Reprogramming In Primordial Germ Cells Research Articles

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells

BackgroundGenome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs.ResultsWe show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine.ConclusionsThe results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells.

Introduction: Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo-embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs). Methods: We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia. Results: In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type. Discussion: This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development.

The paternal environment has been linked to infertility and negative outcomes. Such effects may be transmitted via sperm through histone modifications. To date, in-depth profiling of the sperm chromatin in men has been limited. Here, we use deep sequencing to characterize the sperm profiles of histone H3 lysine 4 tri-methylation (H3K4me3) and DNA methylation in a representative reference population of 37 men. Our analysis reveals that H3K4me3 is localized throughout the genome and at genes for fertility and development. Remarkably, enrichment is also found at regions that escape epigenetic reprogramming in primordial germ cells, embryonic enhancers, and short-interspersed nuclear elements (SINEs). There is significant overlap in H3K4me3 and DNA methylation throughout the genome, suggesting a potential interplay between these marks previously reported to be mutually exclusive in sperm. Comparisons made between H3K4me3 marked regions in sperm and the embryonic transcriptome suggest an influence of paternal chromatin on embryonic gene expression.

Reconstitution of the oocyte transcriptional network with transcription factors.

During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.

The dynamics of DNA methylation during epigenetic reprogramming of primordial germ cells in medaka (Oryzias latipes)

ABSTRACTPrimordial germ cells (PGCs) are precursors of eggs and sperm. How the PGCs epigenetically reprogram during early embryonic development in fish is currently unknown. Here we generated a series of PGC methylomes using whole genome bisulfite sequencing across key stages from 8 days post fertilization (dpf) to 25 dpf coinciding with germ cell sex determination and gonadal sex differentiation in medaka (Oryzias latipes) to elucidate the dynamics of DNA methylation during epigenetic reprogramming in germ cells. Our high-resolution DNA methylome maps show a global demethylation taking place in medaka PGCs in a two-step strategy. The first step occurs between the blastula and 8-dpf stages, and the second step occurs between the 10-dpf and 12-dpf stages. Both demethylation processes are global, except for CGI promoters which remain hypomethylated throughout the stage of PGC specification. De novo methylation proceeded at 25-dpf stage with the process in male germ cells superseding female germ cells. Gene expression analysis showed that tet2 maintains high levels of expression during the demethylation stage, while dnmt3ba expression increases during the de novo methylation stage during sexual fate determination in germ cells. The present results suggest that medaka PGCs undergo a bi-phasic epigenetic reprogramming process. Global erasure of DNA methylation marks peaks at 15-dpf and de novo methylation in male germ cells takes precedence over female germ cells at 25 dpf. Results also provide important insights into the developmental window of susceptibility to environmental stressors for multi- and trans-generational health outcomes in fish.

Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming.

Primordial germ cells (PGCs) are reprogrammed into pluripotent embryonic germ cells (EGCs) under specific culture conditions, but the detailed mechanisms of PGC reprogramming have not yet been fully clarified. Previous studies have demonstrated that AKT, an important intracellular signaling molecule, promotes reprogramming of PGCs into EGCs. Because AKT likely inhibits p53 functions to enhance PGC reprogramming, and p53 negatively regulates cell cycle progression, we analyzed cell cycle changes in PGCs following AKT activation and found that the ratio of PGCs in the G1/G0 phase was decreased while that of PGCs in the G2/M phase was increased after AKT activation. We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1‐S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation. The results suggested that the characteristic cell cycle changes of PGCs by AKT activation are, at least in part, due to decreased expression of p27kip1 . We also investigated changes in histone H3K27 tri-methylation (H3K27me3) by AKT activation in PGCs, because we previously found that decreased H3K27me3 was involved in PGC reprogramming via upregulation of cyclin D1. We observed that AKT activation in PGCs resulted in H3K27 hypomethylation. In addition, DZNeP, an inhibitor of the H3K27 trimethyl transferase Ezh2, stimulated EGC formation. These results together suggested that AKT activation promotes G1-S transition and downregulates H3K27me3 to enhance PGC reprogramming.

