Treatments to repair the human heart following regenerative diseases remain a challenge for medical science. Unlike lower vertebrate species the human heart lacks a regenerative pathway meaning that research has to be focused on cell transplantation. Porcines ( Sus scrofa ) are excellent models for cardiovascular disease and pluripotent stem cells (PSCs) generated from porcines will provide important clinical insights for cardiac cell therapy. This could open a new avenue of research into degenerative conditions as porcine is a more effective human proxy to work with. However, bona fide PSCs are currently available onlyin rodents (mouse, rat) and primates (monkey, human). Attempts to derivepluripotent stem cells (PSCs) from porcine have been going on for more than two decades with slow progress. Despite the fact that the porcine stem cells are under increasing glare of publicity due to milestone achievements in this area of research. Advances in stem cell technology, especially the genetic engineering, innovative cell culturing and induced pluripotency to generate stem cells has dramatically revolutionized the basic and applied investigations and applications of porcine stem cells. This review attempts to summarize the major signaling pathways involved in maintenance of pluripotency and the state of the art conceptual and technical progress for generating bona fide porcine PSCs.
Stem cells have huge potential for alleviating suffering for many diseases of humans for which effective therapy is currently not available. The PSCs are the unicellular equivalent to a whole animal derived from either pre-(em- bryonic stem cells or ESCs) or post-(epiblast stem cells or EpiSCs) implantation embryos [
In the late 1960s, Gordon’s pioneering work on nuclear transfer using amphibian cells demonstrated that differentiation was a reversible process [
While, mouse and human embryonic stem cells have been in the limelight for the last two decades, limited efforts have been on porcine embryonic stem cells despite reports of first isolation in early 1990s [
Recent advances in with reprogramming technology have provided ability to generate porcine iPSCs. Putative porcine iPS cells fulfilling several criteria of true pluripotency were reported after reprogramming fibroblasts with viral vectors [
The paucity of information related to morphology, pluripotency markers, and differentiation capabilities ham- pers a thorough evaluation of the validity of putative stem cell lines. Data from the literature suggests that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. It is envisaged that the understanding of signaling pathways and other subtle, but consequential diversities may endow with valuable information for isolating the genuine porcine PSCs.
The two most important and distinguishing traits that categorize PSCs, are pluripotency and self-renewal that allow the stem cells to divide continually in an undifferentiated manner. Both the traits orchestrate by a complex cascade of signaling pathways. However, literature indicates the involvement of some common signaling pathways among mice, primates and porcine stem cells, though the growth factors equilibrium may differ among different species [
It has already been established that mouse serum and the presence of the leukemia inhibitory factor (LIF: Interleukin 6 class cytokine) is required for maintenance of pluripotency in murine stem cells in vitro [
Modified form of murine stem cell culture conditions were effectively used to derive first monkey [
The LIF is not required for culturing and maintaining human ESCs [
In disparity to TGF-Beta/Activin/Nodal signaling, BMP signaling is incapable to sustain self-renewal and is linked with differentiation. In human PSCs, the BMP2 promotes extra-embryonic endoderm differentiation,
whereas the BMP4 induces differentiation into mesoderm and ectoderm. FGF2 (fibroblast growth factor-2) is proficient in maintaining human PSCs without serum and feeder cells via repression of BMP. FGF2 promotes self-renewal of Human PSCs by activating the PI3K pathway [
Wnts (Wingless-Type MMTV Integration Site Family Members) proteins are also supposed to play significant role in maintaining pluripotency. In Canonical Wnt signaling, Wnt ligands bind to Frizzled receptor, which in turn activates Dsh (Dishevelled) that leads to inactivation of GSK-3β (glycogen synthase kinase-3β). GSK-3β negatively regulates the degradation of β-catenin which accumulates in cell nucleus and forms a transcriptionally active complex with T-cell specific factors (TCF) that activates target genes [
However, human PSCs cannot be maintained during long-term culture by activation of the Wnt pathway solely, and requires other extrinsic signals [
Apart from that, Nanog (NK2-family homeobox transcription factor) is reported to be vital for self-renewal in human PSCs. In comparison to Oct3/4 and Sox2, Nanog expression is elevated in human stem cells and is down regulated as cells undergo differentiation. Oct3/4 and Sox2 bind to the Nanog promoter to regulate Nanog transcription. Human PSCs display various signaling pathways implicated in self-renewal and pluripotency that are mutually dependent and exhibit array of cross-talk mechanisms [
