Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_ AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/ AKT/ mTOR (mammalian target of rapamycin) pathway or GSK3 beta inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells

NaNog: A pluripotency homeobox (master) molecule.

One of the most intriguing aspects of cell biology is the state of pluripotency, where the cell is capable of self-renewal for as many times as deemed necessary , then at a specified time can differentiate into any type of cell. This fundamental process is required during organogenesis in foetal life and importantly during tissue repair in health and disease. Pluripotency is very tightly regulated, as any dysregulation can result in congenital defects, inability to repair damage, or cancer. Fuelled by the relatively recent interest in stem cell biology and tissue regeneration, the molecules implicated in regulating pluripotency have been the subject of extensive research. One of the important molecules involved in pluripotency, is NaNog, the subject of this article

Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, “hyper-pluripotent” state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency

Co-expression networks in generation of induced pluripotent stem cells.

We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation

Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans

Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.

DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1⁻/⁻) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1⁻/⁻;Dnmt3a⁻/⁻;Dnmt3b⁻/⁻ or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1⁻/⁻ and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1⁻/⁻ and TKO EBs. Despite late wild type and Dnmt1⁻/⁻ EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1⁻/⁻ and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation