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1.  The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells 
An in-depth proteomic comparison of human-induced pluripotent stem cells, and their parent fibroblast cells, with embryonic stem cells shows that the reprogramming process comprehensively remodels protein expression levels, creating cells that closely resemble natural stem cells.
We present here a large proteomic characterization of human embryonic stem cells, human-induced pluripotent stem cells and their parental fibroblasts cell lines.Overall, 97.8% of the 2683 quantified proteins in four experiments showed no significant differences in abundance between hESC and hiPSC highlighting the high similarity of these pluripotent cell lines.In total, 58 proteins were found significantly differentially expressed between hiPSCs and hESCs. The observed low overlap of these proteins with previous transcriptomic studies suggests that those differences do no reflect a recurrent molecular signature.
Human embryonic stem cells (hESCs) are capable of self-renewal and multi-lineage differentiation. However, the use of hESCs for clinical treatment entails ethical issues as they are derived from human embryos. Recently, reprogramming of somatic cells to an embryonic stem cell-like state, named induced pluripotent stem cells (iPSCs), was achieved through ectopic expression of defined factors. In addition to their clinical potential, hiPSCs represent a unique tool to develop cellular models for human diseases as well. Although current functional assays (e.g., tetraploid complementation) have confirmed the pluripotency of hiPSCs, there might still be significant differences (e.g., differentiation potential) when compared with their natural hESCs counterparts. Consequently, an extensive molecular characterization to address differences and similarities between these two pluripotent cell lines seems to be a prerequisite before any clinical application is conducted. Despite that great efforts, mainly at the genomic levels, have been made to address how similar hESCs and hiPSCs are, the definite answer to this fundamental question is currently still debated. Direct assessment of protein levels has yet to be incorporated into these integrative systems-level analyses. Protein levels are tuned by intricate mechanisms of gene expression regulation and it has recently been documented that mRNA and protein levels poorly correlate in mouse ESCs. Here, we use in-depth quantitative proteomics to gain insights into the differences and similarities in the protein content of two hiPS cell lines, their precursor IMR90 and 4Skin fibroblast cell lines and one hES cell line, providing novel molecular signatures that may assist in filling a gap in the understanding of pluripotency.
To study the degree of similarity, at the protein level, between hiPSCs and hESCs, four MS-based proteomic experiments were designed that use our in-house developed triplex dimethyl labeling chemistry followed by extensive fractionation by strong cation exchange (SCX) chromatography to reduce the sample complexity. High-resolution LC-MS/MS with dedicated fragmentation schemes (i.e., electron transfer dissociation, collision-induced dissociation and higher-energy collision dissociation) was subsequently used to maximize peptide identification rates. A total of 348 LC-MS/MS analyses (including technical and biological replicates) were performed. We confidently identified 1 593 446 peptide spectrum matches (peptide FDR<1%) corresponding to 10 628 unique protein groups (protein FDR∼4%). Using the extracted ion chromatograms, we also estimated the absolute abundance of the proteins within the samples spanning six orders of magnitude. To the best of our knowledge, the coverage obtained in this study represents the largest achieved by any proteomics screen on pluripotent cells.
Most importantly, our results indicate that the reprogramming process remodeled the proteome of both fibroblast cell lines to a profile that closely resembles the pluripotent hESCs proteome: 97.8% of the quantified proteins (2638 proteins in all four experiments) showed nonsignificant changes. Nevertheless, a small fraction of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly regulated between hiPSCs and hESCs. A comparison of the regulated proteins to previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. On the other side, the inclusion of the two parental fibroblast cell lines in our analysis allowed us to study changes in the proteome at both the starting and end points of the reprogramming process. As expected, the vast majority of the proteins (73.4%) showed differential expression between the parental fibroblasts and the reprogrammed pluripotent cells.
To find out if the differences observed in our study were a consequence of transcriptional or translational regulation, we performed paired genome-wide gene expression analyses on the same six samples that were used for the proteomic profiling. Overall, we observed a good correlation between mRNA and protein levels (r∼0.7). These results further authenticated the proteomic measurements and implied a high degree of control at the transcriptional level. Nevertheless, numerous genes were found uncorrelated highlighting the necessity of complementing transcriptomic-based approaches with proteomics.
Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.
PMCID: PMC3261715  PMID: 22108792
human embryonic stem cells; human-induced pluripotent stem cells; proteomics; quantitation
2.  Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation 
PLoS Genetics  2012;8(5):e1002691.
Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.
Author Summary
The epigenome and chromatin play critical roles in stem cell fate determination. Linker histone H1 is a major chromatin structural protein that facilitates higher-order chromatin folding. By analyzing the differentiation capacity of embryonic stem cells (ESCs) that lack multiple H1 subtypes, we find, for the first time, that H1 and higher-order chromatin compaction are required for proper differentiation and lineage commitment of pluripotent stem cells. Triple-H1 null murine ESCs are impaired in both spontaneous differentiation and embryoid body differentiation. Furthermore, triple-H1 null ESCs are compromised in neural differentiation. Finally, we demonstrate that H1 depletion leads to failure of efficient repression of pluripotency gene expression both in embryos and in ESC differentiation. We present evidence that H1 participates in mediating changes of histone marks and DNA methylation necessary for silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. This finding is important because it provides a mechanistic link by which H1 and chromatin compaction may participate in pluripotent stem cell differentiation through repression of pluripotency gene expression.
PMCID: PMC3349736  PMID: 22589736
3.  MicroRNA Profiling Reveals Distinct Mechanisms Governing Cardiac and Neural Lineage-Specification of Pluripotent Human Embryonic Stem Cells 
Realizing the potential of human embryonic stem cells (hESCs) has been hindered by the inefficiency and instability of generating desired cell types from pluripotent cells through multi-lineage differentiation. We recently reported that pluripotent hESCs maintained under a defined platform can be uniformly converted into a cardiac or neural lineage by small molecule induction, which enables lineage-specific differentiation direct from the pluripotent state of hESCs and opens the door to investigate human embryonic development using in vitro cellular model systems. To identify mechanisms of small molecule induced lineage-specification of pluripotent hESCs, in this study, we compared the expression and intracellular distribution patterns of a set of cardinal chromatin modifiers in pluripotent hESCs, nicotinamide (NAM)-induced cardiomesodermal cells, and retinoic acid (RA)-induced neuroectodermal cells. Further, genome-scale profiling of microRNA (miRNA) differential expression patterns was used to monitor the regulatory networks of the entire genome and identify the development-initiating miRNAs in hESC cardiac and neural lineage-specification. We found that NAM induced nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing, while RA induced silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family to high levels. Genome-scale miRNA profiling indentified that a unique set of pluripotence-associated miRNAs was down-regulated, while novel sets of distinct cardiac- and neural-driving miRNAs were up-regulated upon the induction of lineage-specification direct from the pluripotent state of hESCs. These findings suggest that a predominant epigenetic mechanism via SIRT1-mediated global chromatin silencing governs NAM-induced hESC cardiac fate determination, while a predominant genetic mechanism via silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family governs RA-induced hESC neural fate determination. This study provides critical insight into the earliest events in human embryogenesis as well as offers means for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies.
PMCID: PMC3554249  PMID: 23355957
MicroRNA; Chromatin modification; Chromatin remodeling; Small molecule; Retinoic acid; Nicotinamide; Human embryonic stem cell; Pluripotence; Neural differentiation; Cardiac differentiation; Lineage-specification; Neuroectoderm; Cardiomesoderm; Neuronal progenitor; Cardiac precursor; Neuron; Cardiomyocyte; Defined culture system; SIRT1; MiR-302; MiR-10
4.  Embedding the Future of Regenerative Medicine into the Open Epigenomic Landscape of Pluripotent Human Embryonic Stem Cells 
It has been recognized that pluripotent human embryonic stem cells (hESCs) must be transformed into fate-restricted derivatives before use for cell therapy. Realizing the therapeutic potential of pluripotent hESC derivatives demands a better understanding of how a pluripotent cell becomes progressively constrained in its fate options to the lineages of tissue or organ in need of repair. Discerning the intrinsic plasticity and regenerative potential of human stem cell populations reside in chromatin modifications that shape the respective epigenomes of their derivation routes. The broad potential of pluripotent hESCs is defined by an epigenome constituted of open conformation of chromatin mediated by a pattern of Oct-4 global distribution that corresponds genome-wide closely with those of active chroma tin modifications. Dynamic alterations in chromatin states correlate with loss-of-Oct4-associated hESC differentiation. The epigenomic transition from pluripotence to restriction in lineage choices is characterized by genome-wide increases in histone H3K9 methylation that mediates global chromatin-silencing and somatic identity. Human stem cell derivatives retain more open epigenomic landscape, therefore, more developmental potential for scale-up regeneration, when derived from the hESCs in vitro than from the CNS tissue in vivo. Recent technology breakthrough enables direct conversion of pluripotent hESCs by small molecule induction into a large supply of lineage-specific neuronal cells or heart muscle cells with adequate capacity to regenerate neurons and contractile heart muscles for developing safe and effective stem cell therapies. Nuclear translocation of NAD-dependent histone deacetylase SIRT1 and global chromatin silencing lead to hESC cardiac fate determination, while silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family lead to hESC neural fate determination. These recent studies place global chromatin dynamics as central to tracking the normal pluripotence and lineage progres sion of hESCs. Embedding lineage-specific genetic and epigenetic developmental programs into the open epigenomic landscape of pluripotent hESCs offers a new repository of human stem cell therapy derivatives for the future of regenerative medicine.
