Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure.
Embryonic stem cells; Pluripotency; Self-renewal; Differentiation; Chromatin; Histone modifications; Histone variants; Chromatin remodeling enzymes
Embryonic stem (ES) and induced pluripotent stem (iPS) cells self-renew and are pluripotent. Differentiation of these cells can yield over 200 somatic cell types, making pluripotent cells an obvious source for regenerative medicine. Before the potential of these cells can be maximally harnessed for clinical applications, it will be necessary to understand the processes that maintain pluripotentiality and signal differentiation. Currently, three unique molecular properties distinguish pluripotent stem cells from somatic cells. These include a unique transcriptional hierarchy that sustains the pluripotent state during the process of self-renewal; a poised epigenetic state that maintains chromatin in a form ready for rapid cell fate decisions; and a cell cycle characterized by an extremely short gap 1 (G1) phase and the near absence of normal somatic cell checkpoint controls. Recently, B-MYB (MYBL2) was implicated in the gene regulation of two pluripotency factors and normal cell cycle progression. In this article, the three pluripotency properties and the potential role of B-Myb to regulate these processes will be discussed.
Pluripotency; Stem cells; Transcription Factors; Epigenetics; Cell Cycle; B-Myb
Pluripotency is maintained by a complex system that includes the genetic and
epigenetic levels. Recent studies have shown that the genetic level
(transcription factors, signal pathways, and microRNAs) closely interacts with
the enzymes and other specific proteins that participate in the formation of the
chromatin structure. The interaction between the two systems results in the
unique chromatin state observed in pluripotent cells. In this review, the
epigenetic features of embryonic stem cells and induced pluripotent stem cells
are considered. Special attention is paid to the interplay of the transcription
factors OCT4, SOX2, and NANOG with the Polycomb group proteins and other
molecules involved in the regulation of the chromatin structure. The
participation of the transcription factors of the pluripotency system in the
inactivation of the X chromosome is discussed. In addition, the epigenetic
events taking place during reprogramming of somatic cells to the pluripotent
state and the problem of “epigenetic memory” are considered.
embryonic stem cells; induced pluripotent stem cells; pluripotency; covalent histone modifications; DNA methylation
Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction.
Pluripotency; Embryonic stem cell; Induced pluripotent stem cell; Somatic cell nuclear transfer; Cell fusion hybrid
Somatic cell nuclear transfer and transcription factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. These two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, by different mechanisms and kinetics, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low passage induced pluripotent stem cells (iPSC) derived by factor-based reprogramming harbor residual DNA methylation signatures characteristic of their somatic tissue of origin, which favors their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an “epigenetic memory” of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSC with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSC, consistent with more effective reprogramming. Our data demonstrate that factor-based reprogramming can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modeling or treatment.
Reprogramming somatic cells to pluripotency, especially by the induced pluripotent stem cell (iPSC) technology, has become widely used today to generate various types of stem cells for research and for regenerative medicine. However the mechanism(s) of reprogramming still need detailed elucidation, including the roles played by the leukemia inhibitory factor (LIF) signaling pathway. LIF is central in maintaining the ground state pluripotency of mouse embryonic stem cells (ESCs) and iPSCs by activating the Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) pathway. Characterizing and understanding this pathway holds the key to generate naïve pluripotent human iPSCs which will facilitate the development of patient-specific stem cell therapy. Here we review the historical and recent developments on how LIF signaling pathway regulates ESC pluripotency maintenance and somatic cell reprogramming, with a focus on JAK-STAT3.
JAK; STAT3; LIF; embryonic stem cells; reprogramming; iPSC; epigenetics
Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.
Pluripotent Human Embryonic Stem Cells (hESCs) have the unconstrained capacity for long-term stable undifferentiated growth in culture and unrestricted developmental capacity. Packaging of the eukaryotic genome into chromatin confers higher order structural control over maintaining stem cell plasticity and directing differentiation. We recently reported the establishment of a defined culture system for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards functional lineages. To unveil the epigenetic mechanism in maintaining the epiblast pluripotence of hESCs, in this study, the global chromatin dynamics in the pluripotent hESCs maintained under the defined culture were examined. This study shows that the genomic plasticity of pluripotent hESCs is enabled by an acetylated globally active chromatin maintained by Oct-4. The pluripotency of hESCs that display normal stable expansion is associated with high levels of expression and nuclear localization of active chromatin remodeling factors that include acetylated histone H3 and H4, Brg-1, hSNF2H, HAT p300, and HDAC1; weak expression or cytoplasmic localization of repressive chromatin remodeling factors that are implicated in transcriptional silencing; and residual H3 K9 methylation. A dynamic progression from acetylated to transient hyperacetylated to hypoacetylated chromatin states correlates with loss-of-Oct4-associated hESC differentiation. RNA interference directed against Oct-4 and HDAC inhibitor analysis support this pivotal link between chromatin dynamics and hESC differentiation. These findings reveal an epigenetic mechanism for placing global chromatin dynamics as central to tracking the normal pluripotency and lineage progression of hESCs.