Identification of KLF9 and BCL3 as transcription factors that enhance reprogramming of primordial germ cells.

Primordial germ cells (PGCs) are precursors of eggs and sperm. Although PGCs are unipotent cells in vivo, they are reprogrammed into pluripotent stem cells (PSCs), also known as embryonic germ cells (EGCs), in the presence of leukemia inhibitory factor and basic fibroblast growth factor (bFGF) in vitro. However, the molecular mechanisms responsible for their reprogramming are not fully understood. Here we show identification of transcription factors that mediate PGC reprogramming. We selected genes encoding transcription factors or epigenetic regulatory factors whose expression was significantly different between PGCs and PSCs with in silico analysis and RT-qPCR. Among the candidate genes, over-expression (OE) of Bcl3 or Klf9 significantly enhanced PGC reprogramming. Notably, EGC formation was stimulated by Klf9-OE even without bFGF. G-protein-coupled receptor signaling-related pathways, which are involved in PGC reprogramming, were enriched among genes down-regulated by Klf9-OE, and forskolin which activate adenylate cyclase, rescued repressed EGC formation by knock-down of Klf9, suggesting a molecular linkage between KLF9 and such signaling.

Intrauterine hyperglycemia exposure results in intergenerational inheritance via DNA methylation reprogramming on F1 PGCs

BackgroundThe existing reports about intergenerational or transgenerational effects of intrauterine hyperglycemia have included both intrauterine and postnatal metabolic exposure factors, while the impact of intrauterine hyperglycemia per se has not been assessed alone. A number of studies suggest DNA methylation reprogramming of gametes plays a crucial role in the metabolic inheritance, but it is unclear when and how DNA methylation patterns are altered when exposed to intrauterine hyperglycemia. In this study, we selected nondiabetic F1- and F2-gestational diabetes mellitus (GDM) male mice as founders to examine metabolic changes in the next generation and performed methylome sequencing of day 13.5 primordial germ cells (PGCs) from F1-GDM to explore the underlying epigenetic mechanism.ResultsWe found that intrauterine hyperglycemia exposure resulted in obesity, insulin resistance, and/or glucose intolerance in F2 male mice, but no metabolic changes in F3 male mice at 8 weeks. Using reduced representation bisulfite sequencing, we found DNA methylome of day 13.5 PGCs from F1-GDM fetuses revealed differently methylated genes enriched in obesity and diabetes. Methylation validation of the insulin resistance and fat accumulation gene Fyn showed a consistent hypomethylation status in F1 PGCs, F1 fetal testes, sperm from F1/C-GDM mice, and somatic cells from F2-GDM male mice. In contrast, no methylation alteration was observed in F2-GDM male germ cells and F3-GDM somatic cells.ConclusionWe provide evidence that intrauterine hyperglycemia exposure per se contributes to intergenerational metabolic changes in the F2 but not F3 generation. And the aberrant DNA methylation reprogramming occurs as early as day 13.5 in PGCs of the F1 generation. Our findings suggest that intrauterine exposure alone is sufficient to cause the epigenetic inheritance in F2 offspring, and the epigenetic memory carried by DNA methylation pattern could be erased by the second wave of methylation reprogramming in F2 PGCs during fetal development.

Reprogramming: Methylation patterns in primordial germ cells.

This study describes the molecular mechanisms underlying epigenetic reprogramming in primordial germ cells.

Dnd1-mediated epigenetic control of teratoma formation in mouse.

ABSTRACTSpontaneous testicular teratoma develops from primordial germ cells (PGCs) in embryos; however, the molecular mechanisms underlying teratoma formation are not fully understood. Mutation of the dead-end 1 (Dnd1) gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of Dnd1 mutation-induced teratoma formation, we focused on histone H3 lysine 27 (H3K27) trimethylation (me3), and found that the levels of H3K27me3 and its responsible methyltransferase, enhancer of zeste homolog 2 (Ezh2), were decreased in the teratoma-forming cells of Dnd1 mutant embryos. We also showed that Dnd1 suppressed miR-26a-mediated inhibition of Ezh2 expression, and that Dnd1 deficiency resulted in decreased H3K27me3 of a cell-cycle regulator gene, Ccnd1. In addition, Ezh2 expression or Ccnd1 deficiency repressed the reprogramming of PGCs into pluripotent stem cells, which mimicked the conversion of embryonic germ cells into teratoma-forming cells. These results revealed an epigenetic molecular linkage between Dnd1 and the suppression of testicular teratoma formation.

