects of ZF1 treatment on adipogenic differentiation in hMSCs. The hASCs, hBM-MSCs, hDP-MSCs, and hWJ-MSCs, at the 5th passage, were exposed for 72 h in the presence of 20 μg/mL ZF1, or a solvent as a control (SOLV). An experiment was performed in technical triplicate for each cell type. At the end of treatment, hMSCs were incubated with adipogenic medium for two weeks and then differentiation was assessed using Oil Red O staining. The lipid-rich vacuoles Oil Red O dye was extracted from each well and its absorbance was read at 495 nm with a spectrophotometer. Data are expressed as mean relative lipid content (absorbance at 495 nm) of the ZF1 treated cells over the solvent treated ones (used as control, SOLV = 1) ± SD and statistical significance was evaluated using the Student’s t-test, * p < 0.05, n = 3.

3. Discussion

The hMSCs are virtually present in all human organs and have different regenerative potential among the various tissue districts [47]. The properties of self-maintenance, multilineage differentiation, and trophic signaling that are exhibited by hMSCs make them highly attractive candidates for cell therapy approaches, endowing great advantages and peculiarities. The stem cell population that has been first isolated and characterized is hBM-MSCs. The hASCs and hDP-MSCs are commonly harvested in minimally invasive contexts, being abundant and rapidly proliferating stem cell populations, respectively. The hWJ-MSCs are obtained from discarded tissues and retain high plasticity and remarkable immunomodulatory properties due to their embryological and developmental origin [48,49,50,51]. In addition, the hMSCs from either adult or perinatal origins are not burdened by ethical problems and are supposed to be safer than embryonic stem cells or induced pluripotent stem (iPS) cells in terms of tumorigenesis and genomic modifications [52,53].
In an effort to use hMSCs from different tissue sources as potential tools for cell therapy, these cells are subjected to prolonged in vitro expansion in order to increase their number, and conceivably, their regenerative effect, prior to transplantation [10].
However, despite their diversity [54,55], all types of hMSCs undergo replicative senescence when cultured in vitro, a major drawback from the original assumption that tissue regeneration is promoted in a cell number-dependent fashion.
Here, we first show that the use of a developmentally defined zebrafish embryo extracted (ZF1) at concentrations of 10 and 20 μg/mL did not alter cell morphology and cell proliferation in hASCs as evidence of nontoxicity. Furthermore, ZF1 was able to counteract the hASC expression of a well-established senescence marker (SA β-gal) and it caused a significant increase in both induced and spontaneous adipogenic commitment, reinforcing our previous observations about hASCs [25,26]. In addition, similar effects on the β-gal activity were evident in other hMSCs that were investigated such as hBM-MSCs, hDP-MSCs, and hWJ-MSCs when treated with ZF1 at the defined concentration, however, adipogenic commitment was not significantly influenced. In particular, the different tissue origin could affect the MSC differentiation ability after ZF1 treatment [47,56].
In addition, the observed enhancement of TERT expression induced by ZF1 in all the investigated hMSC populations suggests that the rescue of a telomerase-dependent pathway could represent a common mechanism for the anti-senescence action of this embryo extract in hMSCs, independent of the stem cell source. The over-expression of TERT in hASCs reinforced the previously observed results in long-term cultures [26]. In particular, TERT expression is required to support optimal telomerase activity to counteract progressive telomere shortening and senescence [57]. Nevertheless, the observation, only in hASCs, that ZF1 enhanced the expression of BMI1, and the complex telomerase-dependent and -independent functions exploited by this chromatin remodeler in senescence repression [58,59] suggests that the anti-senescence effect of ZF1 may also entail pleiotropic, which are still unexplored dynamics, depending on the MSC source. Such a hypothesis is further supported by the finding that different from hASCs, in hBM-MSCs, hDP-MSCs, and hWJ-MSCs the over-expression of TERT induced by ZF1 was associated with a down-regulation in TP53 (p53) transcription and in both the gene and protein CDKN1A (p21) expression. In these hMSC types the opposite transcriptional responses induced by ZF1 may converge to potentiate the anti-senescence response as it may be inferred by the fact that the loss of p53 function has been found to accelerate telomerase activity [60], and that senescent cells upregulate cell cycle inhibitors (such as p53 and p21) [61]. Therefore, the observation that p53-dependent transcriptional repression of TERT was mediated by CDKN1A (p21) in both normal and pathologic cells [62,63] indicates that the down-regulation of TP53 (p53) and CDKN1A (p21), concomitant with TERT over-expression, may be part of a delicate circuitry through which ZF1 could affect senescence in hMSCs in a source-dependent manner, since a similar pattern was not observed in hASCs in response to ZF1 treatment. The lack of an effect of ZF1 on CDKN2A (p16) transcription in all investigated hMSCs suggests that ZF1 action may not involve previously reported BMI1/p16/pRB pathways [58,59].
Compounding the complexity of the putative mechanisms underlying the speculated anti-senescence response elicited by ZF1 are recent observations that provide evidence for a novel function of TERT in stem cell activation, which is independent of telomerase activity, as shown by the promoting effect on hair growth by TERT over-expression in a mouse strain that is lacking the RNA component of telomerase [64]. In this regard, it has also been found that TERT has a role in activating stem cells which is mediated by the transcriptional activation of a developmental program converging on Myc and Wnt signaling [65]. Therefore, the ZF1-mediated increase in TERT transcription may also involve a pleiotropic activation of stem cells, counteracting their senescence in a manner which is independent of the synthesis of telomere repeats. Further studies are in progress to discriminate among these putative telomerase-dependent and -independent pathways in hMSC response to ZF1. In fact, to date, we only have evidence about the ZF1 effect on telomerase activity in a long-term culture of hASCs [26]. As a next step, we plan to further investigate telomerase activity in other hMSCs under different experimental conditions. Starting from the information currently available on the presence of defined transcripts and proteins in zebrafish at the developmental stage of 50% epiboly [36,41], we will attempt to identify putative conductors capable of recapitulating the biological program observed in the present study.
On the whole, our results suggest that the anti-senescence action elicited by ZF1 on multisource hMSCs can be unfolded into two major, non-mutually exclusive, biomedical implications. Concerning the first implication, antagonizing stem cell senescence during prolonged in vitro culture may allow stem cells expansion up to the number precisely required for a targeted regenerative setting. As far as the second implication is concerned, the anti-senescence action promoted in vitro by ZF1 could be conceivably deployed in vivo using the extract to target the stem cells where they are in all body tissues and afford a process of tissue rejuvenation without the need for stem cell transplantation.
In future studies we are committed to extending the characterization of ZF1 composition and action, and also to addressing the potential limits, which include alterations in genome stability and eventual tumorigenic drift. We also plan to investigate the ZF1 effect on senescence progression at late passages in multiple hMSC types, with particular emphasis on WJ-hMSCs. In fact, these perinatal hMSCs retain the chance for higher multilineage commitment than other adult hMSCs [66]. For this reason, WJ-hMSCs are particularly amenable for future off-the-shelf approaches of allogeneic cell therapy where cells are conceived for long-lasting banking (years) prior to transplantation, an issue that will require effective and precise control of senescence drift over the banking time.