Human embryonic stem cells; Pluripotency; Epigenetic; Chromatin; Histone modification; Histone acetylation; Histone deacetylation; Histone methylation; Histone acetyltransferase; Histone deacetylase; Histone methytransferase; Chromatin remodeling; Oct-4; Differentiation; Defined culture system
Open chromatin is a hallmark of pluripotent stem cells, but the underlying molecular mechanisms are only beginning to be unraveled. In this review we highlight recent studies that employ embryonic stem cells and induced pluripotent stem cells to investigate the regulation of open chromatin and its role in the maintenance and acquisition of pluripotency in vitro. We suggest that findings from in vitro studies using pluripotent stem cells are predictive of in vivo processes of epigenetic regulation of pluripotency, specifically in the development of the zygote and primordial germ cells. The combination of in vitro and in vivo approaches is expected to provide a comprehensive understanding of the epigenetic regulation of pluripotency and reprogramming.
Embryonic stem (ES) cells are pluripotent cells that can self renew indefinitely or be induced to differentiate into multiple cell lineages, and thus have the potential to be used in regenerative medicine. Pluripotency transcription factors (TFs), such as Oct4, Sox2, and Nanog, function in a regulatory circuit that silences the expression of key TFs required for differentiation and activates the expression of genes important for maintenance of pluripotency. In addition, proteins that remodel chromatin structure also play important roles in determining the ES cell-specific gene expression pattern. Here we review recent studies demonstrating the roles of enzymes that carry out one facet of chromatin regulation, nucleosome remodeling, in control of ES cell self-renewal and differentiation.
The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting.
lineage reprogramming; cell plasticity; cell replacement therapy; disease modeling
The application of stem cells to regenerative medicine depends on a thorough understanding of the molecular mechanisms underlying their pluripotency. Many studies have identified key transcription factor-regulated transcriptional networks and chromatin landscapes of embryonic and a number of adult stem cells. In addition, recent publications have revealed another interesting molecular feature of stem cells— a distinct alternative splicing pattern. Thus, it is possible that both the identity and activity of stem cells are maintained by stem cell-specific mRNA isoforms, while switching to different isoforms ensures proper differentiation. In this review, we will discuss the generality of mRNA isoform switching and its interaction with other molecular mechanisms to regulate stem cell pluripotency, as well as the reprogramming process in which differentiated cells are induced to become pluripotent stem cell-like cells (iPSCs).
alternative splicing; embryonic stem cells; adult stem cells; stem cell maintenance and differentiation; post-transcriptional regulation; epigenetic regulation
An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.
Bioengineered by ectopic expression of stemness factors, induced pluripotent stem (iPS) cells demonstrate embryonic stem cell-like properties and offer a unique platform for derivation of autologous pluripotent cells from somatic tissue sources. In the process of nuclear reprogramming, somatic tissues are converted to a pluripotent ground state, thus unlocking an unlimited potential to expand progenitor pools. Molecular dissection of nuclear reprogramming suggests that a residual memory derived from the original parental source, along with the remnants of the reprogramming process itself, leads to a biased potential of the bioengineered progeny to differentiate into target tissues such as cardiac cytotypes. In this way, iPS cells that fulfill pluripotency criteria may display heterogeneous profiles for lineage specification. Small molecule-based strategies have been identified that modulate the epigenetic state of reprogrammed cells and are optimized to erase the residual memory and homogenize the differentiation potential of iPS cells derived from distinct backgrounds. Here, we describe the salient components of the reprogramming process and their effect on the downstream differentiation capacity of the iPS populations in the context of cardiovascular regenerative applications.
iPS; Epigenetic memory; Differentiation capacity; Memory-free pluripotency
Human pluripotent cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) and their in vitro differentiation models hold great promise for regenerative medicine as they provide both a model for investigating mechanisms underlying human development and disease and a potential source of replacement cells in cellular transplantation approaches. The remarkable developmental plasticity of pluripotent cells is reflected in their unique chromatin marking and organization patterns, or epigenomes. Pluripotent cell epigenomes must organize genetic information in a way that is compatible with both the maintenance of self-renewal programs and the retention of multilineage differentiation potential. In this review, we give a brief overview of the recent technological advances in genomics that are allowing scientists to characterize and compare epigenomes of different cell types at an unprecedented scale and resolution. We then discuss how utilizing these technologies for studies of hESCs has demonstrated that certain chromatin features, including bivalent promoters, poised enhancers, and unique DNA modification patterns, are particularly pervasive in hESCs compared with differentiated cell types. We outline these unique characteristics and discuss the extent to which they are recapitulated in iPSCs. Finally, we envision broad applications of epigenomics in characterizing the quality and differentiation potential of individual pluripotent lines, and we discuss how epigenomic profiling of regulatory elements in hESCs, iPSCs and their derivatives can improve our understanding of complex human diseases and their underlying genetic variants.
