Intelligent Information gives birth to Life: it is the factors existing at 5 stages of stem cells differentiation that determine the fate of healthy and pathological cells.

Youth: a gift that we must achieve

As we get older, the risk of getting all kind of diseases increases progressively. Therefore the goal of research on growth and differentiation factors is to succeed in intervening on aging process and preventing pathologies caused by cellular degeneration.
Aging is not a disease but a natural process triggered by the cells aging. With no telomerase activity the telomere of the chromosomes of the cells in active proliferation shorten progressively; when telomere length falls below a critical threshold, stem cells stop dividing and die. Therefore elderly telomeres are shorter than those of young people. Longevity is a balance between genes and environment, when this relationship is meant as a way in which life habits interact with genetic heritage. In this sense we must see the aging process too, i.e. always bearing in mind that genetic heritage can be modified consecutively.
Today thanks to the growth and differentiation factors we are able to keep the telomeres always long, even when the cell divides. Growth factors allow the cell to make her telomere re-grow as it spontaneously occurs in youth.

Cellular degeneration due to aging is not exactly the same for everyone in the same way, these are the average age for the aging of individual organs:

These are the results produced by a collaboration of 23 Italian universities on new frontiers of cells reprogramming, aimed to determine fate of healthy and pathological stem cells and define incredible regulatory activities of the code that organizes life: the epigenetic code.

The studies on functioning of the epigenetic code made it possible to understand that what had been studied was in fact the code of regulation of gene expression. This code is present in its totality and with all its different functions only at the moment when life is formed. In this moment this code could be studied and understood in its global functioning, even if subdivided and divided into different stages of differentiation. In fact if all the substances present in all the different stages of differentiation had been obtained, we would have had the entire epigenetic code available, that is the code able to regulate all the genes of all the cells that make up an entire organism. In other words we would have had the whole code that regulates life: the “Code of Life”. This possibility of studying the epigenetic code in its entirety exists only in the embryo and only in the moment when all the organs and systems differentiate. In that period from a single totipotent stem cell (the fertilized egg) there is a differentiation of all types of stem cells through various stages: pluripotent, multipotent, oligopotent cells, cells in process of definitive differentiation and finally completely differentiated cells. Once the organogenesis is over, it is no longer possible to study the entire epigenetic code in its whole extent and with its different functions. In fact, when the organogenesis is terminated, the epigenetic code is divided into various organs and tissues and every organ contains that part of the code which serves to control and regulate gene expression of the cells of that specific organ, and there is no longer a possibility of studying all the different and incredible functions of the “Code of Life”. Therefore only in that moment when it was decided to study the epigenetic regulation code, i.e. during the period of differentiation of various organs and apparatuses, it became possible to study all the different and incredible regulatory capacities of this code. And that is what has been done, choosing first of all an embryo as a study model for cell differentiation: i.e. in this case the embryo of Zebrafish.

The study of the entire epigenetic code and its functions has led us to fantastic discoveries. These different functions are here briefly described in order to make it clear what great opportunities the study of the code of life offers.

The different regulatory activities of the epigenetic code are listed below:

  • Anti-Aging Activity
  • Slowing of the multiplication and growth of cancer cells
  • Prevention of Neuro-Degeneration

Anti-Aging Action:

For the first time in the world it has been identified a fraction of the epigenetic code which has proved to be able to naturally maintain, without genetic manipulations, stem genes responsible for preventing cellular aging (these are the same genes that Shinya Yamanaka, who won the Nobel Prize in 2012, had artificially introduced within a retrovirus in a differentiated cell. This cell however cannot be used without risk precisely due to the undergone manipulations, which modify the cellular cyclicity and keep the cell in a phase of continuous multiplication. In our research, instead, the cells increase the duration of their life without being manipulated, based only on a physiological regulation of the stem genes. In fact if the administration of these factors is stopped, the cells return to age, thus demonstrating that they have not lost their cyclical nature and normal physiology). The cellular life increase occurs thanks to the impediment of telomere cutting, that is the terminal part of the chromosome composed of repeated sequences of DNA. The function of telomeres is to protect the termination of chromosomes, to allow cell division, to protect against aging and cancer. The telomere prevents the progressive degradation of chromosomes what could bring to the loss of genetic information: telomeres in this way act as a sort of biological clock, linked to a maximum number of DNA replications, at the end of which the cell becomes too old for being kept alive. At that point the cell undertakes the path of programmed cell death and thus concludes its life cycle. The factors isolated in our studies not only prevent the cutting of telomeres, thus lengthening the life span of cells, but also activate other genes, such as Bmi-1, which induce the synthesis of various proteins responsible for preventing cellular senescence.

Therefore these factors, which can only be obtained in specific and well-defined moments of stem cell differentiation, not only increase the duration of life, but keep the cells young, preventing them from aging. This is a very important point, considering that if we only lengthen the life span, but we leave cells grow old, then the risk is to be at an old age having numerous health problems and a considerable physical decay. Fortunately the factors we isolated do not allow cells to age and thus the increase of life span can be associated with a good psycho-physical condition. At this moment the factors that had shown an anti-aging activity have been used to prepare specific creams. These creams have proved to be highly effective as they have been able to significantly reduce wrinkles on the face and décolletage. These results, which were obtained with the help of factors isolated during the differentiation of specific types of stem cells, are almost impossible to obtain by artificial manipulations of the genetic code. This is because different genetic manipulations can’t transfer complete information able to increase the duration of life and at the same time to block cellular senescence. At this point the limits of scientific reductionism emerge even more clearly.

Stages of embryonic cell differentiation
Identified biological proteins
Existing Scientific publications

Integrative treatment for the prevention of aging: cell growth and differentiation factors


100% Biological and physiological, 100% effective.

As growth and differentiation factors are biological and physiological, they have no side effects.

Protein analysis of Zebrafish embryo extract,


A proteins analysis of embryo extract from zebrafish embryo has been performed. A suspension of the glycerol-alcohol solution was analysed by means of the monodimensional electrophoresis gel (SDS-PAGE). As shown in the figure below, there have been identified three major groups in all 5 phases extracted, distinguished by their molecular weight: over 45 kDa, about 25-35 kDa and below 20 kDa. In any case, the relative amount of proteins is different in the 5-stage samples.

Below is the list of proteins that have been identified by Biava et al. with mass spectrometry analysis. The asterisk (*) lists the proteins never described before in the Zebrafish embryo:

Dr. Pier Mario Biava on Telomeres:

“Telomeres get shorter each time the cells divide. When the telomere becomes too short, the chromosome reaches a critical length and the cell can no longer replicate. This means that the cell ages and dies. However, GROWTH and DIFFERENTIATION FACTORS can be a very effective tool to promote cellular senescence antagonists in a physiological way. The result is a healthy cell and potentially an inversion of the aging process”

TELOMERS are essential parts of human cells that influence the way we age. Telomeres represent sort of a “cap” of DNA that protects cells from errors during their replication. Over time this hood tends to shorten and lose its protective properties. Thanks to the factors of Zebrafish this problem can be prevented by preserving telomers.

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


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


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.


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.


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).


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.


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.


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.


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