Stem cells pluripotency and self-renewal capacity in both mouse and human are regulated by conserved transcription factors. However, downstream regulators are apparently not well conserved as compared to conventional pluripotency factors Oct4/Sox 2 in both mouse and humans [
Particular cell signaling pathways have been revealed to direct pluripotency in mouse and primates. The existing observations in porcine induced pluripotency and maintenance in murine and human stem cells advocate that, similar regulatory pathways might be conserved among them. However, species-related differences in the mechanisms controlling pluripotency are apparent. The cells isolated from primate pre-implantation of blastocyst, presumably originating from naive epiblast, as it occurs in mouse, spontaneously progress to the primed epiblast in vitro before giving rise to stable cell lines that despite the fact that have been named ESCs, are actually EpiSCs. EpiSC are different form typical ES cells in various aspects as listed in
Pig epiblast has been shown to be dependent on activin/nodal signaling for self-renewal, as previously shown for human ESCs, indicating that maintenance of pluripotency by this signaling mechanism is conserved in mammals [
The presence of FGF receptor indicates the ability of porcine cells to bind bFGF and advocates a possible role of the FGF signaling pathway in self-renewal of porcine stem cells. Besides, it has been stated that bFGF can also bind and stimulate the PI3K ⁄AKT cascade [
Culture cell | Naive | Primed |
---|---|---|
Embryonic tissue of origin | Mouse pre-implantation ICM | Mouse post implantation epiblast, Primate pre-implantation ICM |
Cell line | Rodent ESC | hESC, rodent EpiSC |
Pluripotency in vitro | Yes | Yes |
Teratomas | Yes | Yes |
Colony morphology | Domed | Flattened |
Rapid self-renewal | Yes | No |
Slow self-renewal | No | Yes |
Response to LIF/BMP4 | Self-renewal | None |
Response to FGF2/ERK | differentiation | None |
Chimera formation | Yes | No |
Pluripotency markers | Oct4, Nanog, Sox2, Klf2, Klf4 | Oct4, Sox2 ,Nanog |
Single cell dissociation | Yes | No |
Clonigenecity | High | Low |
X-inactivation | No | Yes |
Response to 2i | Self-renewal | Cell death |
potent cells. Altogether these revelations suggested that Nanog might be able to maintain pig pluripotent cells in an undifferentiated state, and may thus serve a vital molecule for self-renewal in porcine [
Evaluation of crucial signaling pathways like WNT, NOTCH, TGFB1, VEGF and JAK-STAT in porcine, murine and human PSCs have indicated that the principal transcriptional network to uphold pluripotency and self-renewal in porcine had noteworthy resemblances to human but were diverse from that in murine. More- over, porcine PSCs were found to be positive for prime state markers of Otx2 and Fabp7, which is the characte- ristic feature of human ESC and murine EpiSCs. However, porcine PSCs were lacking expression of specific naïve state markers like KLF2/4/5 [
Apart from that the bunch of imprinted genes were muzzled in porcine PSCs as earlier witnessed in murine PSCs that have inadequate potential to contribute to chimaeras [
Isolation of putative PSCs have been demonstrated from porcine blastocyst [
Apart from porcine ES cells, Chen and co-workers demonstrated the generation of pESCs from intact, early hatched blastocysts and as well as isolated ICM (inner cell mass) of intermediate and late-hatched blastocysts. Furthermore, in vivo pluripotency of these cells was verified by birth of a chimeric piglet, as well as by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. However, only one live piglet was born from 131 embryos transferred, which was chimeric in different tissues assessed by microsatellite markers. Unfortunately germ line contribution was not observed [
Furthermore, pESCs have also been generated from epiblast of elongating embryos [
While till date there is no established bona fide pESC reported, multiple reports have shown derivation of piPSCs from a variety of somatic tissues as listed in
The piPSC generated from different tissue by using different approaches (
It has been shown that human PSCs in primed state can be transformed to the naive like state by using molecules like 2i, LIF and Forskolin [
Cell type | RF | Mode of delivery | Medium supplement | Transgene activation | Teratoma | Chimeras | References |
---|---|---|---|---|---|---|---|
Foetal fibroblasts | OSKM | Lentiviral vector | bFGF | Not tested | Yes | No | [ |
Embryonic fibroblasts | OSKM | Retroviral vectors | bFGF | Not tested | Yes | No | [ |
Mesenchymal stem cells | OSKMLN | Lentiviral vectors | bFGF | Not tested | Not tested | Yes, 29 offsprings | [ |
Adult fibroblast Bone marrow cells | OSKMLN | Lentivral vectors | Doxicillin inducible Transgene expression | Not tested | Yes | No | [ |
Adult fibroblasts | SKM | Retroviral vectors | bFGF, LIF | Not tested | Yes | No | [ |
Foetal fibroblasts | OSKM | Transposon mediated | bFGF | Not tested | Yes | No | [ |
Embryonic fibroblasts | OSKM | Non-viral | bFGF | Not tested | No | No | [ |
Embryonic fibroblasts | OSKM | Retroviral | Porcine LIF | Not tested | Yes | Yes | [ |
Abbreviations: RF: Reprograming factors; O: Oct4; S: Sox2; K: Klf4; M: c-Myc; L: Lin28; N: Nanog.