Pluripotency is a transient cellular state during early development which can be recreated in vitro by direct reprogramming. The molecular mechanisms driving entry into and exit from the pluripotent state are the subject of intense research interest. Here, we review the role of the homeodomain-containing transcription factor Nanog in mammalian embryology and induced pluripotency. Nanog was originally thought to be confined to the maintenance of pluripotency, but recent insights from genetic studies uncovered a new biological function. Embryonic stem cells deficient in Nanog alleles are more prone to differentiate but do not lose pluripotency per se. Instead, Nanog is transiently required for the specification of the naive pluripotent epiblast and development of primordial germ cells. Nanog is also essential to finalize somatic cell reprogramming during induction of pluripotency. We propose that this unique transcription factor acts as a molecular switch to turn on the naive pluripotent programme in mammalian cells. In this context, the capacity of Nanog to resist differentiation can be regarded as recapitulation of effects normally associated with the specification of pluripotency. Pertinent questions are how Nanog specifies naive pluripotency and whether this mechanism is evolutionarily conserved.
Nanog; pluripotency; reprogramming; ES cells; iPS cells; molecular evolution
Pluripotency, the property of a cell to differentiate into all cellular types of a given organism, is central to the development of stem cell-based therapies and regenerative medicine. Stem cell pluripotency is the result of the orchestrated activation of a complex transcriptional network characterized by the expression of a set of transcription factors including the master regulators of pluripotency Nanog and Oct4. Recently, it has been shown that pluripotency can be induced in somatic cells by viral-mediated expression of the transcription factors Oct3/4, Sox2, Klf4, and c-Myc.
Here we show that 5-Aminoimidazole-4-carboxamide-1-b-riboside (AICAR) is able to activate the molecular circuitry of pluripotency in mouse embryonic stem cells (mESC) and maintain Nanog and Oct4 expression in mESC exposed to the differentiating agent retinoic acid. We also show that AICAR is able to induce Klf4, Klf2 and Myc expression in both mESC and murine fibroblasts.
AICAR is able to activate the molecular circuitry of pluripotency in mESC and to induce the expression of several key regulators of pluripotency in somatic cells. AICAR is therefore a useful pharmacological entity for studying small molecule mediated induction of pluripotency.
Introduction of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, can successfully reprogram somatic cells into embryonic stem (ES)-like cells. These cells, which are referred to as induced pluripotent stem (iPS) cells, closely resemble embryonic stem cells in genomic, cell biologic, and phenotypic characteristics, and the creation of these special cells was a major triumph in cell biology. In contrast to pluripotent stem cells generated by somatic cell nuclear-transfer (SCNT) or ES cells derived from the inner cell mass (ICM) of the blastocyst, direct reprogramming provides a convenient and reliable means of generating pluripotent stem cells. iPS cells have already shown incredible potential for research and for therapeutic applications in regenerative medicine within just a few years of their discovery. In this review, current techniques of generating iPS cells and mechanisms of nuclear reprogramming are reviewed, and the potential for therapeutic applications is discussed. Antioxid. Redox Signal. 15, 1799–1820.
Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells.
We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism.
Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH).
Embryonic stem (ES) cells have the capacity to form every type of cell in our adult bodies due to their pluripotency. The prospective use of ES cells in regenerative therapies for human diseases such as Parkinson’s disease and diabetes has raised the interest in identifying the mechanisms that allow these cells to maintain pluripotent fate and differentiate along many lineages. However, ethical questions regarding the use of human eggs and∕or embryos for medical research have limited the ability of scientists to develop therapies with human ES cells. Three recent papers in Nature and Cell Stem Cell have revealed novel methods of reprogramming somatic cells into cells with the same pluripotent potential as ES cells via the expression of only four transcription factors. These scientific advances illuminate the mechanisms that drive pluripotent fate in embryonic cells. In addition, by giving scientists a model to study ES-like cells that are not derived from embryos, these newly identified models have the potential to progress therapies for regenerative medicine.
Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, hold great promise as a cell source for regenerative therapies to treat many major diseases characterized by an irreversible loss of functional tissues. However, the future clinical application of pluripotent stem cells faces a number of obstacles regarding the safety, efficiency and long-term benefits. Some of these challenges are being addressed by the chemical biology approach using small molecules. In this paper, we review the recent progress and patents on small molecules which promote pluripotent stem cell maintenance, reprogramming, and direct differentiation with a focus on cardiomyogenesis.