PMCID: PMC4190676  PMID: 25309947
Human embryonic stem cell; stem cell; pluripotent; epigenome; chromatin; regenerative medicine; neurological disease; heart disease; cell therapy
5.  ATP Dependent Chromatin Remodeling Enzymes in Embryonic Stem Cells 
Stem cell reviews  2010;6(1):62-73.
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.
PMCID: PMC2862992  PMID: 20148317
Embryonic stem cells; Pluripotency; Self-renewal; Differentiation; Chromatin; Histone modifications; Histone variants; Chromatin remodeling enzymes
6.  Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition 
PLoS Biology  2008;6(10):e253.
Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency—including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras—were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1–2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.
Author Summary
Development of an organism proceeds irreversibly from embryo to adult, with cells differentiating progressively towards specialised final phenotypes. Now, following the pioneering discovery of induced pluripotency by Shinya Yamanaka, it has become possible to reverse developmental time: we can reprogramme an adult cell back to the naïve state of pluripotency found in the early embryo. Induction of pluripotency is an extraordinary phenomenon but is currently poorly understood and inefficient. We investigated stem cells from the mouse brain and found that they reprogrammed faster than other cell types. However, the reprogrammed brain cells arrested on the verge of full pluripotency and did not gain some essential properties of induced pluripotency. Guided by the rationale of reversing a development process, we explored the effect of blocking the signal that initiates loss of pluripotency and entry into differentiation in the embryo. We used a chemical inhibitor of this signal in combination with stimulation of a second pathway known to promote maintenance of pluripotency. This simple treatment allowed the partly converted neural stem cells to complete the transition efficiently and become indistinguishable from embryonic stem cells. Therefore, incompletely reprogrammed cells, which have previously been dismissed as useless by-products of attempts to generate pluripotent stem cells, in fact provide the fastest, most reliable, and most efficient route to obtaining authentic induced pluripotent cells.
Induced reprogramming of stem cells proceeds in two phases via an intermediate that is undifferentiated but not pluripotent. Inhibition of mitogen-activated protein kinase signaling converts this intermediate transitional state to authentic pluripotency.
PMCID: PMC2570424  PMID: 18942890
7.  The Dynamics of Global Chromatin Remodeling are Pivotal for Tracking the Normal Pluripotency of Human Embryonic Stem Cells 
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.
PMCID: PMC3609651  PMID: 23543848
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
8.  Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine 
British biotechnology journal  2013;3(4):424-457.
To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point.
PMCID: PMC4051304  PMID: 24926434
Human embryonic stem cell; stem cell; pluripotent; tissue engineering; cell therapy; regenerative medicine; neurological disease; heart disease
9.  Epigenetic Landscape of Pluripotent Stem Cells 
Antioxidants & Redox Signaling  2012;17(2):205-223.