larly, in pig the Oct4 is present in the ICM, trophectoderm, and during hatching it is expressed in the epiblast component of the embryonic disc [
Like Oct-4, Nanog too shows marked differences in porcine species as compared to human or rodent systems. In porcine, Nanog expression is not only limited to the ICM and epiblast [
Porcine iPS cells derived from embryonic fibroblasts in serum containing medium morphologically resemble human ES cells with flatten, compact colonies that stain positively for AP, SSEA-4, Nanog and Rex1 [
The most challenging criterion of pluripotency was accomplished by West, and his colleagues, where 30 of 36 pigs produced were chimeric, displaying varying levels of chimerism. In this study, the forced expression of transcription factors enabled the derivation and establishment of iPSCs in pig that have displayed pluripotency [
The PSCs derived from embryos and iPSCs generated from embryonic fibroblasts in porcine model has been found to possess a primed pluripotent state analogous to that of mEpiSCs or hESCs, rather than to that of mESCs [
Studies have demonstrated that piPSCs generated from fibroblast using OSKM, displayed mES like morphology when they were maintained in LIF/STAT3-dependent manner and they exhibited potential of generating chimeric and reconstructed NT (Nuclear Transfer) embryos [
Kues et al., displayed the utility of porcine Oct4-EGFP cells and transposon mediated reprogramming for the derivation of porcine iPS cells. However, this study also indicated the limitation of culture conditions and culture media to support pluripotency [
These finding suggest that the endogenous pluripotent genes and their networks could not sustain pluripotency of putative porcine PSCs. Thus, establishment and optimization of culture conditions specific for porcine plu- ripotent stem cells is prerequisite for any further advancement in porcine stem cell research.
It is evident from earlier reports that culture conditions required to sustain pPSCs have not been well established. The stumbling block faced by laboratories worldwide has been that putative PSCs lose their pluripotency over a relatively short number of passages. Bona fide Porcine PSCs in which pluripotency maintained by the endogenous pluripotency factors have not yet been reported [
Over the past few decades, all endeavour’s to generate livestock PSCs have failed. Researchers have used mouse or human protocols as reference for culturing of ungulates PS cells, which include feeders, serum and supplementation with LIF [
Usually for the pPSC culturing, standard human stem cell medium [
Researchers also tried to culture ICM and epiblast in feeder layer and serum-free culture conditions. A study was based on modified TeSR medium and matrigel. Almost half of the primary cultures got differentiated within 3 - 4 passages. They used mechanical passaging for sub-culturing [
Vassiliev et al., cultured ICM cells in MEM medium supplemented with EGF (epidermal growth factor) and activin [
A very different culture regime i.e., 2i/LIF medium led to the first isolation and establishment of bona fide ESCs from rat embryos [
Haraguchi et al., and colleagues were able to generate presumptive porcine ES cell lines from the ICM of porcine embryos by using inhibitors of glycogen synthase kinase 3β and MAPK1. They were able to maintain the cell lines over 100 passages only [
We used the 2i (GSK 3β inhibitor, and MAPK inhibitor)/LIF culture regime (chemically defined feeder-free culture without supplementing exogenous growth factors) for deriving bovine PSCs [
Rudriguez et al., demonstrated that blocking MEK signaling increases the proportion of Nanog expressing cells in the ICM of porcine embryos [