Cardiomyogenesis; chemical biology; differentiation; iPS cells; pluripotent stem cells; small molecules
After the hope and controversy brought by embryonic stem cells two decades ago for regenerative medicine, a new turn has been taken in pluripotent cells research when, in 2006, Yamanaka's group reported the reprogramming of fibroblasts to pluripotent cells with the transfection of only four transcription factors. Since then many researchers have managed to reprogram somatic cells from diverse origins into pluripotent cells, though the cellular and genetic consequences of reprogramming remain largely unknown. Furthermore, it is still unclear whether induced pluripotent stem cells (iPSCs) are truly functionally equivalent to embryonic stem cells (ESCs) and if they demonstrate the same differentiation potential as ESCs. There are a large number of reprogramming experiments published so far encompassing genome-wide transcriptional profiling of the cells of origin, the iPSCs and ESCs, which are used as standards of pluripotent cells and allow us to provide here an in-depth analysis of transcriptional profiles of human and mouse cells before and after reprogramming. When compared to ESCs, iPSCs, as expected, share a common pluripotency/self-renewal network. Perhaps more importantly, they also show differences in the expression of some genes. We concentrated our efforts on the study of bivalent domain-containing genes (in ESCs) which are not expressed in ESCs, as they are supposedly important for differentiation and should possess a poised status in pluripotent cells, i.e. be ready to but not yet be expressed. We studied each iPSC line separately to estimate the quality of the reprogramming and saw a correlation of the lowest number of such genes expressed in each respective iPSC line with the stringency of the pluripotency test achieved by the line. We propose that the study of expression of bivalent domain-containing genes, which are normally silenced in ESCs, gives a valuable indication of the quality of the iPSC line, and could be used to select the best iPSC lines out of a large number of lines generated in each reprogramming experiment.
In mammals, one of the two X chromosomes of female cells is inactivated for dosage compensation between the sexes. X chromosome inactivation is initiated in early embryos by the noncoding Xist RNA. Subsequent chromatin modifications on the inactive X chromosome (Xi) lead to a remarkable stability of gene repression in somatic cell lineages. In mice, reactivation of genes on the Xi accompanies the establishment of pluripotent cells of the female blastocyst and the development of primordial germ cells. Xi reactivation also occurs when pluripotency is established during the reprogramming of somatic cells to induced pluripotent stem cells. The mechanism of Xi reactivation has attracted increasing interest for studying changes in epigenetic patterns and for improving methods of cell reprogramming. Here, we review recent advances in the understanding of Xi reactivation during development and reprogramming and illustrate potential clinical applications.
X chromosome inactivation; Dosage compensation; Mammalian development; Reprogramming
Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally ‘naïve’, with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy.
Epigenetics; Reprogramming; X-chromosome inactivation; Pluripotency; Stem cells; Noncoding RNAs
Somatic cell nuclear transfer (SCNT) has been proved capable of reprogramming various differentiated somatic cells into pluripotent stem cells. Recently, induced pluripotent stem cells (iPS) have been successfully derived from mouse and human somatic cells by the over-expression of a combination of transcription factors. However, the molecular mechanisms underlying the reprogramming mediated by either the SCNT or iPS approach are poorly understood. Increasing evidence indicates that many tumor pathways play roles in the derivation of iPS cells. Embryonal carcinoma (EC) cells have the characteristics of both stem cells and cancer cells and thus they might be the better candidates for elucidating the details of the reprogramming process. Although previous studies indicate that EC cells cannot be reprogrammed into real pluripotent stem cells, the reasons for this remain unclear. Here, nuclei from mouse EC cells (P19) were transplanted into enucleated oocytes and pluripotent stem cells (P19 NTES cells) were subsequently established. Interestingly, P19 NTES cells prolonged the development of tetraploid aggregated embryos compared to EC cells alone. More importantly, we found that the expression recovery of the imprinted H19 gene was dependent on the methylation state in the differential methylation region (DMR). The induction of Nanog expression, however, was independent of the promoter region DNA methylation state in P19 NTES cells. A whole-genome transcriptome analysis further demonstrated that P19 NTES cells were indeed the intermediates between P19 cells and ES cells and many interesting genes were uncovered that may be responsible for the failed reprogramming of P19 cells. To our knowledge, for the first time, we linked incomplete reprogramming to the improved pluripotency of EC cell-derived pluripotent stem cells. The candidate genes we discovered may be useful not only for understanding the mechanisms of reprogramming, but also for deciphering the transition between tumorigenesis and pluripotency.