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells

Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.

Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation.

SummaryThe establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.

Metabolic Reprogramming, Autophagy, and Reactive Oxygen Species Are Necessary for Primordial Germ Cell Reprogramming into Pluripotency.

Cellular reprogramming is accompanied by a metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Previous results from our laboratory showed that hypoxia alone is able to reprogram primordial germ cells (PGCs) into pluripotency and that this action is mediated by hypoxia-inducible factor 1 (HIF1). As HIF1 exerts a myriad of actions by upregulating several hundred genes, to ascertain whether the metabolic switch toward glycolysis is solely responsible for reprogramming, PGCs were cultured in the presence of a pyruvate kinase M2 isoform (PKM2) activator, or glycolysis was promoted by manipulating PPARγ. Conversely, OXPHOS was stimulated by inhibiting PDK1 activity in normoxic or in hypoxic conditions. Inhibition or promotion of autophagy and reactive oxygen species (ROS) production was performed to ascertain their role in cell reprogramming. Our results show that a metabolic shift toward glycolysis, autophagy, and mitochondrial inactivation and an early rise in ROS levels are necessary for PGC reprogramming. All of these processes are governed by HIF1/HIF2 balance and strict intermediate Oct4 levels. Histone acetylation plays a role in reprogramming and is observed under all reprogramming conditions. The pluripotent cells thus generated were unable to self-renew, probably due to insufficient Blimp1 downregulation and a lack of Klf4 and cMyc expression.

Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells

Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function.

BFGF signaling-mediated reprogramming of porcine primordial germ cells.

Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming.

Similarity of epigenetic reprogramming in primordial germ cells between human and mouse

Germ cells have a unique ability to produce a new individual and are paramount for transmitting intact genome and appropriate epigenome from generation to generation. Primordial germ cells (PGCs), the first germ cell population established in the embryo, undergo sequential epigenetic reprogramming to ensure the fidelity of this transmission. Although the developmental and epigenetic processes in mouse PGCs are well understood, antithetically our knowledge in human PGCs remains elusive. Recently, three research teams coincidently discovered the transcriptome and DNA methylome of human PGCs by single-cell RNA-sequencing and whole-genome bisulfite sequencing, providing a fundamental framework for understanding human germline epigenetic reprogramming during embryonic development and its biological outcome [1–3]. One of the teams, led by Fuchou Tang and Jie Qiao at Peking University in China, analyzed the transcriptome and DNAmethylome of PGCs from 4to 19week-old human embryos and provided four key insights [2]. First, the transcriptomes of early human PGCs were generally stable between 4 and 11 weeks of development. Only after 17 weeks the heterogeneity of gene expressionoccured in meiotic female PGCs, indicating entry into meiosis asynchronously. However, the inactivated X chromosome was reactivated in human PGCs as early as 4 weeks. Second, global DNA demethylation in human PGCs was much more thorough than that of preimplantation embryos, and happened in gene body regions, the surrounding intergenic regions, functional genomic elements and CpG islands. However, the evolutionarily younger and more active transposable elements evaded global DNA demethylaion in human PGCs and retained high levels of residual DNA methylation, implying potential role for transgenerational epigenetic inheritance as the other group suggested [3].Third, extensive erasure of the methylation of imprinted genes occurred in early humanPGCs and wasmaintaineduntil 19weeks.Lastly, the study showed that there was strong enrichment of the base-excision mismatch repair (BER) pathway in human PGCs, denoting the possibility of global DNA demethylation being contributed by the BER pathway. A comparison of the methylomes between human and mouse PGCs reveals high conservation of epigenetic reprograming, with overall similar genomewide DNA demethylation dynamics in human migrating and gonadal PGCs and those of mouse PGCs at comparable stages.Mechanistically, BER pathway enrichment has been also observed at similar stages of PGCs in mouse [4,5], implying human and mouse may share same BER-involved DNA demethylation mechanisms. With the detailed map of transcriptome and DNA methylome, the next challenge will be to elucidate the underlying mechanisms behind epigenetic regulation during human PGC development. It remains to be clarified how global DNA demethylation does not result in deregulation gene transcription in PGCs. The functional link between regions resistant to DNA demethylation and transgenerational epigenetic inheritanceneeds tobe further defined in future work.More specifically,what is themechanism for driving escape from global erasure of DNA methylation during human PGCdevelopment?As humanPGCsamples remain scarce, achieving a mechanistic understanding of epigenetic regulation in human PGCs will be difficult. However, given the similarity between DNA methylation dynamics in mouse and human PGCs, mouse PGCs should provide a valuable model for uncovering themechanismsof epigenetic reprogramming in human PGCs.

Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Generation of Rat Embryonic Germ Cells via Inhibition of TGFß and MEK Pathways.

Embryonic germ (EG) cells are the results of reprogramming primordial germ cells (PGC) in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-β (TGFβ) and mitogen-activated protein kinase kinase (MEK) signaling pathways. In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor (SCF) as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through G-banding technique. Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 (GSK3β) and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3β molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFβ signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFβ instead of GSK3β significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities. Rat EG cells with higher karyotype stability establish from PGCs by inhibiting TGFβ and MEK signaling pathways.

Mechanisms of pluripotency and epigenetic reprogramming in primordial germ cells: lessons for the conversion of other cell types into the stem cell lineage

Primordial germ cells (PGCs) provide an excellent tool to better understand ancestor?descendent relationships as well as the efficiency and molecular mechanisms governing pluripotency in the reprogramming of somatic cells, since the latter type of cells have a relatively lower efficiency of conversion to pluripotent cells. This kind of comparison has gained credence from the commonalities regarding the expression of key transcription factors such as octamer-binding transcriptionhttp://en.wikipedia.org/wiki/Transcription_factor factor-4 (Oct3/4), SRY-related HMG box (Sox2), myelocytomatosis (c-Myc), and Nanog, as well as redundancy in terms of Kruppel-like factor 2 (Klf2), Kruppel-like factor 5 (Klf5), estrogen-related receptor beta (Esrrb), and estrogen-related receptor gamma (Esrrg) compensating for the absence of Kruppel-like factor 4 (Klf4). However, the exogenous addition of any one of these factors was found to be important, thereby implying that the expression level is important. L-Myelocytomatosis (L-myc) was shown to improve reprogramming efficiency without affecting tumorigenic potential. Molecular aspects of epigenetic reprogramming during the acquisition of pluripotency, as well as tumorigenic potential, have also been discussed, thus providing an understanding of the factors that can improve the former without increasing the possibility of neoplastic transformation. An improved understanding of the molecular events would pave the way for the development and use of endogenous biomolecules as well as currently available chemical reprogrammers for improving the efficiency of conversion of PGCs into cells of the stem cell lineage. Such chemicals, when adequately tested, can possibly be an alternative to viral vectors, since the introduced transgenes can become oncogenic.

Primordial germ cell specification: a context-dependent cellular differentiation event [corrected

During embryonic development, the foundation of the germline is laid by the specification of primordial germ cells (PGCs) from the postimplantation epiblast via bone morphogenetic protein (BMP) and WNT signalling. While the majority of epiblast cells undergo differentiation towards somatic cell lineages, PGCs initiate a unique cellular programme driven by the cooperation of the transcription factors BLIMP1, PRDM14 and AP2γ. These factors synergistically suppress the ongoing somatic differentiation and drive the re-expression of pluripotency and germ cell-specific genes accompanied by global epigenetic changes. However, an unresolved question is how postimplantation epiblast cells acquire the developmental competence for the PGC fate downstream of BMP/WNT signalling. One emerging concept is that transcriptional enhancers might play a central role in the establishment of developmental competence and the execution of cell fate determination. Here, we discuss recent advances on the specification and reprogramming of PGCs thereby highlighting the concept of enhancer function.