4. Materials and Methods

4.1. Ethics Statement

All the tissue samples were obtained from subjects that gave their informed consent for inclusion before their participation in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local ethical committees (CE) (S. Orsola-Malpighi University Hospital, project identification code: n.1645/2014, ref. 35/2014/U/Tess and Villalba Hospital, project identification code: 16076 of Bologna, Italy).

4.2. hMSCs Harvesting and Culture

hASCs Human subcutaneous adipose tissue samples (obtained from 3 subjects) were isolated from lipoaspiration procedures and processed by using the Lipogems device (PCT/IB2011/052204), as previously described [43]. A volume of 1.5 mL of Lipogems product containing hASCs was seeded in a T75 flask (Falcon BD, Bedford, MA, USA) precoated with human fibronectin (0.55 μg/cm2) (Sigma-Aldrich Co., St. Louis, MO, USA) and human collagen I–III (0.50 μg/cm2) (ABCell-Bio, Paris, France) and cultured in alfa-minimal essential medium (α-MEM, Carlo Erba Reagents, Milano, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 1% penicillin-streptomycin solution, 1% l-glutamine 200 mM (Carlo Erba Reagents).
hDP-MSCs Vital human molars were obtained from 3 adult subjects during routine dental extraction. Dental pulp tissue fragments were recovered and digested as previously described [67]. The hDP-MSCs, slipped down from the explants, were isolated and cultured in Dulbecco’s modified Eagle’s medium high glucose (DMEM, BioWhittaker Cambrex, Walkersville, MD, USA) in the presence of 10% FBS (Gibco) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B) (Carlo Erba Reagents).
hBM-MSCs Bone marrow was collected from 3 healthy adult volunteers and treated as previously described [68]. The isolated hBM-MSCs were plated at 1 × 106/cm2 in culture flasks in DMEM high glucose (BioWhittaker Cambrex) supplemented with 20% FBS (Gibco) and antibiotics (200 U/mL penicillin, 200 μg/mL streptomycin) (Carlo Erba Reagents).
hWJ-MSCs Umbilical cords from 3 healthy donor mothers obtained from caesarean sections were rapidly transferred to the laboratory and treated as previously described [69]. Cord fragments with an approximate diameter of 2 mm were seeded onto the surface of a culture dish with DMEM low glucose (BioWhittaker Cambrex) supplemented with 10% FBS (Gibco), penicillin and streptomycin 200 μg/mL (Carlo Erba Reagents).
All investigated cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO2. The non-adherent cells were removed, and the medium was changed subsequently every 2–4 days. Confluent cells were detached by treatment with trypsin-EDTA (Sigma-Aldrich Co.), maintained and expanded until the desired culture passages.
Before their experimental use, mesenchymal stem cells obtained from all investigated culture tissues at the same doubling passage were observed under the optical microscope (at 40× magnification and bright field illumination). Therefore, cells were analyzed with flow cytometry to confirm the presence of minimal criteria for defining multipotent mesenchymal stem cells, in accordance with the International Society for Cellular Therapy [8].

4.3. Zebrafish Embryo Extract, Dose Analysis and Treatments

Zebrafish embryos were harvested and processed as previously described [25,70]. The 50% epiboly (5 h and 15 min post-fertilization, hpf) developmental stage (named here ZF1) was chosen and the extract containing eggs at the density of 100/mL was prepared in a glycero-alcoholic solution (60% glycerol, 5% ethanol, 0.12% potassium sorbate and 0.08% sodium benzoate) and stored at 4 °C until use, according to the manufacturer’s standard protocol (Aurora Biosearch, Bollate, Milano, Italy). The ZF1 extract was previously analysed on a one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its proteic content was characterized by using liquid chromatography–tandem mass spectrometry (LC-MS/MS) [41]. In order to evaluate the eligible ZF1 dose to use in the present work, hASCs were first treated for 72 h with ZF1 at five different concentrations (0.01, 10, 20, 40, and 100 μg/mL), analyzed under a light microscope (at 200× magnification and bright field illumination), and then submitted to preliminary analyses. On the basis of the results from these treatments, investigated hMSCs were cultured with 20 μg/mL ZF1 for 72 h in all experiments. An equal amount of ZF1 solvent (a glycero-alcoholic solution) was used as a control in all experiments except in the resazurin-based assay where both control and solvent were used.

4.4. BCA Protein Assay

The protein content of ZF1 was determined using a BCA protein assay kit, following the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA). As previously described, the protein content of the extract was determined using a Nanodrop instrument (Nanodrop ND 1000 v.3.8.1, Wilmington, DE, USA), with serial dilution of bovine serum albumin as a standard [25].