Significance: Derived from the inner cell mass of the preimplantation embryo, embryonic stem cells are prototype pluripotent stem (PS) cells that have the ability of self-renewal and differentiation into almost all cell types. Exploration of the mechanisms governing this pluripotency is important for understanding reprogramming mechanisms and stem cell behavior of PS cells and can lead to enhancing reprogramming efficiency and other applications. Recent Advances: Induced pluripotent stem cells are recently discovered PS cells that can be derived from somatic cells by overexpression of pluripotency-related transcription factors. Recent studies have shown that transcription factors and their epigenetic regulation play important roles in the generating, maintaining, and differentiating these PS cells. Recent advances in sequencing technologies allow detailed analysis of target epigenomes and microRNAs (miRs), and have revealed unique epigenetic marks and miRs for PS cells. Critical Issues: Epigenetic modifications of genes include histone modifications, DNA methylation, and chromatin remodeling. Working closely with epigenetic modifiers, miRs play an important role in inducing and maintaining pluripotency. Future Directions: The dynamic changes in epigenetic marks during reprogramming and their role in cell fate changes are being uncovered. This review focuses on these new advances in the epigenetics of PS cells. Antioxid. Redox Signal. 17, 205–223.
PMCID: PMC3353817  PMID: 22044221
10.  Stem Cell Pluripotency: A Cellular Trait that Depends on Transcription Factors, Chromatin State and a Checkpoint Deficient Cell Cycle 
Journal of Cellular Physiology  2009;221(1):10-17.
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.
PMCID: PMC3326661  PMID: 19562686
Pluripotency; Stem cells; Transcription Factors; Epigenetics; Cell Cycle; B-Myb
11.  Epigenetic memory in induced pluripotent stem cells 
Nature  2010;467(7313):285-290.
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.
PMCID: PMC3150836  PMID: 20644535
12.  CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells 
PLoS Genetics  2006;2(11):e181.
During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.
Chromatin is the support of our genetic information. It is composed of numerous repeated units called nucleosomes, in which DNA wraps around a core of histone proteins. Modifications in the composition and biochemical properties of nucleosomes play major roles in the regulation of genome function. Such modifications are termed “epigenetic” when they are inherited across cell divisions and confer new information to chromatin, in addition to the genetic information provided by DNA. It is usually believed that during genome replication, the basic chromatin assembly machinery builds up “naïve” nucleosomes, and, in a subsequent step, nucleosomes are selectively modified by a series of enzymes to acquire epigenetic information. Here, the authors studied the role of a basic chromatin assembly factor (CAF-1) in mouse embryonic stem cells and early embryos. Surprisingly, they show that CAF-1 confers epigenetic information to specific genomic regions. In addition, this study revealed that CAF-1 is required for the proper spatial organization of chromosomes in the nucleus. This new knowledge may contribute to better understanding the role of chromatin in the maintenance of embryonic stem cell identity and plasticity.
PMCID: PMC1630711  PMID: 17083276
13.  Long Noncoding RNAs: New Players in the Molecular Mechanism for Maintenance and Differentiation of Pluripotent Stem Cells 
Stem Cells and Development  2013;22(16):2240-2253.
Maintenance of the pluripotent state or differentiation of the pluripotent state into any germ layer depends on the factors that orchestrate expression of thousands of genes through epigenetic, transcriptional, and post-transcriptional regulation. Long noncoding RNAs (lncRNAs) are implicated in the complex molecular circuitry in the developmental processes. The ENCODE project has opened up new avenues for studying these lncRNA transcripts with the availability of new datasets for lncRNA annotation and regulation. Expression studies identified hundreds of long noncoding RNAs differentially expressed in the pluripotent state, and many of these lncRNAs are found to control the pluripotency and stemness in embryonic and induced pluripotent stem cells or, in the reverse way, promote differentiation of pluripotent cells. They are generally transcriptionally activated or repressed by pluripotency-associated transcription factors and function as molecular mediators of gene expression that determine the pluripotent state of the cell. They can act as molecular scaffolds or guides for the chromatin-modifying complexes to direct them to bind into specific genomic loci to impart a repressive or activating effect on gene expression, or they can transcriptionally or post-transcriptionally regulate gene expression by diverse molecular mechanisms. This review focuses on recent findings on the regulatory role of lncRNAs in two main aspects of pluripotency, namely, self renewal and differentiation into any lineage, and elucidates the underlying molecular mechanisms that are being uncovered lately.
PMCID: PMC3730374  PMID: 23528033
14.  The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells 
eLife  null;3:e03573.
Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.
eLife digest
The stem cells found in an embryo are able to develop into any of the cell types found in the body of the animal: an ability called pluripotency. When a cell becomes a specialized cell type, such as a nerve cell or a muscle cell, it loses this ability. However, mature cells can be reprogrammed back to a pluripotent state by artificially introducing certain proteins (known as ‘reprogramming factors’) into the mature cells.
