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 [14] . LIF binds to specific LIF receptor (LIFR-α) at cellular surface, which forms a heterodimer with GP130 signal transducing unit (Figure 1). This leads to the activation of the JAK/STAT ( Janus kinase/signal transducer and activator of transcription) and mitogen-activated protein kinase (MAPK) cascade. Once activated, the STAT3 is phosphorylated and subjected to dimerization, before being translocated to the nucleus where it acts as a transcription factor [15] . Withdrawal of LIF in culture medium pushes stem cells en route for differentiation, but they somehow maintain pluripotency. Hence, it is necessary to supplement the culture media with LIF to maintain murine stem cells in undifferentiated state. Genetically manipulated stem cells may remain undifferentiated in absence of LIF. For mouse ES cell pluripotency and self-renewal, STAT3 is indispensable [16] [17] , and the downstream target of STAT3 is c-Myc [18] . LIF can sustain self-renewal only in the presence of serum (cheap proxy for BMP4). It means that key factor controlling this process is BMP (Bone Morphogenic Protein, a polypeptide related to the TGF-β superfamily of proteins). BMP4 binds to two distinct types of serine-threonine kinase receptors known as BMPR1 and BMPR2. Signal transduction via these receptors takes place via Smad and MAP kinase pathways to upshot transcription of its target genes. The downstream effectors of BMP4 are well-characterized SMAD1, 5 and 8 proteins. The role of BMP4 has been usually expressed more as controlling cell differentiation rather than promoting self-renewal [19] [20] .

Modified form of murine stem cell culture conditions were effectively used to derive first monkey [21] and then human ESCs [22] . But so far, only a limited success has been achieved in other mammalian species. To understand the molecular mechanisms that contribute to pluripotency in vitro, mouse and primate PSCs are used as the main reference.

The LIF is not required for culturing and maintaining human ESCs [23] . However, LIF phosphorylates STAT3 in primate ES cells as in mouse ESCs [24] . In human PSCs, the TGF-β signaling pathway plays a key role in maintaining pluripotency and self-renewal (Figure 1). They signal via SMAD1/5 and activin/nodal branch which signal through Smad 2/3/4. SMAD 1/5 branch transduces via type I receptors [Anaplastic lymphoma kinase (ALK) also known as ALK tyrosine kinase receptor], ALK1, ALK2, ALK3 and ALK6 (ACVERL1, ACVER1, BMPRIA and BMPR1B). TGF-β/activin/nodal involves activation of SMAD2/3 via ALK4, ALK5 and ALK7 (TGFBR1 and ACVR 1C ). On activation, via phosphorylation and alliance with common SMAD4, the receptor-activated SMADs enters into the nucleus where they regulate transcription of downstream genes [25] .

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 [26] [27] .

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 [28] .

However, human PSCs cannot be maintained during long-term culture by activation of the Wnt pathway solely, and requires other extrinsic signals [29] .

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 [30] .

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 [31] . Substantial dissimilarities occur in the transcriptional networks and signaling pathways that regulates mouse and human PSC self-renewal and lineage development.

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 Table 1.

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 [32] . These authors demonstrated that pig EpiSCs express core pluripotency markers and maintain pluripotency via activin/nodal signaling pathway. Moreover, these cells could be induced to differentiate toward trophectoderm and to germ cell precursors in response to BMP4 [32] . Blomberg et al., 2008, demonstrated inconsistent expression of LIF receptors in porcine epiblast cells cultured for 24 hr after separating them from inner cell mass (ICM) of the embryo [33] . Hall et al., 2009, confirmed that LIFR and bFGF are not expressed in epiblast, but within the trophectoderm [34] . These findings revealed that cell signaling associated with maintaining pluripotency in human ESCs indicates the dispensability of LIF in supporting pluripotency in porcine species. However, Brevini et al., have shown that LIF is a key factor and it supports both attachment and self- renewal of stem cells [35] . They further hypothesized that LIF is unlikely to act through the gp130/LIFR/ STAT3 signaling pathway, but rather via an alternative cascade involving phosphoinositide-3 kinase (PI 3K), serine/threonine protein kinase (AKT) (a key effector in the PI3K pathway) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (a negative regulator of the same pathway), known to be responsive to LIF and has been previously shown to trigger the expression of NANOG and to facilitate efficient proliferation and survival of murine ESCs [36] . bFGF has been reported to be indispensable for proliferation, self-renewal and pluripotency of pig epiblast cells [37] .