Apart from mRNA and episomal vector approach to safely reprogram somatic cells, another approach is usage of concoction of small molecules that are allied with epigenetic modifiers and key signaling pathways listed in
Lee et al., demonstrated that Sirtuin 1 (SIRT1) (a member of the sirtuin family of NAD+-dependent protein deacetylases) a class III HDAC assists in generation of iPSCs from MEFs via miR -34a and p53 Pathways [
Korean scientists discovered a new molecular compound, RSC133 (inhibits histone deacetylase and DNA methyltransferase) that could enhance the reprogramming efficiency of human adult cell to iPSCs. It was inferred that compound derivative acts as the “booster of pluripotency”, and it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells [
Even in livestock, there are several reports that suggest that the reprogramming process is mired by faulty epigenetic modifications during the reprogramming process, resulting into low survival rates among clones [
Kim et al., have shown that the usage of decitabine (5-aza-2’-deoxycytydine) in ESC culture improves the number of colonies obtained [
Ever since the first report of the existence of miRNA [
Names | Mode of action | References |
---|---|---|
Forskolin | Stimulates adenylate cyclase activity and increases cAMP | [ |
Valproic acid | Histone deacetylase inhibitor | [ |
BIO (6-Bromoindirubin -3’ -oxime) | Inhibitor for GSK -3 a /β in the Wnt signaling pathway. | [ |
Lithium | GSK3 Inhibition (Wnt+) | [ |
CHIR99021 | GSK3 inhibitor | [ |
Trichostatin | A histone deacetylase inhibitor | [ |
Sodium Butyrate | Sodium butyrate is a known inhibitor of histone deacetylases and inhibits both the mRNA and protein content of cyclin D1 | [ |
Parnate | Histone demethylase inhibitor | [ |
5-azacytidine | DNA methyltransferase inhibitor | [ |
SC1 (Pluripotin) | Can act as a LIF replacement molecule for mouse embryonic (mES) cell self-renewal | [ |
RG108 | DNA methyltransferase inhibitor | [ |
PD0325901 | Selectively binds and inhibits MEK | [ |
SIRT1 | A class III HDAC assists in Generation of iPSCs | [ |
RSC133 | Inhibits histone deacetylase and DNA methyltransferase | [ |
RepSox | Tgfbr1 kinase inhibitor | [ |
Etoposide | Notch1 upregulation | [ |
Cytochalasin D | Wnt 5a upregulation | [ |
Vitamin C | Nutrient vital that lower reactiveoxygen species | [ |
Chaetocin | Histone Methyltransferase Inhibition | [ |
Pifithrin-α | Inhibitor of p53-dependent apoptosis. Moreover, reduced p53 activity augments reprogramming efficiency of human and mouse somatic cells | [ |
Pifithrin-μ | Inhibitor of p53 mitochondrial pathway by reducing its affinity for antiapoptotic proteins Bcl-2 and Bcl-XL but does not affect any additional transcriptional functions of p53 | [ |
DNMT1 | Inhibition promote fully reprogramming | [ |
HDACs | Inhibition increase the efficiency of reprogramming | [ |
G 9a | Increase the efficiency of reprogramming | [ |
modifiers, the miRNAs play a significant part in the reprogramming of terminally differentiated cells.
Several miRNAs have been shown to enhance iPSC reprogramming when expressed along with combinations of the OSKM factors. Introduction of miRNAs specific to ESCs improve the generation of iPSCs in mouse. The miRNAs miR-291-3p, miR-294 and miR-295 augment the reprogramming efficiency [
Oct-4 and Sox2 are the decisive factors requisite for iPSC reprogramming and the miR302/367 clusters has been revealed to be the direct target of these two key genes [
Despite non-stabilized defined state of pluripotency of pPSCs [
It is clear that pig and humans share far more anatomical, histological, biochemical and physiological properties than do the mice and humans. As a result, the pig has emerged as an excellent model for cardiovascular research and many other basic and applied biomedical applications.
