5-Aminoimidazole-4-carboxyamide ribonucleoside induces G(1)/S arrest and Nanog downregulation via p53 and enhances erythroid differentiation

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling energy metabolism, is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest, without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers, Nanog and stage-specific embryonic antigen-1, in the presence of leukemia inhibitory factor, without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently, an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation, an effect inhibited by MG132, a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs, it increased expression levels of erythroid cell lineage markers (Ter119, GATA1, Klf1, Hbb-b, and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR, endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs.     

In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins

Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.     

Embryoid body-mediated differentiation of mouse embryonic stem cells along a hepatocyte lineage: insights from gene expression profiles

Pluripotent embryonic stem (ES) cells represent a promising renewable cell source for the generation of functional differentiated cells. Previous studies incorporating embryoid body (EB)-mediated stem cell differentiation have, either spontaneously or after growth factor and extracellular matrix protein supplementation, yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB). In an effort to promote ES cell commitment to the hepatocyte lineage, we have evaluated the effects of four culture conditions on albumin and gene expression in differentiating ES cells. Quantitative in situ immunofluorescence and cDNA microarray analyses were used to describe not only lineage specificity but also to provide insights into the effects of disparate culture environments on the mechanisms of differentiation. The results of these studies suggest that spontaneous and collagen-mediated differentiation induce cells with the highest levels of ALB expression but mature liver specific genes were only expressed in the spontaneous condition. Further analysis of gene expression profiles indicated that two distinct mechanisms may govern spontaneous and collagen-mediated differentiation.     

Efficient hematopoietic redifferentiation of induced pluripotent stem cells derived from primitive murine bone marrow cells

Heterogeneity among induced pluripotent stem cell (iPSC) lines with regard to their gene expression profile and differentiation potential has been described and at least partly linked to the tissue of origin. Here, we generated iPSCs from primitive [lineage negative (Lin(neg))] and nonadherent differentiated [lineage positive (Lin(pos))] bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and embryonic stem cells (ESC). In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction toward the hematopoietic lineage. All 3 BM-iPSC lines derived from undifferentiated Lin(neg) cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41 and in 2 of these lines high proportions of CD41+/ CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in 4 Lin(pos) BM-iPSC and 3 Fib-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in 2 of the Lin(neg) BM-iPSCs only. Thus, in conclusion, our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for Lin(neg) BM-iPSC lines, thereby supporting the notion of an epigenetic memory in iPSCs.     

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.     

Generation of retinal cells from mouse and human induced pluripotent stem cells

We previously reported a technique for generating retinal pigment epithelia (RPE) and putative photoreceptors from embryonic stem (ES) cells. Here we tested whether our procedure can promote retinal differentiation of mouse and human induced pluripotent stem cells (iPSCs). Treating iPSCs with Wnt and Nodal antagonists in suspension culture induced expression of markers of retinal progenitor cells and generated RPE cells. Subsequently, treatment with retinoic acid and taurine generated cells positive for photoreceptor markers in all but one human cell lines. We propose that iPSCs can be induced to differentiate into retinal cells which have a possibility to be used as patient-specific donor cells for transplantation therapies.     

质粒重编程诱导多潜能干细胞及定向分化神经干细胞

目的 　探讨游离质粒载体重编程诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)为非整合型诱导多潜能干细胞,并在体外定向分化为神经干细胞(neural stem cells, NSCs), 为神经干细胞移植治疗神经损伤提供稳定、安全的细胞来源。 方法　使用电转仪将质粒pEP4-EO2S-ET2K转入小鼠MEFs, 经诱导培养重编程为诱导多潜能干细胞(induced pluripotent stem cells, iPSCs), iPSCs在不同诱导培养基中经2次悬浮及贴壁培养分化为NSCs,在体内及体外实验鉴定iPSCs多向分化潜能特性及NSCs特性。 结果　体内外实验显示iPSCs具有与胚胎干细胞(embryonic stem cells, ESCs)相似的多向分化潜能, 且不整合外源性基因。iPSCs进一步分化的NSCs其相关标志基因表达与野生型NSCs相近,且较iPSCs显著增加,免疫荧光显示NSCs高表达NSC标志物 NESTIN及PAX6,在体外存活能分化为神经元、少突胶质细胞及星形胶质细胞。 结论　游离质粒能重编程诱导非整合型iPSCs, 并定向分化为神经干细胞及神经元, 是神经损伤修复的理想种子细胞。     

Surface Marker Epithelial Cell Adhesion Molecule and E-cadherin Facilitate the Identification and Selection of Induced Pluripotent Stem Cells

The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1 (SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation.     

Embryonic stem cell models of development.