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 [38] and there is a possibility that bFGF and LIF acts in tandem to exert its pluripotency-related effect through this pathway as well as through the canonical one. Particularly, the PI3K/AKT cascade is known to modulate several regulatory pathways and to exert a potent triggering effect on Nanog expression which, in contrast to OCT4, is persistently transcribed and steadily detectable in pig pluri-

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 [32] .

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 [39] .These finding indicates that porcine PS cells are more like prime state cells.

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 [39] . These significant dissimilarities in imprinting and naïve state gene expression advocates that so far recognized poricne PS cell lines could be more analogous to primed state cells. Hence, the porcine PS cells seem to have the less potential to give rise to chimeras and cloned offspring’s [39] .

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 [12] . In other words, the incessant expression of the exogenous transgenic transcription factors is still obligatory to sustain the pluripotent phenotype of porcine iPS- like cells [9] - [11] .

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 [45] [75] [76] or FGF2 [75] , epidermal growth factor and stem cell factor (SCF) [76] .

Usually for the pPSC culturing, standard human stem cell medium [69] or mouse and human standard culturing medium are mixed in 1:1 ratio [8] [77] because of the paucity of the information related to presence of receptors on pPSCs [35] demonstrated that porcine pluripotent cell lines do not express LIF receptors [13] . However, they do express FGFR-2 [13] . Apart from that their finding also suggests that LIF is not essential for the maintenance of pluripotency, but its presence inhibits the differentiation process. They also indicated that LIF do not act through LIF receptor signaling pathway.

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 [64] . However, mTeSR1 based on Ludwig et al., is most widely used feeder and serum free culture medium for hESCs and iPSCs [78] .

Vassiliev et al., cultured ICM cells in MEM medium supplemented with EGF (epidermal growth factor) and activin [51] . They were able to generate chimeras by using putative porcine ES cells. But it was not vivid from their study whether they were able to keep the putative pESCs in undifferentiated self-renewal state beyond 14 passages [51] . Various type of culture conditions were used to culture ICM/epiblast primary out growths [26] . But, none of the culture conditions supported to keep the putative porcine ES cells in undifferentiated state after certain passages.

A very different culture regime i.e., 2i/LIF medium led to the first isolation and establishment of bona fide ESCs from rat embryos [53] [54] , and noncompliant mouse strains [79] . Human embryo-derived stem cells cannot be maintained in 2i/LIF alone [80] . However, it facilitated to establish mouse ESC-like cells in presence of forskolin [55] . Consequently, human cells with an epigenetic and transcriptional profile analogous to naïve ESC lines could be established.

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 [52] .

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 [46] . Under these minimal conditions, Verma et al., investigated the subsistence of a pluripotent ground state, stabilized through shielding from FGF/MAPK signaling and Wnt activation, in bovine ICM-derived cell cultures. The GSK 3β facilitate Wnt 3a signaling pathway, and the deficiency of the Wnt signal roots GSK3β to inactivate targets, such as β-catenin and c-Myc. CHIR99021, a GSK3β inhibitor, effects Wnt signaling activation [81] [82] . These findings were in conjugation with several previous reports, where they have shown that CHIR99021 augments the reprogramming efficiency [82] .

Rudriguez et al., demonstrated that blocking MEK signaling increases the proportion of Nanog expressing cells in the ICM of porcine embryos [83] . From their findings, it is apparent that FGF signaling participates in the formation of the founders of the ICM. PDO325901 (MEK pathway inhibitor), CHIR (GSK3β inhibitior) and LIF supplementation were used to modulate pluripotency in piPSCs cells to acquire features of naive/ground pluripotency. It was demonstrated that small molecule inhibitors can be used to increase the homogeneity of iPSC cultures [83] . Oxygen tension is also an imperative trait for PSC’s maintenance and differentiation. The iPSCs induction executed in hypoxic conditions (5% O₂) demonstrated augmentation of the reprogramming efficiency [84] .

