Low microRNA-199a expression in human amniotic epithelial cell feeder layers maintains human-induced pluripotent stem cell pluripotency via increased leukemia inhibitory factor expression

Human-induced pluripotent stem (iPS) cells share the same key properties as embryonic stem cells, and may be generated from patient- or disease-specific sources, which makes them attractive for personalized medicine, drug screens, or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state is a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, as they express endogenous leukemia inhibitory factor (LIF) at high levels. Here, we examined the effect of exogenous microRNA-199a regulation on endogenous LIF expression in HuAECs, and in turn on human iPS cell pluripotency. We found that HuAECs feeder cells transfected with microRNA-199a mutant expressed LIF at high levels, allowing iPS to maintain a high level of alkaline phosphatase activity in long-term culture and form teratomas in severe combined immunodeficient mice. The expression of stem cell markers was increased in iPS cultured on HuAECs feeder cells transfected with the microRNA-199a mutant, compared with iPS cultured on HuAECs transfected with microRNA-199a or mouse embryo fibroblasts. Taken together, these results suggested that LIF expression might be regulated by microRNA-199a, and LIF was a crucial component in feeder cells, and also was required for maintenance of human iPS cells in an undifferentiated, proliferative state capable of self-renewal.     

Proliferation Rate of Somatic Cells Affects Reprogramming Efficiency

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.     

c-Myc对诱导性多能干细胞的诱导效率及全能性影响的研究

该研究探讨转录因子c—Myc对多能诱导干细胞（inducedpluripotentstemcells,iPS）诱导效率及形成的iPS克隆全能性的影响。将Yam,dnaka四因子（Oct4、Sox2、Klf4和C—Myc,OSKM）和不舍C。Myc的三因子（OSK）病毒分别感染OG2小鼠成纤维细胞（mouseembryofibroblast,MEF）,诱导成为iPSN胞,通过计数iPS克隆形成数目和流式细胞仪分析iPS克隆的绿色荧光蛋白（GFP）阳性比例。比较OSKM和OSK诱导iPS的效率。分别挑取三株OSK和OSKM诱导的iPS克隆,采用荧光定量PCR法、碱性磷酸酶fAP）染色法和免疫荧光法检测iPS克隆的干性相关蛋白的表达;采用畸胎瘤实验、嵌合体形成实验和四倍体补偿实验检测iPS克隆的全能性。结果显示：在该实验室诱导体系里,OSK病毒感染MEF后形成的iPS克隆数目明显增多,GFP阳性克隆的比例增加。OSK和OSKM诱导iPS克隆的干细胞基本特征,包括形态、AP染色、干细胞特异性基因表达、三胚层形成均没有明显差异。然而,OSK诱导iPS克隆在形成嵌合体小鼠的全能性评估实验中的阳性比例高于OSKM克隆,且只有OSK克隆株能够获得生殖系传递小鼠和四倍体补偿小鼠。该研究表明,在实验诱导体系里去除C—Myc的三因子OSK诱导iPS的效率显著高于OSKM四因子,且三因子诱导iPS克隆具有更好的全能性。     

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder‐free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round‐shaped cells resembling immature oocytes. Mvh⁺ cells formed clumps by co‐aggregation with differentiation‐supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency. Mol. Reprod. Dev. 77: 802–811, 2010. © 2010 Wiley‐Liss, Inc.     

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem(iPS)cells can be derived from human somatic cells by cellular reprogramming.This technology provides a potential source of non-controversial therapeutic cells for tissue repair,drug discovery,and opportunities for studying the molecular basis of human disease.Normally,mouse embryonic fibroblasts(MEFs)are used as feeder layers in the initial derivation of iPS lines.The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cells reprogrammed from somatic cells using lentivirai expressed reprogramming factors.In our study,iPS cells expressed common pluripotency markers,displayed human embryonic stern cells(hESCs)morphology and unmethylated promoters of NANOG and OCT4.These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.     

Inhibition of the integrin signal constitutes a mouse iPS cell niche

Stem cells are regulated by their surrounding microenvironments, called niche, such as cell–cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS‐MEF‐Ng‐20D‐17, iPS‐MEF‐Ng‐178B‐5 and iPS‐MEF‐Fb/Ng‐440A‐3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder‐less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells.     

定量分析诱导山羊体细胞重编程过程中端粒酶的表达变化

动物体细胞重编程为诱导多能干细胞(iPS)是目前干细胞生物学研究的热点。文中重点对山羊体细胞重编程过程中端粒酶(TERT)基因的相对表达量进行了检测,探讨了山羊重编程细胞的形成与端粒酶基因表达的关系。从关中奶山羊胎儿皮肤分离得到的胎儿成纤维细胞(GEF),其增殖能力较强,核型正常(60条XY),通过转录因子在体外诱导得到山羊重编程细胞。利用Real-timeRT-PCR方法首先对关中奶山羊胎儿各种组织的TERT表达进行了检测,结果表明睾丸组织中TERT的表达显著高于上皮组织(P0.01),在山羊胎儿的其他组织中TERT也有不同程度的表达。对原代重编程细胞和4株不完全重编程细胞株的TERT表达检测结果发现,碱性磷酸酶(AP)阳性的重编程细胞端粒酶表达量要显著高于AP阴性的重编程细胞(P0.01)。这一结果揭示,激活端粒酶活性并使其保持较高的表达水平对体细胞的重编程至关重要。     

哺乳动物DNA甲基化及其在体细胞诱导重编程中的作用

诱导多能干细胞(Induced pluripotent stem cell, iPS)技术提供了将终末分化的细胞逆转为多潜能干细胞的可能, 在干细胞基础理论研究和再生医学中具有重要意义。然而, 目前体细胞诱导重编程方法效率极低, 常发生不完全的重编程。研究表明, 在不完全重编程的细胞中存在体细胞的表观遗传记忆, 而DNA甲基化作为相对长期和稳定的表观遗传修饰, 是影响重编程效率和iPS细胞分化能力的重要因素之一。哺乳动物DNA甲基化是指胞嘧啶第五位碳原子上的甲基化修饰, 常发生于CpG位点。DNA甲基化能够调节体细胞特异基因和多能性基因的表达, 因此其在哺乳动物基因调控、胚胎发育和细胞重编程过程中发挥着重要作用。此外, 异常DNA甲基化可能导致iPS细胞基因印记的异常和X染色体的失活。文章重点围绕DNA甲基化的机制、分布特点、及其在体细胞诱导重编程中的作用进行了综述。     

Regulation of somatic cell reprogramming through inducible mir-302 expression

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.     

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem(iPS) cells can be derived from human somatic cells by cellular reprogramming.This technology provides a potential source of non-controversial therapeutic cells for tissue repair,drug discovery,and opportunities for studying the molecular basis of human disease.Normally,mouse embryonic fibroblasts(MEFs) are used as feeder layers in the initial derivation of iPS lines.The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cell...     

Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.