Pluripotent mouse embryonic stem (ES) cell lines have provided a means to analyze gene function in development via gene targeting. At the same time, they provide an opportunity to directly probe gene function by assessing the in vitro differentiation capacity of the ES cells themselves. In addition to providing direct data on lineage decisions not accessible in the complex three-dimensional milieu of the early mouse embryo, controlled differentiation of ES into specific lineages may provide a source of cells for transplantation and gene therapy.     

Reprogramming induced pluripotent stem cells in the absence of c-Myc for differentiation into hepatocyte-like cells

Induced pluripotent stem cells (iPSCs) with four reprogramming factors (Oct-4/Sox2/Klf-4/c-Myc) have been shown to differentiate into hepatic lineages. However, it was unclear whether obviation of the c-Myc oncogene in iPSCs affected hepatic differentiation or inhibited in?vivo tumor formation. In this study, we demonstrated that iPSCs without c-Myc had the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) with biological functions. As detected using planar-radionuclide imaging and Hoechst labeling assays, these iPSCs and iPSC-Heps tended to mobilize to the injured liver area in thioacetamide (TAA)-treated mice. Intravenous transplantation of both iPSCs and iPSC-Heps but not mouse embryonic fibroblasts (MEFs) reduced the hepatic necrotic area, improved liver functions, and rescued TAA-treated mice from lethal acute hepatic failure (AHF). In addition, microarray-based bioinformatics and quantitative RT-PCR showed high expression of antioxidant genes in iPSCs and iPSC-Heps compared to MEFs. In vivo and in?vitro studies of NAC pretreatment confirmed that iPSCs and iPSC-Heps potentially suppressed ROS production and activated antioxidant enzymes in TAA-injured livers. Six months after transplantation in TAA-treated mice, tumor formation was not seen in non-c-Myc iPSC grafts. Therefore, reprogramming adult somatic cells without c-Myc may prevent oxidative stress-induced damage and provide a safer alternative for hepatic regeneration in AHF.     

配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析

目的 建立配对盒基因6（Pax6）/小鼠胚胎干细胞（mESCs）细胞系并鉴定其干细胞生物学特性。 方法 体外培养mESCs,将重组载体pEF1α-Pax6-IRES-AcGFP和空载体pEF1α-IRES-AcGFP分别用脂质体法转染mESCs,经G418梯度及荧光蛋白双筛选后,使用细胞免疫荧光染色、免疫印迹法及RT-PCR技术检测Pax6的表达情况,流式细胞术检测Pax6/mESCs阳性细胞的比例。将获得正确的细胞系分别采用细胞免疫荧光染色对其干细胞标志物阶段特异性胚胎抗原1（SSEA1）、八聚体结合转录因子4（OCT4）进行检测,碱性磷酸酶（AP）染色法对其多能性进行检测,流式细胞术检测增殖指数Ki67。将Pax6/mESCs进行肾背囊下移植,移植物行HE染色观察其分化能力。 结果 Pax6成功在mESCs内表达,经G418筛选后,获得Pax6/mESCs细胞系,流式结果显示,Pax6阳性率为90%,免疫荧光显示,干细胞标志物SSEA1、OCT4表达阳性且AP染色阳性,并且在体内移植后能向3个胚层分化。 结论 Pax6成功在mESCs内表达,经G418筛选后,获得细胞系Pax6/mESCs并维持良好的干细胞特性。     

Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells

The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra-Web) whose three-dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self-renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self-renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3-kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three-dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self-renewal.     

Differentiation and transplantation of functional pancreatic Beta cells generated from induced pluripotent stem cells derived from a type 1 diabetes mouse model

The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.     

In vitro direct osteogenesis of murine embryonic stem cells without embryoid body formation

Embryonic stem cells (ESCs) posses the ability to self-renew and differentiate into a multitude of lineages, including the osteogenic lineage in vitro. Currently, most approaches have focused on embryonic body (EB)-mediated osteogenic differentiation, which relies on formation of all three germ layers resulting in limited yields and labour-intensive culture processes. Our study aimed at developing an efficient culture strategy resulting in the upregulated in vitro osteogenic differentiation of murine ESCs (mESCs), which completely avoided EB formation. Specifically, mESCs were cultured in HepG2 conditioned medium for 3 days and then directed into osteogenic differentiation for 21 days without prior EB formation. The mineralised bone nodules generated were characterized by Alizarin red S-staining, phenotypic alkaline phosphatase expression, time-course analysis of ALPase activity, the presence of type I collagen and osteopontin, and osteocalcin, cbfa-1/runx-2, and osterix gene expression. Our method of direct osteogenic differentiation of mESCs represents a novel and efficient approach that results in enhanced yields and could have significant applications in bone tissue engineering.