4.5. In Vitro Resazurin-Based Toxicology Assay

To evaluate the ZF1 cell proliferation as a function of metabolic activity of the cells, In Vitro Toxicology Assay kit, resazurin-based, (Sigma-Aldrich Co.) was preliminarily used on the hASCs at the 5th culture passage, as representative of the investigated mesenchymal cells. These cells were seeded in technical quadruplicate in a 96-well plate at 4000 cells/cm2. After 24 h in standard conditions, cells were treated for 72 h with ZF1 at three different concentrations (0.01, 10, and 20 μg/mL), or with a solvent (in equal amount), or were untreated (control cells) and were cultured at 37 °C in a complete medium with resazurin reagent (at the ratio of 10:1, respectively). In each experiment, negative (blue resazurin solution with medium) and positive (red totally-reduced resazurin with medium) controls in the absence of cells were added. Fluorescence (correlated to the presence of reduced resazurin as marker of cell metabolic activity over time) was measured after 4, 24, 48, and 72 h from the beginning of the treatment with the Wallac 1420 Victor2 multilabel counter (Perkin Elmer, Waltham, MA, USA) at an emission wavelength of 590 nm and an excitation wavelength of 560 nm. The number of viable cells correlating with the magnitude of dye reduction was expressed as a percentage of resazurin reduction according to this formula: (FI 590 of test agent–FI 590 of negative control)/(FI 590 of 100% reduced of resazurin–FI 590 negative control) × 100, where FI means fluorescence intensity.
Then, the same experiments were extended to hMSCs isolated from four sources (hASCs, hDP-MSCs, hBM-MSCs, and hWJ-MSCs), and ZF1 at the concentration of 20 μg/mL was used. Each treatment was performed in quadruplicate and the whole experiment was repeated with cells obtained from three different subjects (culture passages spanning from the 5th to the 7th).

4.6. Senescence-Associated β-Galactosidase Staining

Cell staining was performed using a SA β-gal kit (Cell Signaling, Danvers, MA, USA). Briefly, all investigated hMSCs were seeded at culture passages between 5th and 7th in 6-well plates (Falcon BD) at the density of 2000 cells/cm2. After 24 h in standard conditions, cells were treated for 72 h with ZF1 or with a solvent as a control. The preliminary experiments were conducted by using three ZF1 different concentrations (0.01, 10, and 20 μg/mL) in hASCs (n = 3). Then the experiments were extended to hMSCs isolated from four sources (hASCs, hDP-MSCs, hBM-MSCs, and hWJ-MSCs) using ZF1 only at the concentration of 20 μg/mL for 72 h and at the different density of 1770 cells/cm2 for hWJ-MSCs. Each treatment was performed in duplicate and the whole experiment was repeated with cells obtained from three different subjects. Cells were fixed and processed according to the manufacturer’s instructions. The number of positive (blue) and negative (not colored) cells was counted in each sample in at least three random fields under the microscope (at 200× magnification and bright field illumination) and the percentage of SA β-gal-positive cells was calculated as the number of positive cells divided by the total number of cells counted multiplied by 100 [71,72].

4.7. Adipogenic Differentiation

The hASCs at culture passages between 5th and 7th (n = 3) were tested for their ability to differentiate into adipogenic lineage. The hASCs were cultured on 24-well plates at the density of 15,000 cells/well. After 24 h in standard conditions, cells were treated for 72 h with ZF1 at three different concentrations (0.01, 10, and 20 μg/mL), or with a solvent as a control. Each treatment (ZF1 at different doses and solvent) was performed in six wells. At the end of the treatment, 4 wells were incubated with adipogenic medium (hMSC adipogenic differentiation medium, Lonza, Walkersville, MD, USA), which was changed twice a week for two weeks. The other two wells were not induced with adipogenic medium but cultured in a standard medium. At the end of the two weeks, differentiation was assessed using Oil Red O staining, as previously described [73]. In particular, cells were fixed in 10% formalin at room temperature for 15 min, washed in distilled water and incubated for 15 min with Oil Red O solution. Then, the cell monolayer was washed three times with demineralized H2O. Finally, Oil Red O was extracted by incubation with isopropanol for 10 min in moderate agitation. For each well, the dye was aliquoted and transferred in triplicate to a 96-well plate prior to reading absorbance at 495 nm using a spectrophotometer (Victor 2, Perkin Elmer Wallac, Milan, Italy).
Furthermore, the adipogenic differentiation assay was extended to hMSCs isolated from four sources (hASCs, hDP-MSCs, hBM-MSCs, and hWJ-MSCs) at the 5th culture passage. A ZF1 concentration of 20 μg/mL was used, and for each investigated hMSC type a technical triplicate was performed (n = 3).

4.8. RNA Extraction and RT-PCR

The hMSCs obtained from the investigated sources, were seeded in T25 flasks at the density of 3500 cells/cm2 (culture passages 5th–7th) and incubated in standard conditions for 24 h before treatments. The hASCs were treated for 72 h with 0.01, 10, and 20 μg/mL ZF1, or with the solvent as a control in a preliminary experiment (technical triplicate), and subsequently, each type of MSCs was treated for 72 h with 20 μg/mL ZF1, or with the solvent as a control. For each type of MSCs, the whole experiment was repeated in biological triplicate. Total RNA was extracted from all investigated cell lineages using the RNeasy mini kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. The genomic DNA contamination was removed by digestion with RNase-free deoxyribonuclease I (RNase-free DNase set, QIAGEN). The reverse transcription of the extracted RNA was performed as previously described except for the temperature of the reaction that was 37 °C instead of 42 °C [74]. The success of the reaction was verified by the amplification of the human Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, using specific primers (forward sequence: 5′-GAAATCCCATCACCATCTTCCAG-3′ and reverse sequence 5′-GCTACACTGAGCACCAGGTGGTCTCCT-3′). The GAPDH amplification was performed as previously described [75], for 25 cycles instead of 45. The amplicon detection was performed by gel electrophoresis as previously described [25].