A core group of reprogramming factors are known to activate networks of genes that are normally only expressed in stem cells, and by doing so trigger and maintain a pluripotent state. Other proteins help these core factors to regulate these networks of genes. In 2011, researchers discovered that a protein complex called XPC—which is normally involved in DNA repair—also helps two core reprogramming factors to activate an important gene related to pluripotency.
Now, Fong et al., including several of the researchers involved in the 2011 work, have identified another unexpected partner for the same two core reprogramming factors. The protein complex, called DKC1, has a number of known functions related to the processing of RNA molecules. This complex has also been linked to a fatal, rare human disorder called dyskeratosis congenita—a condition that affects many parts of the body, including the skin and bone marrow. Fong et al. found that when embryonic stems cells from mice are depleted of the DKC1 complex, the activation of important pluripotency-related genes by two of the core reprogramming factors is markedly reduced.
The XPC and DKC1 protein complexes were found to interact in pluripotent cells, and together they can activate a pluripotency-related gene to a greater extent than either can individually. Fong et al. propose that DKC1 binds to XPC, which in turn binds to two of the core reprogramming factors.
The DKC1 complex also binds to RNA molecules, and Fong et al. found that when the DKC1 complex binds to certain RNAs it is more able to help reprogramming factors activate pluripotency-related genes. On the other hand, other RNA molecules seem to inhibit the complex's ability to activate these genes.
Mutations identified in people with dyskeratosis congenita can prevent the DKC1 complex from binding to a subset of human RNA molecules. Moreover, the activity of stem cells is impaired in people with this developmental condition. As such, one of the next challenges will be to investigate if these mutations and RNA binding could be linked to problems with the activation of genes related to pluripotency in stem cells.
PMCID: PMC4270071  PMID: 25407680
transcriptional coactivators; pluripotency; embryonic stem cells; non-coding RNA; induced pluripotent stem cells; priviledged somatic cell state; human; mouse
15.  Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes 
PLoS Genetics  2008;4(6):e1000116.
DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.
Author Summary
Large scale epigenetic reprogramming occurs in mammalian germ cells and the early embryo. The biological purpose of this reprogramming is largely unknown, although it has been suggested that it may be required for the embryonic genome to return to a pluripotent state. We carried out a genome-wide screen of promoter methylation in the mouse, comparing germ cells with pluripotent cells, multipotent cells, and more differentiated cell types. We find that promoter methylation is an epigenetic signature of developmental potency. Genes linked to pluripotency are generally hypomethylated in stem cells and hypermethylated (and silenced) in more differentiated cell types, and our genome-wide screen provides new candidates for the regulation of pluripotency. Importantly, germ cells resemble pluripotent cell types in that most promoters have been reprogrammed. However, a small group of key pluripotency regulators (including Nanog), are methylated in mature germ cells, presumably in order to suppress pluripotency at critical stages of germ cell differentiation. Indeed, methylation in these genes becomes reprogrammed after fertilisation so that the embryo can regain totipotency. This work, therefore, shows for the first time that epigenetic reprogramming is crucial for maintaining the pluripotency of germ and embryonic stem cells.
PMCID: PMC2432031  PMID: 18584034
16.  Role of Oct4 in maintaining and regaining stem cell pluripotency 
Pluripotency, a characteristic of cells in the inner cell mass of the mammalian preimplantation blastocyst as well as of embryonic stem cells, is defined as the ability of a cell to generate all of the cell types of an organism. A group of transcription factors is essential for the establishment and maintenance of the pluripotent state. Recent studies have demonstrated that differentiated somatic cells could be reverted to a pluripotent state by the overexpression of a set of transcription factors, further highlighting the significance of transcription factors in the control of pluripotency. Among these factors, a member of the POU transcription factor family, Oct4, is central to the machinery governing pluripotency. Oct4 is highly expressed in pluripotent cells and becomes silenced upon differentiation. Interestingly, the precise expression level of Oct4 determines the fate of embryonic stem cells. Therefore, to control the expression of Oct4 precisely, a variety of regulators function at multiple levels, including transcription, translation of mRNA and post-translational modification. Additionally, in cooperation with Sox2, Nanog and other members of the core transcriptional regulatory circuitry, Oct4 activates both protein-coding genes and noncoding RNAs necessary for pluripotency. Simultaneously, in association with transcriptional repressive complexes, Oct4 represses another set of targets involved in developmental processes. Importantly, Oct4 can re-establish pluripotency in somatic cells, and proper reprogramming of Oct4 expression is indispensable for deriving genuine induced pluripotent stem cell lines. In the past several years, genome-wide identification of Oct4 target genes and Oct4-centered protein interactomes has been reported, indicating that Oct4 exerts tight control over pluripotency regulator expression and protects embryonic stem cells in an undifferentiated state. Nevertheless, further investigation is required to fully elucidate the underlying molecular mechanisms through which Oct4 maintains and reinitiates pluripotency. Systemic and dynamic exploration of the protein complexes and target genes associated with Oct4 will help to elucidate the role of Oct4 more comprehensively.