The supply of human organs and tissues will be scarce to satisfy burgeoning demands of organ transplantation, making xenotransplantation a viable alternative. In this concern, pig is a promising effective translational model, and represents a candidate species for studying CVD, besides several other applications in regenerative medicine. Bona fide stem cells generated from porcine could be used in multiple applications such as creating models for human genetic diseases, engineering organs for transplantation therapies.
Understanding and unravelling the basic cellular and molecular mechanisms that regulate cell-mediated tissue regeneration is the key to harness this potential. Future research in the field could open novel prospects of research into degenerative conditions as pig is a more effective human proxy to work with. The research on porcine stem cells will lay a foundation for future studies of stem cell transplantation and regenerative medicine. In vitro culturing of porcine stem cell-derived cardiomyocytes could be transplanted into porcine MI model, which would give the researchers and clinicians valuable insights for cell therapy. There is an urgent need to develop validated reagents and cell culture methods to enhance use of porcine stem cells in regenerative medicine. To bestow important clinical insights for cell therapy or transplantation, necessary investigation is required in large animals.
The general ethical and logistical issues impede the use of humans or human-derived tissues for research and discovery pertaining to biomedical applications, which highlight the need for research model that closely mimic human anatomy, physiology, disease and injury processes. In view of significant differences between rodents and humans, the murine (rat and mice) confound or prohibit their use in translational studies.
The pig will continue to be ideal species to serve this purpose. The pigs conserve immunological and physio- logical attributes of human hearts apart from resemblance with the human heart. Hence, they can provide a potent research tool for pre-clinical study and particularly for transplantation medicine.
The Failure to identify or validate bona fide porcine/livestock PSCs is still a bewildering issue because of the several discrepancies in the presumed markers reported in various studies as listed in
Mode of delivery and characterization of porcine iPSCs | [ | [ | [ | [ | [ | [ | [ | [ | [ | |
---|---|---|---|---|---|---|---|---|---|---|
Delivery method | Viral | Viral | Viral | Viral | Plasmid | Viral | Viral | Viral | Viral | |
Pluripotency markers | Oct4 | ND | + | + | + | + | + | + | + | + |
Sox2 | + | + | + | + | + | + | + | + | + | |
Nanog | + | + | + | + | + | + | + | + | + | |
Rex1 | + | + | + | ND | + | ND | ND | ND | + | |
CDH1 | ND | + | + | ND | ND | ND | ND | ND | + | |
Surface markers | SSEA1 | ND | + | − | + | − | + | ND | − | − |
SSEA3 | ND | − | + | + | − | − | ND | ND | ND | |
SSEA4 | + | − | + | − | + | + | + | + | − | |
Tra-1-60 | ND | ± | + | + | + | ND | + | ND | ND | |
Tra-1-81 | ND | − | + | − | − | ND | + | ND | ND | |
Functional assays | Teratoma | + | + | + | + | ND | + | + | − | − |
EBs | ND | + | + | + | + | + | + | + | + | |
Chimera | ND | ND | ND | + | ND | ND | ND | ND | ND |
*Tet O gp; #Ef 1a and CAG gp; ND: Not Determined.
the globe. So far, generation of bona fide porcine PSCs has proven elusive, despite overwhelming knowledge and availability of numerous types of epigenetic modifiers, small molecules, specialized reagents and culture details from mouse and human stem cells work. Presumably, this information cannot directly extrapolate to other species as such. Building on the available knowledge, we can innovate on techniques and ideas very specific to porcine species.
The establishment of methods for generating porcine stem cells for clinical applications is still a work in pro- gress. Porcine pre and peri-implantation process as well as mechanism behind different cell fate specification is considerably dissimilar from mouse and human ones, that’s why presumptive porcine PS cells behave differently from existing human and mouse stem cell lines. Consequently, a comprehensive understanding of species specific developmental processes, signaling pathways and other related transcription factors would tender a roadmap for the generation of bona fide PSCs and their in vitro differentiation into cardiomyocytes. Until now none of the research group has documented successful differentiation of pPSCs into cardiomyocytes. To recapitulate, porcine PSCs will fill the vacuum between mouse and human pluripotent stem cells. However, noteworthy technical obstacles for the generation of bona fide porcine PS cells still persists that will only prevail over via ages of exhaustive research.
The authors indicate no potential conflicts of interest.
This work is supported by a grant from DST (SERB/LS-310/2013).