4.9. Real-Time PCR

A relative quantitative real-time PCR (qPCR) was performed in a Bio-Rad CFX96 real-time thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) as previously described [76]. Briefly, 25 ng of cDNA were amplified using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories) in technical triplicate for every cDNA sample. Primers for TERT were purchased from Invitrogen (forward sequence: 5′-AAGTTCCTGCACTGGCTGATG-3′ and reverse sequence 5′-GCTTTGCAACTTGCTCCAGAC-3′) (Invitrogen, Carlsbad, CA, USA) and were used as 0.2 μM each in the qPCR reactions. Specific primers for BMI1, TP53, CDKN1A (p21), and CDKN2A (p16) (unique assay ID: qHsaCED0046537, qHsaCID0013658, qHsaCID0014498 and qHsaCED0056722, respectively, Bio-Rad Laboratories) were designed by Bio-Rad and used following the manufacturer’s instructions (20×, Bio-Rad Laboratories).
Relative gene expression was determined using CFX Manager Software version 3.1 (Bio-Rad Laboratories) with the “delta-delta CT method” [77] and hypoxanthine phosphoribosyl transferase 1 (HPRT1), TATA box binding protein (TBP), GAPDH (20×, unique assay ID: qHsaCID0016375, qHsaCID0007122, qHsaCED0038674, respectively, Bio-Rad Laboratories) were used as reference genes. The investigated mRNA levels in hASCs, hDP-MSCs, hBM-MSCs, and hWJ-MSCs treated with ZF1 20 μg/mL were expressed as fold of change (2−ΔΔCt), relative to mRNA levels evaluated in respective hMSC lines treated with a solvent as a control. A preliminary gene expression analysis with qPCR was conducted to exclude effects of the treatment with solvent. Gene expression analysis was performed in biological triplicate for each cell type.

4.10. Protein Extraction and Western Blot

All investigated hMSCs were seeded at culture passages between 5th and 7th in T25 flasks (Falcon BD) at the density of 3500 cells/cm2. After 24 h in standard conditions, cells were treated for 72 h with ZF1 20 μg/mL or with a solvent as a control.
At the end of the treatment, cells were scraped from the flasks and pelleted at 1200 rpm for 5 min. Pellets were lysed with the mammalian protein extraction reagent (M-PER, Thermo-Fisher Scientific, Waltham, MA, USA) supplemented with 100× protease and phosphatase inhibitors (Sigma-Aldrich). Then, cell lysates were subjected to 3 cycles of 5 s of sonication followed by 2 min in ice and then centrifuged at the maximum speed to remove the cellular debris. The protein content of each sample was determined using a Bradford kit (VWR International, Radnor, Pennsylvania, USA).
Before electrophoresis, every sample was mixed with 4× Laemmly sample buffer (Bio-Rad) supplemented with a 10% of 2-mercaptoethanol (Sigma-Aldrich) and then boiled at 95 °C for 5 min.
An amount of 10 µg of proteins were loaded in Mini-PROTEAN TGX stain-free precast protein gels (Bio-Rad) and electrophoresis was performed in a 10× tris/glycine/SDS buffer (Bio-Rad) with the Mini-PROTEAN Tetra System (Bio-Rad). Gels and total protein amount were analyzed with ChemiDoc imaging system (Bio-Rad). Blotting was performed using a Trans-Blot Turbo Transfer Pack and a Trans-Blot Turbo Transfer System (Bio-Rad). After a blocking step of 1 h in a 3% solution of milk (Blotting Grade Blocking; Bio-Rad) in TBS-Tween 0.1%, membranes were incubated overnight at 4 °C with the p21 (Santa Cruz Biotechnologies, Dallas, Texas, USA) primary antibody used 1:1000 and 1 h at room temperature with the appropriate secondary antibody, diluted 1:5000 (Santa Cruz Biotechnologies). Protein detection was performed after 1 min of incubation with Clarity Max Western ECL substrate (Bio-Rad) and image acquired using ChemiDoc Touch Imaging System (Bio-Rad). The detected signal for each sample was normalized to the total protein detected in stain-free acquisitions. The Western blot analysis was performed in biological triplicate for each cell type.

4.11. Statistical Analysis

Data obtained from in vitro toxicology assay were investigated with ANOVA analysis followed by the Tukey HSD test. Data obtained from the SA β-gal assay were analyzed using the Student’s t-test in preliminary experiment and Z-test for proportional in the study of the four types of hMSCs treated with ZF1 20 μg/mL. Data obtained from the adipogenesis assay and the Western blot were analyzed using the Student’s t-test. Data obtained from qPCR were analyzed using the CFX Manager Software version 3.1 (Bio-Rad Laboratories) and the Student’s t-test. The results were considered statistically significant with a p-value < 0.05 and highly significant with a p-value < 0.01.

Author Contributions

F.F. and F.A. conceived and executed the experimental plan; S.C., E.B., and M.R. performed the experiments; F.F., F.A., S.C., E.B., and C.V. analyzed the data; C.V. designed/supervised the project; C.V., F.F., F.A., and S.C. wrote the manuscript; L.B., R.C., and P.M.B. collaborated in conceiving the experimental plan; all authors reviewed the manuscript.

Funding

This research was funded by the Eldor Lab, via Vittor Pisani 16, 20124 Milan, Italy.

Acknowledgments

The statisticians Barbara Bordini and Umberto Santoro are gratefully acknowledged for their contribution.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

α-MEMalfa-Minimal Essential Medium
BMI1BMI1 proto-oncogene, polycomb ring finger
BMbone marrow
CDKN1A (p21)cyclin dependent kinase inhibitor 1A
CDKN2A (p16)cyclin dependent kinase inhibitor 2A
c-Mycv-myc avian myelocytomatosis viral oncogene homolog
CTRcontrol
DPdental pulp
DMEMDulbecco’s Modified Eagle’s Medium
FBSFetal Bovine Serum
GAPDHglyceraldehyde 3-phosphate dehydrogenase
hhour
hpfhours post fertilization
hMSCshuman mesenchymal stem cells
hASCshuman adipose tissue-derived stem cells
HPRT1hypoxanthine phosphoribosyl transferase 1
iPSinduced pluripotent stem
LC-MS/MSliquid chromatography-tandem mass spectrometry
OCT-4POU domain class 5 homeobox 1 (POU5F1), alias Oct-4
qPCRquantitative relative real-time PCR
SA β-galSenescence-Associated β-Galactosidase
SDS-PAGESodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
SOLVsolvent
TBPTATA box binding protein
TERTtelomerase reverse transcriptase
TP53tumor protein p53
WJWharton’s Jelly
ZF1Zebrafish extract