PMCID: PMC3025441  PMID: 21156086
17.  HMGA1 Reprograms Somatic Cells into Pluripotent Stem Cells by Inducing Stem Cell Transcriptional Networks 
PLoS ONE  2012;7(11):e48533.
Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear.
Methods/Principal Findings
We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC – OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state.
Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.
PMCID: PMC3499526  PMID: 23166588
18.  Pivots of pluripotency: the roles of non-coding RNA in regulating embryonic and induced pluripotent stem cells 
Biochimica et biophysica acta  2012;1830(2):2385-2394.
Induced pluripotent stem cells (iPSC) derived from reprogrammed patient somatic cells possess enormous therapeutic potential. However, unlocking the full capabilities of iPSC will require an improved understanding of the molecular mechanisms which govern the induction and maintenance of pluripotency, as well as directed differentiation to clinically relevant lineages. Induced pluripotency of a differentiated cell is mediated by sequential cascades of genetic and epigenetic reprogramming of somatic histone and DNA CpG methylation marks. These genome-wide changes are mediated by a coordinated activity of transcription factors and epigenetic modifying enzymes. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are now recognized as an important third class of regulators of the pluripotent state.
This review surveys the currently known roles and mechanisms of ncRNAs in regulating the embryonic and induced pluripotent states.
Through a variety of mechanisms, ncRNAs regulate constellations of key pluripotency genes and epigenetic regulators, and thus critically determine induction and maintenance of the pluripotent state.
A further understanding of the roles of ncRNAs in regulating pluripotency may help assess the quality of human iPSC reprogramming. Additionally, ncRNA biology may help decipher potential transcriptional and epigenetic commonalities between the self renewal processes that govern both ESC and tumor initiating cancer stem cells (CSC).
PMCID: PMC3552091  PMID: 23104383
19.  Pursuing Self-Renewal and Pluripotency with the Stem Cell Factor Nanog 
Stem cells (Dayton, Ohio)  2013;31(7):1227-1236.
Pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise for future use in tissue replacement therapies due to their ability to self-renew indefinitely and to differentiate into all adult cell types. Harnessing this therapeutic potential efficiently requires a much deeper understanding of the molecular processes at work within the pluripotency network. The transcription factors Nanog, Oct4, and Sox2 reside at the core of this network, where they interact and regulate their own expression as well as that of numerous other pluripotency factors. Of these core factors, Nanog is critical for blocking the differentiation of pluripotent cells, and more importantly, for establishing the pluripotent ground state during somatic cell reprogramming. Both mouse and human Nanog are able to form dimers in vivo, allowing them to preferentially interact with certain factors and perform unique functions. Recent studies have identified an evolutionary functional conservation among vertebrate Nanog orthologs from chick, zebrafish, and the axolotl salamander, adding an additional layer of complexity to Nanog function. Here we present a detailed overview of published work focusing on Nanog structure, function, dimerization, and regulation at the genetic and post-translational levels with regard to the establishment and maintenance of pluripotency. The full spectrum of Nanog function in pluripotent stem cells and in cancer is only beginning to be revealed. We therefore use this evidence to advocate for more comprehensive analysis of Nanog in the context of disease, development, and regeneration.
PMCID: PMC3706551  PMID: 23653415
ESCs; iPSCs; pluripotency; Nanog; self-renewal; reprogramming
20.  Zfp322a Regulates Mouse ES Cell Pluripotency and Enhances Reprogramming Efficiency 
PLoS Genetics  2014;10(2):e1004038.
Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotency maintenance of mouse ES cells. Here, we report that Zfp322a is required for mES cell identity since depletion of Zfp322a directs mES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Pou5f1 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of mES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated mES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits mES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka's factors (OSKM). It can be even used in combination with Yamanaka's factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of mouse ES cells.