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 Table 3. Small molecules that alter chromatin and gene expression enhance the reprogramming efficiency in iPSC generation. Inhibitors of histone deacetylases (HDACs), histone demethylases (HDMs), and histone methyltransferases (HMTs), which regulate chromatin remodeling, have been recognized as small molecules for terminally differentiated cells reprogramming. For instance, Huangfu et al., used chromatin modification inhibitors, the 5’ -azacytidine (5-aza) (DNA methyltransferase inhibitor), dexamethazone (synthetic glucocorticoid) and valproic acid (VPA, a histone deacetylase inhibitor) to investigate their effects on reprogramming efficiency [85] . It was inferred that 5-aza, dexamethazone and VPA enhanced the efficiency of cellular reprogramming by 10-fold, 2.6-fold and >100-fold respectively. The effect of VPA was found to be much stronger than that of 5’ -azacytidine and other HDAC inhibitors tested [85] . Other noticeable epigenetic modifiers which are used during the reprogramming are, parnate, a histone demethylase inhibitor [86] , 5-aza and RG108, a DNA methyltransferase inhibitors [87] . Butyrate (a HDAC inhibitor) also improves iPSC generation by augumenting acetylation of histone H3 and demethylation of genes promoter, related to of pluripotency [88] [89] .

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 [90] . It was hypothesized that SIRT1 works as a positive epigenetic regulator in reprogramming somatic cells.

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 [91] .

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 [92] and inadequacy of iPS reprogramming. Histone deacetylation is considered to be an unwanted event in nuclear reprogramming assays. Trichostatin A (TSA) has been used to improve cloning efficiency by as much as 75% in various species [92] .

Kim et al., have shown that the usage of decitabine (5-aza-2’-deoxycytydine) in ESC culture improves the number of colonies obtained [93] . Treatment of early pre-implantation murine and bovine embryos with 5-AzaC exhibited a simple and efficient method for isolation of putative bovine PS cells [94] . However, there are still safety concerns regarding the usage of epigenetic modifiers or cocktail of small molecules as they could activate cell growth pathway or inhibit tumor suppressor pathways.

Ever since the first report of the existence of miRNA [95] , approximately thousand types of microRNAs (miRNAs) documented till date, small RNAs have been identified in humans. The typical feature of stem cells is self-re- newal and abnormal pluripotency was reported in ES cells lacking DGCR8 or Dicer [96] [97] which implies their vital function in the biosynthetic pathway of miRNA. Moreover, it’s been showed that miRNA have reduced expression in differentiated ES cells. In addition reprogramming factors and chemicals as epigenetic

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 [98] . The miRNAs did not improve reprogramming efficiency in the presence of c-Myc transgene; it suggests that they could be downstream target of c-Myc. These miRNA seems to have the ability to control cell cycle, to effect the augmentation of iPSC reprogramming [98] .

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 [99] . Morrisey’s group demonstrated that expression of the miR302/367 cluster reprograms mouse and human somatic cells to pluripotent state very efficiently, in the absence of exogenous transcription factors or Yamanaka factors [100] . This miRNA-based reprogramming regime was found to be more efficient than conventional OSKM-mediated methods. Moreover, mouse and human miR302/367 iPSCs demonstrated features including pluripotency marker expression, teratoma formation and for mouse cells, chimera contribution and germline contribution was found to be analogous to Oct4/Sox2/Klf4/c- Myc iPSCs [100] . It was also shown that miR367 expression is requisite for miR302/367-mediated reprogramming and stimulates expression of Oct4 gene, and that suppression/degradation of Hdac2 by using Valproic acid is also vital [100] . As far as usage of miRNA for reprogramming is concerned, there is no authentic report available.

Despite non-stabilized defined state of pluripotency of pPSCs [74] , in vitro differentiation of presumptive porcine iPSC into rod photoreceptors and their integration into the retinas of recipient pigs has been reported [101] . The impending therapeutic benefits of stem cell-based treatment to recover the cardiac function have been customary in small animal models of myocardial infarction (MI) [102] - [106] . Human PSCs transplanted to treat immune- deficient porcine or murine models of acute myocardial infarction (AMI) [107] , but intra-myocardial injection of livestock stem cells, particularly porcine PS cells in a pig model of AMI has not yet been gauged. Animal models that imitate human cardiac disease, such as MI, AMI and ischemia-reperfusion (IR) that induces heart failure are useful models to study cardiovascular disease (CVD). Recently, a group of researchers examined the possibility of using porcine PS cells to prevent LV dysfunction and post in infarction left ventricular remodelling in hearts suffering from AMI. They demonstrated that porcine PS cell could be an effective treatment for myocardial infarction (MI). In this study, engrafted porcine PS cells showed the differentiation potential by differentiating into vessel cells, which further augmented the formation of new vessels in infarcted heart [108] . However, no observe myocyte generation was observed. piPSCs may have the prospective to be used in ischemic cardiac diseases. This study had some serious limitations and further research is required to translate cell therapy form bench to bed side. However, a porcine acute myocardial infarction (AMI) model can fill the space between treatment for small animals and treatment for humans.