Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E-ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ cells as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency.     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

Accessing naïve human pluripotency

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: na?ve and primed. Na?ve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the na?ve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether na?ve human pluripotent stem cells with bona fide features of both a pluripotent state and na?ve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of na?ve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture na?ve pluripotency in human cells. Unraveling how to stably induce na?ve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.     

Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles

Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.     

家畜胚胎干细胞多能性候选信号通路及分子标志

家畜胚胎干细胞具有重要的生物学意义和广阔的应用前景。以下对比了小鼠、人胚胎干细胞多能性调控信号通路的异同,阐述了小鼠、人胚胎干细胞与家畜胚胎干细胞在多能性分子标志上的差异,并结合本实验室开展绵羊胚胎干细胞研究的实际经验,对目前家畜胚胎干细胞建系中可能存在的多能性候选信号通路及分子标志进行了探讨。     

Gene expression profiles of human inner cell mass cells and embryonic stem cells

Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of preimplantation human blastocysts obtained on days 5–6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM cells isolated from the blastocyst and embryonic stem cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem cells indicated that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types. Genes of the isolated human inner cell mass that are upregulated and downregulated are involved in numerous cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.     

Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.     

单细胞测序分析人类胚胎干细胞神经分化的分子机制

目的 探讨人类胚胎干细胞(ESCs)分化为神经细胞的关键性靶基因及分子机制,为临床靶向治疗神经康复患者提供分子理论依据.方法 基于GEO数据平台芯片,采用单细胞测序方法(scRNA-seq),利用R语言从多分子维度(单细胞差异基因、蛋白互作网络和基因通路等)分析人类ESCs分化过程中的关键Marker基因并利用质控和数...     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells

Embryonic stem cells (ESCs) are progenitor cells that retain the ability to differentiate into various cell types and are necessary for tissue repair. Improving cell culture conditions to maintain the pluripotency of ESCs in vitro is an urgent problem in the field of regenerative medicine. Here, we reveal that Spautin-1, a specific small-molecule inhibitor of ubiquitin-specific protease (USP) family members USP10 and USP13, promotes the maintenance of self-renewal and pluripotency of mouse ESCs in vitro. Functional studies reveal that only knockdown of USP13, but not USP10, is capable of mimicking the function of Spautin-1. Mechanistically, we demonstrate that USP13 physically interacts with, deubiquitinates, and stabilizes serine/threonine kinase Raf1 and thereby sustains Raf1 protein at the posttranslational level to activate the FGF/MEK/ERK prodifferentiation signaling pathway in naïve mouse ESCs. In contrast, in primed mouse epiblast stem cells and human induced pluripotent stem cells, the addition of Spautin-1 had an inhibitory effect on Raf1 levels, but USP13 overexpression promoted self-renewal. The addition of an MEK inhibitor impaired the effect of USP13 upregulation in these cells. These findings provide new insights into the regulatory network of naïve and primed pluripotency.     

人类及小鼠胚胎干细胞的不同多能性状态

胚胎干细胞(embryonic stem cells,ESCs)是来源于早期胚胎的全能性细胞,在合适条件下具有分化为任何一类成体细胞的潜力。在小鼠中,根据细胞来源的胚胎发育时间,ESCs可以被分为原始态多能性(na(?)ve pluripotency)和始发态多能性(primed pluripotency)两种状态。这两种状态的细胞在发育上相互联系,具有不同的形态、信号依赖、发育性质、基因表达及表观遗传学性质,并且在特定的条件下可以相互转化。人类胚胎干细胞(human embryonic stem cells,hESCs)的发育潜能曾一度被认为低于小鼠胚胎干细胞(mouse embryonic stem cells,mESCs),直到人类原始态胚胎干细胞的发现证明了hESCs可以表现出与mESCs相似的性质。这对于人类胚胎发育的研究及ESCs在临床治疗上的实际应用都具有重要的意义。