Author Summary
Embryonic stem (ES) cells are featured by their ability to self-renew and by their potential to differentiate into many different cell types. Recent studies have revealed that the unique properties of mouse ES cells are governed by a specific transcription regulatory network, including master regulators Oct4/Sox2/Nanog and other pluripotency factors. The importance of these factors was highlighted by the subsequent finding that combination of several transcription factors can reprogram differentiated fibroblasts back to pluripotent stem cells. Here, we report that Zfp322a is a novel factor which is required for mES cell identity. We revealed that Zfp322a can regulate the key pluripotency genes Pou5f1 and Nanog and functions as a repressor of MAPK/ERK pathway in mES cells, therefore preventing mES cell differentiation. Furthermore, we discovered that Zfp332a can promote the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Our results reveal that Zfp322a is a novel essential transcription factor which not only regulates ES cell pluripotency but also enhances iPSC formation.
PMCID: PMC3923668  PMID: 24550733
21.  Global Chromatin Architecture Reflects Pluripotency and Lineage Commitment in the Early Mouse Embryo 
PLoS ONE  2010;5(5):e10531.
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.
PMCID: PMC2866533  PMID: 20479880
22.  Chromatin Regulatory Mechanisms in Pluripotency 
Stem cells of all types are characterized by a stable, heritable state permissive of multiple developmental pathways. The past five years have seen remarkable advances in understanding these heritable states and the ways that they are initiated or terminated. Transcription factors that bind directly to DNA and have sufficiency roles have been most easy to investigate and, perhaps for this reason, are most solidly implicated in pluripotency. In addition, large complexes of ATP-dependent chromatin-remodeling and histone-modification enzymes that have specialized functions have also been implicated by genetic studies in initiating and/or maintaining pluripotency or multipotency. Several of these ATP-dependent remodeling complexes play non-redundant roles, and the esBAF complex facilitates reprogramming of induced pluripotent stem cells. The recent finding that virtually all histone modifications can be rapidly reversed and are often highly dynamic has raised new questions about how histone modifications come to play a role in the steady state of pluripotency. Another surprise from genetic studies has been the frequency with which the global effects of mutations in chromatin regulators can be largely reversed by a single target gene. These genetic studies help define the arena for future mechanistic studies that might be helpful to harness pluripotency for therapeutic goals.
PMCID: PMC3085914  PMID: 20624054
epigenetics; chromatin remodeling; BAF complexes; stem cells; lineage specificity
23.  Epigenetics of Pluripotent Cells  
Acta Naturae  2012;4(4):28-46.
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.
PMCID: PMC3548172  PMID: 23346378
embryonic stem cells; induced pluripotent stem cells; pluripotency; covalent histone modifications; DNA methylation
24.  Histone variant macroH2A marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced pluripotency 
Journal of Cell Science  2012;125(24):6094-6104.
How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.
PMCID: PMC3585521  PMID: 23077180
Cell commitment; Epigenetic stability; Induced pluripotency; macroH2A; Nuclear reprogramming
25.  Spatial Pattern Dynamics of 3D Stem Cell Loss of Pluripotency via Rules-Based Computational Modeling 
PLoS Computational Biology  2013;9(3):e1002952.
Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.
Author Summary
Pluripotent embryonic stem cells can differentiate into all cell types making up the adult body; however, this process occurs in a complex three dimensional environment with many different parameters present that are capable of influencing cell fate decisions. A model that can accurately predict the strengths of factors influencing cell fate would allow examination of spatial and temporal patterns of cell phenotype. For this study, we focused on the earliest fate transition that occurs in 3D clusters of embryonic stem cells by monitoring the presence of a transcription factor (Oct4) associated with stem cell pluripotency. After experimentally classifying patterns that arise en route to a fully differentiated aggregate in vitro, we constructed a computational model to deduce how stem cells integrate cues from their surrounding environment to give rise spatial patterns. We used a rules-based modeling approach in which information exchanged by cells with their nearest neighbors regulated cell fate decisions. This parsimonious model captured the spatial dynamics of early cell lineage commitment in a 3D multicellular structure. In combination with strategies to modulate cellular environments, our model provides a flexible tool for elucidating the extra- and intercellular interactions regulating spatially organized differentiation of stem cells in 3D.
PMCID: PMC3597536  PMID: 23516345

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