Generation Of Pluripotent Cells Research Articles

Oct4:Sox2 binding is essential for establishing but not maintaining active and silent states of dynamically regulated genes in pluripotent cells.

Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states.

CCCTC-binding factor is an upstream regulator of the pluripotency factor Oct4 and functions in active transcription of linc1253 and linc1356 genes in pluripotent cells

The embryonic stem cell- (ESC) specific transcription factor Oct4 is a well-known master regulator of pluripotency. The aim of this study was to identify upstream regulators of Oct4 and explore their functional link in regulating lincRNA expression in ESCs. By quantitative real-time PCR (RT-qPCR) analysis upon CCCTC-binding factor (CTCF) or Oct4 knockdown, here, we found that the chromatin insulator CTCF transcriptionally controls Oct4 gene expression in mouse ES cells. Furthermore, co-immunoprecipitation assays showed that CTCF physically interacts with Oct4. By analyzing CTCF and Oct4 ChIP-seq datasets in mouse ES cells and investigating their genomic occupancies, we demonstrated that CTCF and Oct4 share overlapping regulatory functions and are required for active transcription of long intergenic non-coding RNAs (lincRNAs) linc1253 and linc1356, which were reported to repress cellular lineage programs and maintain a pluripotent state. In summary, we propose an integrated model of transcriptional control mediated by CTCF, the master weaver of the genome, for the upstream regulation of Oct4-and ESC-associated genes. These results connect the chromatin insulator CTCF and the pluripotency factor Oct4 in the regulation of lincRNAs in pluripotent cells.

TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2.

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

Combinations of Growth Factors Regulating LIF/STAT3, WNT, and FGF2 Pathways Sustain Pluripotency-Related Proteins in Cat Embryonic Cells.

Propagation of pluripotent cells from early stage embryos in mouse and human highly depend on leukemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (STAT3) and FGF2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. However, mechanisms for maintaining pluripotency in embryonic stem cells using various combinations of growth factors (targeting LIF or FGF2 pathways) and inhibitors (targeting WNT/GSK3 or FGF2 pathways) still have to be deciphered in other models, including the domestic cat. Our objective was to understand how cytokines influence pluripotency in the cat inner cell mass (ICM) outgrowths. Cat ICM was isolated from in vitro-produced embryos and outgrowths were cultured for up to 6 days with single or combined cytokines. Cell proliferation was enhanced with almost all single growth factors and cytokine combinations. Based on gene expression and presence of NANOG, POU5F1, and Sex-determining region Y box 2 (SOX2) as cell state markers, single growth factors could not maintain similar levels in outgrowths as in the original ICMs, which is different from the response in mouse and human. In our conditions, cytokine combinations involving LIF, GSK3 inhibitor, and MEK inhibitor resulted in the most robust expression levels and allowed single-cell dissociation and propagation. However, further characterization of embryonic cells derived from ICM indicated that the pluripotent state was not fully preserved. The absence of detectable transcripts for BMP2-receptor and SMAD4, and very low levels of LIF-receptor and STAT3 in the cat ICM indicated that pluripotency regulatory machinery appear to be different in the cat from the predominant mouse and human models.

Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b

BackgroundHistone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes.ResultsWe report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C.ConclusionsThese results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.

Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation

BackgroundStructural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Changes in the expression and genomic distributions of H1 variants during cell differentiation appear to contribute to phenotypic differences between cell types, but few details are known about the roles of individual H1 variants and the significance of their disparate capacities for phosphorylation. In this study, we investigated the dynamics of interphase phosphorylation at specific sites in individual H1 variants during the differentiation of pluripotent NT2 and mouse embryonic stem cells and characterized the kinases involved in regulating specific H1 variant phosphorylations in NT2 and HeLa cells.ResultsHere, we show that the global levels of phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and H1.4-Ser187 (pS187-H1.4) are regulated differentially during pluripotent cell differentiation. Enrichment of pS187-H1.4 near the transcription start site of pluripotency factor genes in pluripotent cells is markedly reduced upon differentiation, whereas pS187-H1.4 levels at housekeeping genes are largely unaltered. Selective inhibition of CDK7 or CDK9 rapidly diminishes pS187-H1.4 levels globally and its enrichment at housekeeping genes, and similar responses were observed following depletion of CDK9. These data suggest that H1.4-S187 is a bona fide substrate for CDK9, a notion that is further supported by the significant colocalization of CDK9 and pS187-H1.4 to gene promoters in reciprocal re-ChIP analyses. Moreover, treating cells with actinomycin D to inhibit transcription and trigger the release of active CDK9/P-TEFb from 7SK snRNA complexes induces the accumulation of pS187-H1.4 at promoters and gene bodies. Notably, the levels of pS187-H1.4 enrichment after actinomycin D treatment or cell differentiation reflect the extent of CDK9 recruitment at the same loci. Remarkably, the global levels of H1.5-S18 and H1.2/H1.5-S173 phosphorylation are not affected by these transcription inhibitor treatments, and selective inhibition of CDK2 does not affect the global levels of phosphorylation at H1.4-S187 or H1.5-S18.ConclusionsOur data provide strong evidence that H1 variant interphase phosphorylation is dynamically regulated in a site-specific and gene-specific fashion during pluripotent cell differentiation, and that enrichment of pS187-H1.4 at genes is positively related to their transcription. H1.4-S187 is likely to be a direct target of CDK9 during interphase, suggesting the possibility that this particular phosphorylation may contribute to the release of paused RNA pol II. In contrast, the other H1 variant phosphorylations we investigated appear to be mediated by distinct kinases and further analyses are needed to determine their functional significance.

Investigation of the cellular reprogramming phenomenon referred to as stimulus-triggered acquisition of pluripotency (STAP).

In January 2014, it was reported that strong external stimuli, such as a transient low-pH stressor, was capable of inducing the reprogramming of mammalian somatic cells, resulting in the generation of pluripotent cells. This cellular reprograming event was designated ‘stimulus-triggered acquisition of pluripotency’ (STAP) by the authors of these reports. However, after multiple instances of scientific misconduct in the handling and presentation of the data were brought to light, both reports were retracted. To investigate the actual scientific significance of the purported STAP phenomenon, we sought to repeat the original experiments based on the methods presented in the retracted manuscripts and other relevant information. As a result, we have concluded that the STAP phenomenon as described in the original studies is not reproducible.

Prolonged proteasome inhibition cyclically upregulates Oct3/4 and Nanog gene expression, but reduces induced pluripotent stem cell colony formation.

There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells.

Morphological analysis of human induced pluripotent stem cells during induced differentiation and reverse programming.

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming.

Stress and pluripotency.

Stress and pluripotency.

RETRACTED ARTICLE: Stimulus-triggered fate conversion of somatic cells into pluripotency

Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.

False-positive antibody signals for the pluripotency factor OCT4A (POU5F1) in testis-derived cells may lead to erroneous data and misinterpretations

Octamer-binding protein 4 (OCT4) is a key player in pluripotent embryonic stem (ES) cells and is essential for the generation of induced pluripotent stem cells. Recently, several reports indicated the spontaneous recovery of pluripotency in cultured adult human testis-derived cells. This was evidenced also by the detection of OCT4 using antibodies. However, the soundness of some data was recently put into question. During our attempts to derive pluripotent cells from the common marmoset monkey (Callithrix jacchus) testis, we obtained inconsistent data which prompted us to analyze deeper the characteristics of three independent OCT4 antibodies that were used in numerous published studies that received greatest attention. All antibodies detected OCT4 by immunofluorescence (IF) in a marmoset monkey ES cell line. Two of the three OCT4 antibodies also gave robust nuclear signals in testis-derived cells. However, the latter cells expressed no OCT4 mRNA as revealed by quantitative RT–PCR and turned out to be mesenchymal cells. When tested in western blot analyses, all antibodies detected heterologously expressed marmoset monkey OCT4 protein. But, importantly, those antibodies that resulted in non-specific signals in IF also showed additional non-specific bands in western blots. In summary, some commercially available OCT4 antibodies result in false-positive signals which may provoke erroneous conclusions when used in studies aiming at the generation of pluripotent cells in vitro. We conclude that (i) antibodies must be carefully characterized before use to prevent misleading observations and (ii) OCT4 expression must be monitored by a second antibody-independent method.

Factor mediated gene priming in pluripotent stem cells sets the stage for lineage specification

Priming of lineage-specific genes in pluripotent embryonic stem cells facilitates rapid and coordinated activation of transcriptional programmes during differentiation. There is growing evidence that pluripotency factors play key roles in priming tissue-specific genes and in the earliest stages of lineage commitment. As differentiation progresses, pluripotency factors are replaced at some primed genes by related lineage-specific factors that bind to the same sequences and maintain epigenetic priming until the gene is activated. Polycomb and trithorax group proteins bind many genes in pluripotent cells generating bivalent domains that contain both active and repressive histone modifications. The properties of polycomb proteins suggest that they act as gatekeepers, helping to maintain silencing in pluripotent stem cells while establishing a chromatin environment that is permissive for priming by sequence-specific factors. The overall effect of factor-mediated priming is to initiate the input of information required for cell differentiation before the first lineage choices have been made.

Pluripotency and its layers of complexity.

Pluripotency is depicted by a self-renewing state that can competently differentiate to form the three germ layers. Different stages of early murine development can be captured on a petri dish, delineating a spectrum of pluripotent states, ranging from embryonic stem cells, embryonic germ cells to epiblast stem cells. Anomalous cell populations displaying signs of pluripotency have also been uncovered, from the isolation of embryonic carcinoma cells to the derivation of induced pluripotent stem cells. Gaining insight into the molecular circuitry within these cell types enlightens us about the significance and contribution of each stage, hence deepening our understanding of vertebrate development. In this review, we aim to describe experimental milestones that led to the understanding of embryonic development and the conception of pluripotency. We also discuss attempts at exploring the realm of pluripotency with the identification of pluripotent stem cells within mouse teratocarcinomas and embryos, and the generation of pluripotent cells through nuclear reprogramming. In conclusion, we illustrate pluripotent cells derived from other organisms, including human derivatives, and describe current paradigms in the comprehension of human pluripotency.

Enhancing the Outcome of Cell Therapy for Cardiac Repair

Cell therapy has emerged as a promising option to treat myocardial infarction or heart failure; more than 1500 patients with cardiovascular diseases are treated with adult progenitor cells worldwide. The treatment of acute and chronic myocardial infarction with adult bone marrow-derived cells provided a modest benefit in most but not all studies. A number of plausible reasons have been discussed to explain the modest effects, sending researchers back to the bench to elucidate strategies to overcome the limitations of cell therapy and to develop more efficient approaches. Such strategies include the use of other sources to isolate adult progenitor cells (eg, adipose or cardiac tissue) or the generation of pluripotent cells by the reprogramming of somatic cell types. Successful cardiac cell therapy in clinical practice also depends on the efficient delivery and the appropriate integration and alignment of injected or infused cells. The pretreatment of cells with activators to augment cell homing and survival or the improvement of cell delivery tools may provide an opportunity to increase the number of active cells in ischemic or diseased tissues, thereby increasing their therapeutic potential. Finally, the identification of molecules guiding cardiac differentiation provides novel tools to enforce the formation of new functionally active cardiomyocytes to augment cardiac regeneration in its pure sense. This section provides a short overview of the different cell types that have been or are currently being tested for the treatment of patients with acute myocardial infarction or chronic heart failure (Figure 1). Most of the clinical studies performed so far have used bone marrow-derived mononuclear cells (BMCs) for treating patients with acute or chronic infarcts (for review, see elsewhere1). Overall, a modest but significant benefit was seen in meta-analyses of all published studies.2,3 Particularly in the acute setting, the infusion of BMCs via balloon catheters into …

Control of Phenotypic Plasticity of Smooth Muscle Cells by Bone Morphogenetic Protein Signaling through the Myocardin-related Transcription Factors

Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate, and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis, and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for bone morphogenetic proteins (BMPs), in patients with pulmonary arterial hypertension (PAH) provided an indication that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induces SMC-specific genes in pluripotent cells and prevents dedifferentiation of arterial SMCs. The BMP-induced phenotype switch requires intact RhoA/ROCK signaling but is not blocked by inhibitors of the TGFbeta and PI3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a smooth muscle alpha-actin promoter is observed in response to BMP treatment. Thus, BMP signaling modulates VSMC phenotype via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with PAH and other obliterative vascular diseases.

Differentiation of embryonic stem cells after transplantation into peritoneal cavity of irradiated mice and expression of specific germ cell genes in pluripotent cells

Permanent embryonic stem cell lines (ES cells) are considered as one of the most promising cellular sources for regenerative medicine. ES cells have a high proliferative potency and ability to differentiate into all kinds of somatic and germ cells. However, transplantation of undifferentiated ES cells into adult recipient tissue results in the formation of teratomas. To understand the mechanisms underlying self-renewal and determination of pluripotent cells, we investigated differentiation potencies of undifferentiated ES cells and differentiating embryoid bodies (EB). ES cells and EBs growing on acetate-cellulose membranes were transplanted into the peritoneal cavity of irradiated mice. Behavior and differentiation of transplanted cells were studied within 1, 2, 3, and 6 weeks after transplantation. No differences in the cell composition were found in the teratomas formed by ES cells and differentiating EBs. The pattern of expression of the genes specific for pluripotent and germ cells was studied in all types of experimental teratomas. The expression of oct4, stella, fragilis was detected in the teratomas, but nanog was not expressed. We conclude that pluripotent cells are retained in the experimental teratomas formed after transplantation of ES cells and EBs but the pattern of expression of the studied genes underwent changes.

Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control.

We have studied the initial development of pluripotent gut endoderm to hepatocytes using a tissue explant system from mouse embryos. We not only find cellular interactions that specify hepatic differentiation but also those that block hepatogenesis in regions of the endoderm that normally give rise to other tissues. The results implicate both positive and negative signaling in early hepatic specification. In vivo footprinting of the albumin enhancer in precursor gut endoderm shows that the transcriptionally silent but potentially active chromatin is characterized by occupancy of an HNF-3 site. Upon hepatic specification, a host of other factors bind nearby sites as the gene becomes active. Genes in pluripotent cells therefore may be marked for potential expression by entry points in chromatin, where additional factors bind during cell type specification. The findings also provide insight into the evolutionary origin of different endodermal cell types.