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.     

Discovery of nonsteroidal anti-inflammatory drug and anticancer drug enhancing reprogramming and induced pluripotent stem cell generation

Recent breakthroughs in creating induced pluripotent stem cells (iPSCs) provide alternative means to obtain embryonic stem-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. iPSCs are versatile tools for investigating early developmental processes and could become sources of tissues or cells for regenerative therapies. Here, for the first time, we describe a strategy to analyze genomics datasets of mouse embryonic fibroblasts (MEFs) and embryonic stem cells to identify genes constituting barriers to iPSC reprogramming. We further show that computational chemical biology combined with genomics analysis can be used to identify small molecules regulating reprogramming. Specific downregulation by small interfering RNAs (siRNAs) of several key MEF-specific genes encoding proteins with catalytic or regulatory functions, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, and TGFβ3, greatly increased reprogramming efficiency. Based on this rationale, we screened only 17 small molecules in reprogramming assays and discovered that the nonsteroidal anti-inflammatory drug Nabumetone and the anticancer drug 4-hydroxytamoxifen can generate iPSCs without Sox2. Nabumetone could also produce iPSCs in the absence of c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPSCs. In summary, we report a new concept of combining genomics and computational chemical biology to identify new drugs useful for iPSC generation. This hypothesis-driven approach provides an alternative to shot-gun screening and accelerates understanding of molecular mechanisms underlying iPSC induction.     

诱导多功能干细胞的研究进展

背景：由于胚胎干细胞移植存在致瘤性和伦理学争议,有关胚胎干细胞的研究及临床应用存在较大的限制。2006年Yamanaka实验室利用Oct3/4、Sox2、Klf4、c-Myc 4 种因子将鼠成纤维细胞重编程为诱导多功能干细胞,标志着一种新型类胚胎干细胞的问世。 目的：了解诱导多功能干细胞的研究进展和应用前景。 方法：由第一作者检索2006/2010 PubMed 数据库(http://www.ncbi.nlm.nih.gov/PubMed)及万方数据库(http://g.wanfangdata.com.cn/)有关诱导多功能干细胞的产生、细胞特征、产生技术的研究进展及应用前景等方面的文章,英文检索词为“induced pluripotent stem cells,defined factors,reprogramming,vectors,disease”,排除重复性研究,共保留其中的69篇进行归纳总结。 结果与结论：诱导多功能干细胞研究在诱导因子种类,因子导入方式,重编程效率及应用研究等诸多方面取得进展。然而体细胞重编程为诱导多功能干细胞仍存在一定的风险,重编程效率还非常低。一旦解决诱导多功能干细胞的安全性和重编程效率问题,诱导多功能干细胞就可被广泛应用于疾病模型,药物测试,细胞移植及患者和疾病特异性多功能干细胞的建立等诸多方面。     

MT1-MMP 基因敲除细胞的诱导多能干细胞系的构建

目的: 观察野生型鼠和膜1型基质金属蛋白酶( MT1-MMP )基因敲除鼠的成纤维细胞重新编程为诱导多能干细胞(iPSCs)后,其细胞特性是否具有胚胎干细胞(ESCs)相似的潜能。方法: 利用逆转录病毒介导 Oct3/4、Sox2、c-Myc和Klf4 四种基因,转染到野生型鼠和 MT1-MMP 基因敲除鼠的成纤维细胞中。iPSCs克隆形成后,用免疫细胞化学检测ESCs特异性标志的表达情况。体外将iPSCs通过"悬滴法"向内皮细胞和心肌细胞分化,以检测其分化能力。结果: 转染野生型鼠和 MT1-MMP 基因敲除鼠的成纤维细胞2周后,可见ESCs样克隆开始形成。iPSCs明显表达ESCs特异性标志物碱性磷酸酶(AP)、阶段特异性胚胎抗原-1(SSEA-1)和八聚体结合转录因子3/4(OCT3/4)。诱导分化的内皮细胞表达早期内皮标志物Flk-1/KDR;诱导分化的心肌细胞出现跳动,表达心肌标志物肌钙蛋白I。结论: 野生型鼠和 MT1-MMP 基因敲除鼠的成纤维细胞可被重新编程为iPSCs,iPSCs具有ESCs的特征及增殖分化能力,因此可能为再生医学的研究和临床上进行细胞移植治疗提供理想的种子细胞来源。     

可利用小鼠诱导多能干细胞的建立及鉴定

目的 建立小鼠诱导多能性干细胞(iPSCs)模型,为干细胞和iPSCs研究提供可利用资源。 方法 取C57BL/6J小鼠12.5d的胚胎成纤维细胞,感染含有Sox2、Oct4、Klf4和c-Myc的慢病毒进行重编程。病毒感染后12d挑取iPSCs的克隆进行扩大培养,进行碱性磷酸酶染色鉴定。体外悬滴培养检测拟胚体（EB）的形成,并皮下接种BABL/c裸鼠检测畸胎瘤形成。全反式维甲酸诱导iPSCs克隆株定向分化。PCR法进行种属鉴定并检测支原体。 结果 得到了12个碱性磷酸酶阳性的小鼠iPSCs克隆株,体外可以形成拟胚体,其中9个克隆株可以在BALB/c裸鼠体内形成畸胎瘤。全反式维甲酸可诱导iPSCs定向分化为平滑肌细胞。iPSCs克隆株种属鉴定为鼠源性,无支原体的污染,细胞资源中心入库保藏。结论 成功建立了小鼠iPSCs模型,为干细胞、iPSCs重编程机制及定向分化研究提供可利用的资源。     

Analysis of C-MYC function in normal cells via conditional gene-targeted mutation

Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.     

Development of histocompatible primate-induced pluripotent stem cells for neural transplantation

Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. To develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CMs by microsatellite polymerase chain reaction analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson's disease and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to 6 months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the preclinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.     

Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system

PiggyBac (PB) transposition of reprogramming factors (Oct3/4 (O), Sox2 (S), Klf4 (K) and c-Myc) is a safe, nonviral method for generating induced pluripotent stem cells (iPSCs). However, compared with retroviral methods, the reprogramming efficiency of the PB-mediated methods is relatively low. In this study, we describe a simple and efficient system for generating high-quality iPSCs by a single transfection of multiple plasmids that does not require the use of a virus, special instruments or skilled techniques. To improve reprogramming efficiency, we modified the components of the polycistronic 2A vectors used in this study and also investigated the combination of another reprogramming-related factor (L-Myc). By simultaneous transposition of multiple PB vectors containing an EOS (early transposon promoter and Oct3/4 and Sox2 enhancers) reporter and modified polycistronic doxycycline (Dox)-inducible factors, we reprogrammed mouse somatic cells with an efficiency higher than is usually obtained with retroviral methods and we established some iPSC lines that contributed highly to chimeras. By using the Dox-inducible system, we also showed that the appropriate elimination of exogenous-factor expression at appropriate time accelerated the induction of Oct3/4 when a combination of OKS and c-Myc vectors were used.     

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.     

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.     

阿尔茨海默病患者尿液细胞向诱导多能干细胞及神经细胞的诱导转化

目的:获得阿尔茨海默病(Alzheimer disease,AD)患者来源的诱导多能干细胞(induced pluripotent stem cells,i PSCs)。方法:选取3例临床诊断明确的AD病例,收集病人尿液,分离出尿路上皮细胞,对所得细胞进行原代培养。利用电转染的方法将带有Oct4、Sox2、Klf4和SV40LT的质粒导入原代细胞内,将其重编程为i PS细胞。随后利用双向抑制Smad通路的方式继续诱导其神经分化。结果:AD病人尿液来源的细胞(以下称为尿液细胞)均成功诱导成i PS细胞,其诱导效率与正常人来源的细胞无明显差异。且病人的i PS细胞可成功分化为神经细胞,分化效率亦与正常人来源细胞相近。结论:阿尔茨海默症患者来源的尿液细胞可重编程为i PS细胞,所得到的i PS细胞可成功分化成有功能的神经元以及神经胶质细胞。     

Percutaneous intrarenal transplantation of differentiated induced pluripotent stem cells into newborn mice

The in vivo engraftment of induced pluripotent stem cell (iPSC)-derived podocytes following allogeneic transplantation into host kidneys remains a challenge. Here we investigate the survival and engraftment of human dermal fibroblasts-derived differentiated iPSCs using a newborn mouse model, which represents a receptive immunoprivileged host environment. iPSCs were generated from skin biopsies of patients using Sendai virus reprogramming. Differentiation of nephrin (NPHS1)-green fluorescent protein (GFP) iPSCs into kidney podocytes (iPSC-PODs) was performed by the addition of Activin A, bone morphogenetic protein 7 (BMP7), and retinoic acid over 10 days of culture. To assess the in vivo incorporation of cells, undifferentiated iPSCs or day 10 iPSC-PODs, were labeled with either carboxyfluorescein succinimidyl ester (CFSE) or Qdot nanocrystals (Q705). Thereafter, 1 × 10⁵ differentiated iPSC-PODs were injected directly into the kidneys of mouse pups at postnatal day one (P1). Using co-expression analysis of glomerular and podocyte-specific markers, Day 10 differentiated iPSC-PODs that were positive for podocin, were detected following direct kidney injection into newborn mice up to 1 week after transplantation. Undifferentiated iPSC-PODs were not detected at the same timepoint. The transplanted cells were viable and located in the outer nephrogenic zone where they were found to colocalize with, or sit adjacent to, cells positive for glomerular-specific markers including podocin, synaptopodin, and Wilms' tumor 1 (WT1). This study provides proof-of-principle that transplanted iPSC–POD can survive in recipient newborn mouse kidneys due to the immature and immunoprivileged nature of the developing postnatal kidneys.     

Crucial role of c-Myc in the generation of induced pluripotent stem cells

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.     

Induced Pluripotent Stem Cells from Human Hair Follicle Mesenchymal Stem Cells

Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs.