转录因子Oct-4、Nanog与胚胎干细胞的自我更新

胚胎干细胞自我更新分子机制是胚胎干细胞研究的前沿及热点课题。除外源性信号如LIF、BMP、Wnt能维持胚胎干细胞的未分化状态外,转录因子Oct-4和Nanog特异性表达于全能胚胎干细胞,并与其它转录因子如Sox2一起构成调控网络,共同调控与胚胎干细胞多能性相关的一系列重要分子,是保持胚胎干细胞自我更新和多潜能性的关键分子。     

A molecular basis for human embryonic stem cell pluripotency

Embryonic stem cells (ESCs) are able to generate a wide array of differentiated cell fates while maintaining self-renewal. Understanding the biology of these choices may be central to the use of human embryonic stem cells (HESCs), both as a model for early human development as well as a resource for cell based therapies. Efforts to dissect the molecular mechanisms that mediate stem cell identity are underway, and in this review we summarize recent progress in defining the markers and pathways involved in these decisions. We discuss recent efforts to assess the molecular signature of pluripotent HESCs and highlight work demonstrating a set of genes, including representatives from the FGF, TGFβ, and Wnt signaling pathways, that consistently mark the undifferentiated state. In addition, we describe experiments in which signaling of HESCs is augmented by chemical probing with small molecule compounds. Using these compounds, we have demonstrated an important role for Wnt signaling in HESC pluripotency and shown a requirement for TGFβ signaling in the maintenance of the undifferentiated state. These experiments have revealed some molecular aspects of the pluripotent state and demonstrated clear differences between mouse and human ESCs in the maintenance of this identity.     

Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain "stemness" in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas.     

Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells

Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.     

Human amniotic fluid stem cells support undifferentiated propagation and pluripotency of human embryonic stem cell without b-FGF in a density dependent manner

Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P < 0.05). The hESCs were propagated more than 30 passages on hAF-AFSCs layer with exogenous b-FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10⁴/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.     

CDK及Myc等对干细胞周期和多能性的调控作用

干细胞具有自我更新和分化能力,其中自我更新与在持续增殖的细胞周期保持其多能性密切相关.因此,干细胞的周期改变和调节方式均能从多方面影响其多能性,如细胞周期蛋白在干细胞多能性调节中发挥着重要作用,其中,周期蛋白依赖性激酶（CDK）等对干细胞的增殖和分化具有举足轻重的影响;S期是遗传和表观遗传学调控影响干细胞多能性的重要时期;转录因子Myc平衡着干细胞多能性和定向分化能力等.从CDK及Myc分子对干细胞周期的调节和对多能性的影响等方面进行综述和探讨,以促进细胞周期对干细胞相关调控机制的研究,为再生医学后续干细胞的深入研究提供理论依据.     

The Effects of Nuclear Reprogramming on Mitochondrial DNA Replication

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.     

Maintenance of murine embryonic stem cells' self-renewal and pluripotency with increase in proliferation rate by a bovine granulosa cell line-conditioned medium

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications.     

Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation

Recent studies from our laboratory have shown that acellular substrates generated from human fibroblasts successfully maintained human pluripotent stem cells (hPSCs) in their undifferentiated state for extended periods. Aiming at better characterization, we conducted proteomic analyses to identify the extracellular matrix (ECM) proteins in mouse embryonic- and two human fibroblast-derived acellular substrates. Our studies identified heparan sulfate proteoglycan (HSPG) as a core component of these substrates and immunocytochemical analyses confirmed the presence of HSPG as well as other ECM proteins identified through proteomic analyses. In our attempt to develop surfaces that mimic fibroblast-deposited ECM and their self-renewal capabilities, substrates comprising HSPG and other core ECM proteins were formulated and assessed for the function of hPSC self-renewal. WA09 and BG01v hPSCs maintained on these substrates exhibit multiple characteristics of pluripotency, including (i) tight colony formation with typical stem cell morphology; (ii) positive expression of alkaline phosphatase, (iii) positive expression of SSEA3, SSEA4 and Oct4 based on immunocytochemical analyses; (iv) POU5F1, NANOG and SOX2 mRNA expression; and (v) in vitro differentiation and expression of germ-layer-specific markers. Our studies also reveal that although HSPG by itself-does not support hPSC self-renewal, a substrate that combines HSPG and fibronectin is sufficient for undifferentiated propagation of hPSCs. These studies form the basis for identification of appropriate ECM components in a substrate that synergistically promotes activation of adhesion and signaling pathways responsible for hPSC self-renewal.     

Standing on the shoulders of giants: The changing landscape of pluripotent stem cells in research

Stem cells have the remarkable property of self-renewal and differentiation. These two fundamental aspects have excited scientists and clinicians for decades. Stem cells are defined by their potency, with pluripotency being the most permissive and unipotency being the most restricted. In mammals, pluripotency represents cell types found in the preimplantation and early postimplantation embryo. However, these pluripotent cells are not stem cells per se, because they do not meet the criteria of self-renewal. Therefore, pluripotent stem cells are exclusively in vitro cell types that have provided scientists and clinicians with unprecedented power to study the fundamental cell and molecular properties of pluripotency, as well as providing a window into cellular differentiation and a source of cells for regenerative medicine including cell types that could be used to regenerate the kidney.     

Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells

Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a self-renewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.