References

  1. Bryder, D.; Rossi, D.J.; Weissman, I.L. Hematopoietic stem cells: The paradigmatic tissue-specific stem cell. Am. J. Pathol. 2006, 169, 338–346. [Google Scholar] [CrossRef] [PubMed]
  2. Asahara, T.; Kawamoto, A. Endothelial progenitor cells for postnatal vasculogenesis. Am. J. Physiol. Cell Physiol. 2004, 287, C572–C579. [Google Scholar] [CrossRef]
  3. Murry, C.E.; Field, L.J.; Menasche, P. Cell-based cardiac repair: Reflections at the 10-year point. Circulation 2005, 112, 3174–3183. [Google Scholar] [CrossRef]
  4. Lindvall, O.; Kokaia, Z.; Martinez-Serrano, A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat. Med. 2004, 10, S42–S50. [Google Scholar] [CrossRef]
  5. Bonner-Weir, S.; Weir, G.C. New sources of pancreatic beta-cells. Nat. Biotechnol. 2005, 23, 857–861. [Google Scholar] [CrossRef] [PubMed]
  6. Bianco, P.; Riminucci, M.; Gronthos, S.; Robey, P.G. Bone marrow stromal stem cells: Nature, biology, and potential applications. Stem Cells 2001, 19, 180–192. [Google Scholar] [CrossRef]
  7. Crisan, M.; Yap, S.; Casteilla, L.; Chen, C.W.; Corselli, M.; Park, T.S.; Andriolo, G.; Sun, B.; Zheng, B.; Zhang, L.; et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008, 3, 301–313. [Google Scholar] [CrossRef]
  8. Dominici, M.; Le Blanc, K.; Mueller, I.; Slaper-Cortenbach, I.; Marini, F.; Krause, D.; Deans, R.; Keating, A.; Prockop, D.J.; Horwitz, E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006, 8, 315–317. [Google Scholar] [CrossRef] [PubMed]
  9. Wei, X.; Yang, X.; Han, Z.P.; Qu, F.F.; Shao, L.; Shi, Y.F. Mesenchymal stem cells: A new trend for cell therapy. Acta Pharmacol. Sin. 2013, 34, 747–754. [Google Scholar] [CrossRef] [PubMed]
  10. Mahla, R.S. Stem cells applications in regenerative medicine and disease therapeutics. Int. J. Cell Biol. 2016, 2016, 6940283. [Google Scholar] [CrossRef]
  11. Bai, L.; Lennon, D.P.; Caplan, A.I.; DeChant, A.; Hecker, J.; Kranso, J.; Zaremba, A.; Miller, R.H. Hepatocyte growth factor mediates mesenchymal stem cell-induced recovery in multiple sclerosis models. Nat. Neurosci. 2012, 15, 862–870. [Google Scholar] [CrossRef]
  12. Chang, C.; Wang, X.; Niu, D.; Zhang, Z.; Zhao, H.; Gong, F. Mesenchymal stem cells adopt beta-cell fate upon diabetic pancreatic microenvironment. Pancreas 2009, 38, 275–281. [Google Scholar] [CrossRef]
  13. Kuo, Y.R.; Goto, S.; Shih, H.S.; Wang, F.S.; Lin, C.C.; Wang, C.T.; Huang, E.Y.; Chen, C.L.; Wei, F.C.; Zheng, X.X.; et al. Mesenchymal stem cells prolong composite tissue allotransplant survival in a swine model. Transplantation 2009, 87, 1769–1777. [Google Scholar] [CrossRef]
  14. Souza, B.S.; Nogueira, R.C.; de Oliveira, S.A.; de Freitas, L.A.; Lyra, L.G.; Ribeiro dos Santos, R.; Lyra, A.C.; Soares, M.B. Current status of stem cell therapy for liver diseases. Cell Transplant. 2009, 18, 1261–1279. [Google Scholar] [CrossRef]
  15. Quevedo, H.C.; Hatzistergos, K.E.; Oskouei, B.N.; Feigenbaum, G.S.; Rodriguez, J.E.; Valdes, D.; Pattany, P.M.; Zambrano, J.P.; Hu, Q.; McNiece, I.; et al. Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. Proc. Natl. Acad. Sci. USA 2009, 106, 14022–14027. [Google Scholar] [CrossRef]
  16. Facchin, F.; Bianconi, E.; Canaider, S.; Basoli, V.; Biava, P.M.; Ventura, C. Tissue Regeneration without Stem Cell Transplantation: Self-Healing Potential from Ancestral Chemistry and Physical Energies. Stem Cells Int. 2018, 2018, 7412035. [Google Scholar] [CrossRef]
  17. Castorina, A.; Szychlinska, M.A.; Marzagalli, R.; Musumeci, G. Mesenchymal stem cells-based therapy as a potential treatment in neurodegenerative disorders: Is the escape from senescence an answer? Neural Regen. Res. 2015, 10, 850–858. [Google Scholar] [CrossRef]
  18. Szychlinska, M.A.; Stoddart, M.J.; D’Amora, U.; Ambrosio, L.; Alini, M.; Musumeci, G. Mesenchymal Stem Cell-Based Cartilage Regeneration Approach and Cell Senescence: Can We Manipulate Cell Aging and Function? Tissue Eng. Part. B Rev. 2017, 23, 529–539. [Google Scholar] [CrossRef]
  19. Tal, T.L.; Franzosa, J.A.; Tanguay, R.L. Molecular signaling networks that choreograph epimorphic fin regeneration in zebrafish—A mini-review. Gerontology 2010, 56, 231–240. [Google Scholar] [CrossRef]
  20. Shi, W.; Fang, Z.; Li, L.; Luo, L. Using zebrafish as the model organism to understand organ regeneration. Sci. China Life Sci. 2015, 58, 343–351. [Google Scholar] [CrossRef]
  21. Goessling, W.; North, T.E. Repairing quite swimmingly: Advances in regenerative medicine using zebrafish. Dis. Model. Mech. 2014, 7, 769–776. [Google Scholar] [CrossRef]
  22. Brockerhoff, S.E.; Fadool, J.M. Genetics of photoreceptor degeneration and regeneration in zebrafish. Cell Mol. Life Sci. 2011, 68, 651–659. [Google Scholar] [CrossRef]
  23. Becker, T.; Becker, C.G. Axonal regeneration in zebrafish. Curr. Opin. Neurobiol. 2014, 27, 186–191. [Google Scholar] [CrossRef]
  24. Pozzoli, O.; Vella, P.; Iaffaldano, G.; Parente, V.; Devanna, P.; Lacovich, M.; Lamia, C.L.; Fascio, U.; Longoni, D.; Cotelli, F.; et al. Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish. Arterioscler. Thromb. Vasc. Biol. 2011, 31, 1589–1597. [Google Scholar] [CrossRef]
  25. Canaider, S.; Maioli, M.; Facchin, F.; Bianconi, E.; Santaniello, S.; Pigliaru, G.; Ljungberg, L.; Burigana, F.; Bianchi, F.; Olivi, E.; et al. Human stem cell exposure to developmental stage zebrafish extracts: A novel strategy for tuning stemness and senescence patterning. CellR4 2014, 2, e1226. [Google Scholar]
  26. Facchin, F.; Canaider, S.; Bianconi, E.; Maioli, M.; Santoro, U.; Santaniello, S.; Basoli, V.; Biava, P.M.; Ventura, C. Zebrafish embryo extract counteracts human stem cell senescence. Front. Biosci. (Schol Ed.) 2019, 11, 89–104. [Google Scholar] [CrossRef]
  27. Mathavan, S.; Lee, S.G.; Mak, A.; Miller, L.D.; Murthy, K.R.; Govindarajan, K.R.; Tong, Y.; Wu, Y.L.; Lam, S.H.; Yang, H.; Ruan, Y.; et al. Transcriptome analysis of zebrafish embryogenesis using microarrays. PLoS Genet. 2005, 1, 260–276. [Google Scholar] [CrossRef]
  28. Yao, Y.; Ma, L.; Jia, Q.; Deng, W.; Liu, Z.; Zhang, Y.; Ren, J.; Xue, Y.; Jia, H.; Yang, Q. Systematic characterization of small RNAome during zebrafish early developmental stages. BMC Genom. 2014, 15, 117. [Google Scholar] [CrossRef]
  29. Palmblad, M.; Henkel, C.V.; Dirks, R.P.; Meijer, A.H.; Deelder, A.M.; Spaink, H.P. Parallel deep transcriptome and proteome analysis of zebrafish larvae. BMC Res. Notes 2013, 6, 428. [Google Scholar] [CrossRef]
  30. Haggard, D.E.; Noyes, P.D.; Waters, K.M.; Tanguay, R.L. Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists. Reprod. Toxicol. 2018, 77, 80–93. [Google Scholar] [CrossRef]
  31. Huang, X.; Agrawal, I.; Li, Z.; Zheng, W.; Lin, Q.; Gong, Z. Transcriptomic Analyses in Zebrafish Cancer Models for Global Gene Expression and Pathway Discovery. Adv. Exp. Med. Biol. 2016, 916, 147–168. [Google Scholar] [CrossRef]
  32. Li, C.; Tan, X.F.; Lim, T.K.; Lin, Q.; Gong, Z. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human. Sci. Rep. 2016, 6, 24329. [Google Scholar] [CrossRef]
  33. Tay, T.L.; Lin, Q.; Seow, T.K.; Tan, K.H.; Hew, C.L.; Gong, Z. Proteomic analysis of protein profiles during early development of the zebrafish, Danio rerio. Proteomics 2006, 6, 3176–3188. [Google Scholar] [CrossRef]
  34. Link, V.; Shevchenko, A.; Heisenberg, C.P. Proteomics of early zebrafish embryos. BMC Dev. Biol. 2006, 6, 1–9. [Google Scholar] [CrossRef]
  35. Lucitt, M.B.; Price, T.S.; Pizarro, A.; Wu, W.; Yocum, A.K.; Seiler, C.; Pack, M.A.; Blair, I.A.; Fitzgerald, G.A.; Grosser, T. Analysis of the zebrafish proteome during embryonic development. Mol. Cell Proteom. 2008, 7, 981–994. [Google Scholar] [CrossRef]
  36. Vesterlund, L.; Jiao, H.; Unneberg, P.; Hovatta, O.; Kere, J. The zebrafish transcriptome during early development. BMC Dev. Biol. 2011, 11, 30. [Google Scholar] [CrossRef]
  37. Forné, I.; Abián, J.; Cerdà, J. Fish proteome analysis: Model organisms and non-sequenced species. Proteomics 2010, 10, 858–872. [Google Scholar] [CrossRef]
  38. Saxena, S.; Singh, S.K.; Lakshmi, M.G.; Meghah, V.; Sundaram, C.S.; Swamy, C.V.; Idris, M.M. Proteome profile of zebrafish kidney. J. Proteom. 2011, 74, 2937–2947. [Google Scholar] [CrossRef]
  39. Smidak, R.; Aradska, J.; Kirchberger, S.; Distel, M.; Sialana, F.J.; Wackerlig, J.; Mechtcheriakova, D.; Lubec, G. A detailed proteomic profiling of plasma membrane from zebrafish brain. Proteom. Clin. Appl. 2016, 10, 1264–1268. [Google Scholar] [CrossRef]
  40. Carneiro, M.; Gutiérrez-Praena, D.; Osório, H.; Vasconcelos, V.; Carvalho, A.P.; Campos, A. Proteomic analysis of anatoxina acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress. Ecotoxicol. Environ. Saf. 2015, 120, 93–101. [Google Scholar] [CrossRef]
  41. Biava, P.M.; Canaider, S.; Facchin, F.; Bianconi, E.; Ljungberg, L.; Rotilio, D.; Burigana, F.; Ventura, C. Stem cell differentiation stage factors from zebrafish embryo: A novel strategy to modulate the fate of normal and pathological human (stem) cells. Curr. Pharm. Biotechnol. 2015, 16, 782–792. [Google Scholar] [CrossRef] [PubMed]
  42. Paradisi, M.; Alviano, F.; Pirondi, S.; Lanzoni, G.; Fernandez, M.; Lizzo, G.; Giardino, L.; Giuliani, A.; Costa, R.; Marchionni, C.; Bonsi, L.; Calza, L. Human mesenchymal stem cells produce bioactive neurotrophic factors: Source, individual variability and differentiation issues. Int. J. Immunopathol. Pharmacol. 2014, 27, 391–402. [Google Scholar] [CrossRef]
  43. Bianchi, F.; Maioli, M.; Leonardi, E.; Olivi, E.; Pasquinelli, G.; Valente, S.; Mendez, A.J.; Ricordi, C.; Raffaini, M.; Tremolada, C.; Ventura, C. A new nonenzymatic method and device to obtain a fat tissue derivative highly enriched in pericyte-like elements by mild mechanical forces from human lipoaspirates. Cell Transplant. 2013, 22, 2063–2077. [Google Scholar] [CrossRef] [PubMed]
  44. Li, X.; Song, Y.; Liu, D.; Zhao, J.; Xu, J.; Ren, J.; Hu, Y.; Wang, Z.; Hou, Y.; Zhao, G. MiR-495 promotes senescence of mesenchymal stem cells by targeting Bmi-1. Cell Physiol. Biochem. 2017, 42, 780–796. [Google Scholar] [CrossRef] [PubMed]
  45. Molofsky, A.V.; Pardal, R.; Iwashita, T.; Park, I.K.; Clarke, M.F.; Morrison, S.J. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003, 425, 962–967. [Google Scholar] [CrossRef] [PubMed]
  46. Rando, T.A. Stem cells, ageing and the quest for immortality. Nature 2006, 441, 1080–1086. [Google Scholar] [CrossRef]
  47. Da Silva, M.L.; Chagastelles, P.C.; Nardi, N.B. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J. Cell Sci. 2006, 119, 2204–2213. [Google Scholar] [CrossRef]
  48. Neirinckx, V.; Coste, C.; Rogister, B.; Wislet-Gendebien, S. Concise review: Adult mesenchymal stem cells, adult neural crest stem cells, and therapy of neurological pathologies: A state of play. Stem Cells Transl. Med. 2013, 2, 284–296. [Google Scholar] [CrossRef]
  49. Gronthos, S.; Mankani, M.; Brahim, J.; Robey, P.G.; Shi, S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. USA 2000, 97, 13625–13630. [Google Scholar] [CrossRef] [PubMed]
  50. Parolini, O.; Alviano, F.; Bagnara, G.P.; Bilic, G.; Bühring, H.J.; Evangelista, M.; Hennerbichler, S.; Liu, B.; Magatti, M.; Mao, N.; et al. Concise review: Isolation and characterization of cells from human term placenta: Outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells 2008, 26, 300–311. [Google Scholar] [CrossRef]
  51. La Rocca, G.; Lo Iacono, M.; Corsello, T.; Corrao, S.; Farina, F.; Anzalone, R. Human Wharton’s jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic, adipogenic and chondrogenic differentiation in vitro: New perspectives for cellular therapy. Curr. Stem Cell Res. Ther. 2013, 8, 100–113. [Google Scholar] [CrossRef] [PubMed]
  52. Laurent, L.C.; Ulitsky, I.; Slavin, I.; Tran, H.; Schork, A.; Morey, R.; Lynch, C.; Harness, J.V.; Lee, S.; Barrero, M.J.; et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 2011, 8, 106–118. [Google Scholar] [CrossRef] [PubMed]
  53. Chatgilialoglu, A.; Rossi, M.; Alviano, F.; Poggi, P.; Zannini, C.; Marchionni, C.; Ricci, F.; Tazzari, P.L.; Taglioli, V.; Calder, P.C.; Bonsi, L. Restored in vivo-like membrane lipidomics positively influence in vitro features of cultured mesenchymal stromal/stem cells derived from human placenta. Stem Cell Res. Ther. 2017, 8, 31. [Google Scholar] [CrossRef]
  54. Hass, R.; Kasper, C.; Böhm, S.; Jacobs, R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Comm Signal. 2011, 9, 12. [Google Scholar] [CrossRef] [PubMed]
  55. Samsonraj, R.M.; Raghunath, M.; Nurcombe, V.; Hui, J.H.; van Wijnen, A.J.; Cool, S.M. Concise Review: Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine. Stem Cells Transl. Med. 2017, 6, 2173–2185. [Google Scholar] [CrossRef] [PubMed]
  56. Xu, L.; Liu, Y.; Sun, Y.; Wang, B.; Xiong, Y.; Lin, W.; Wei, Q.; Wang, H.; He, W.; Wang, B.; Li, G. Tissue source determines the differentiation potentials of mesenchymal stem cells: A comparative study of human mesenchymal stem cells from bone marrow and adipose tissue. Stem Cell Res. Ther. 2017, 8, 275. [Google Scholar] [CrossRef] [PubMed]
  57. Cong, Y.; Shay, J.W. Actions of human telomerase beyond telomeres. Cell Res. 2008, 18, 725–732. [Google Scholar] [CrossRef] [PubMed]
  58. Bhattacharya, R.; Mustafi, S.B.; Street, M.; Dey, A.; Dwivedi, S.K. Bmi-1: At the crossroads of physiological and pathological biology. Genes Dis. 2015, 2, 225–239. [Google Scholar] [CrossRef]
  59. Oh, J.; Lee, Y.D.; Wagers, A.J. Stem cell aging: Mechanisms, regulators and therapeutic opportunities. Nat. Med. 2014, 20, 870–880. [Google Scholar] [CrossRef]
  60. Stampfer, M.R.; Garbe, J.; Nijjar, T.; Wigington, D.; Swisshelm, K.; Yaswen, P. Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines. Oncogene 2003, 22, 5238–5251. [Google Scholar] [CrossRef]
  61. Artandi, S.E.; Attardi, L.D. Pathways connecting telomeres and p53 in senescence, apoptosis, and cancer. Biochem. Biophys. Res. Commun. 2005, 331, 881–890. [Google Scholar] [CrossRef]
  62. Shats, I.; Milyavsky, M.; Tang, X.; Stambolsky, P.; Erez, N.; Brosh, R.; Kogan, I.; Braunstein, I.; Tzukerman, M.; Ginsberg, D.; Rotter, V. p53-dependent down-regulation of telomerase is mediated by p21waf1. J. Biol. Chem. 2004, 279, 50976–50985. [Google Scholar] [CrossRef]
  63. Liu, T.M.; Ng, W.M.; Tan, H.S.; Vinitha, D.; Yang, Z.; Fan, J.B.; Zou, Y.; Hui, J.H.; Lee, E.H.; Lim, B. Molecular basis of immortalization of human mesenchymal stem cells by combination of p53 knockdown and human telomerase reverse transcriptase overexpression. Stem Cells Dev. 2013, 22, 268–278. [Google Scholar] [CrossRef]
  64. Sarin, K.Y.; Cheung, P.; Gilison, D.; Lee, E.; Tennen, R.I.; Wang, E.; Artandi, M.K.; Oro, A.E.; Artandi, S.E. Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature 2005, 436, 1048–1052. [Google Scholar] [CrossRef]
  65. Choi, J.; Southworth, L.K.; Sarin, K.Y.; Venteicher, A.S.; Ma, W.; Chang, W.; Cheung, P.; Jun, S.; Artandi, M.K.; Shah, N.; Kim, S.K.; Artandi, S.E. TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program. PLoS Genet. 2008, 4, e10. [Google Scholar] [CrossRef]
  66. Bieback, K.; Brinkmann, I. Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy. World J. Stem Cells. 2010, 2, 81–92. [Google Scholar] [CrossRef]
  67. Marchionni, C.; Bonsi, L.; Alviano, F.; Lanzoni, G.; Di Tullio, A.; Costa, R.; Montanari, M.; Tazzari, P.L.; Ricci, F.; Pasquinelli, G.; et al. Angiogenic potential of human dental pulp stromal (stem) cells. Int. J. Immunopathol. Pharmacol. 2009, 22, 699–706. [Google Scholar] [CrossRef]
  68. Pierdomenico, L.; Bonsi, L.; Calvitti, M.; Rondelli, D.; Arpinati, M.; Chirumbolo, G.; Becchetti, E.; Marchionni, C.; Alviano, F.; Fossati, V.; et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 2005, 80, 836–842. [Google Scholar] [CrossRef]
  69. La Rocca, G.; Anzalone, R.; Corrao, S.; Magno, F.; Loria, T.; Lo Iacono, M.; Di Stefano, A.; Giannuzzi, P.; Marasà, L.; Cappello, F.; et al. Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: Differentiation potential and detection of new markers. Histochem. Cell Biol. 2009, 131, 267–282. [Google Scholar] [CrossRef]
  70. Livraghi, T.; Meloni, F.; Frosi, A.; Lazzaroni, S.; Bizzarri, T.M.; Frati, L.; Biava, P.M. Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: An open randomized clinical trial. Oncol. Res. 2005, 15, 399–408. [Google Scholar] [CrossRef]
  71. Rinaldi, S.; Maioli, M.; Pigliaru, G.; Castagna, A.; Santaniello, S.; Basoli, V.; Fontani, V.; Ventura, C. Stem cell senescence. Effects of REAC technology on telomerase-independent and telomerase-dependent pathways. Sci. Rep. 2014, 4, 6373. [Google Scholar] [CrossRef]
  72. Facchin, F.; Bianconi, E.; Romano, M.; Impellizzeri, A.; Alviano, F.; Maioli, M.; Canaider, S.; Ventura, C. Comparison of Oxidative Stress Effects on Senescence Patterning of Human Adult and Perinatal Tissue-Derived Stem Cells in Short and Long-term Cultures. Int. J. Med. Sci. 2018, 15, 1486–1501. [Google Scholar] [CrossRef]
  73. Rossi, M.; Alviano, F.; Ricci, F.; Vignoli, F.; Marchionni, C.; Valente, S.; Zannini, C.; Tazzari, P.L.; Vignoli, M.; Bartoletti, E.; Bonsi, L. In vitro multilineage potential and immunomodulatory properties of adipose derived stromal/stem cells obtained from nanofat lipoaspirates. CellR4 2016, 4, e2212. [Google Scholar]
  74. Casadei, R.; Piovesan, A.; Vitale, L.; Facchin, F.; Pelleri, M.C.; Canaider, S.; Bianconi, E.; Frabetti, F.; Strippoli, P. Genome-scale analysis of human mRNA 5’ coding sequences based on expressed sequence tag (EST) database. Genomics 2012, 100, 125–130. [Google Scholar] [CrossRef] [PubMed]
  75. Facchin, F.; Vitale, L.; Bianconi, E.; Piva, F.; Frabetti, F.; Strippoli, P.; Casadei, R.; Pelleri, M.C.; Piovesan, A.; Canaider, S. Complexity of bidirectional transcription and alternative splicing at human RCAN3 locus. PLoS ONE 2011, 6, e24508. [Google Scholar] [CrossRef]
  76. Beraudi, A.; Bianconi, E.; Catalani, S.; Canaider, S.; De Pasquale, D.; Apostoli, P.; Bordini, B.; Stea, S.; Toni, A.; Facchin, F. In vivo response of heme-oxygenase-1 to metal ions released from metal-on-metal hip prostheses. Mol. Med. Rep. 2016, 14, 474–480. [Google Scholar] [CrossRef] [PubMed]
  77. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; Vandesompele, J.; Wittwer, C.T. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622. [Google Scholar] [CrossRef]
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