多顺反子质粒重编程人脂肪干细胞为诱导多能干细胞

为建立多顺反子质粒载体转染技术获得人脂肪干细胞(adipose stem cells,ASCs)来源的诱导多能干细胞(induced pluripotency stem cells,iPSCs),应用2A元件连接Oct4/Sox2/KLF4/c-Myc四因子基因,构建为单一开放阅读框的多顺反子质粒载体．使用该质粒对ASCs进行转染及重编程为iPSC．采用形态学观察、特异性抗体免疫荧光鉴定、体外拟胚体诱导分化和体内畸胎瘤形成等方法进行鉴定．结果显示,ASCs成功重编程为iPSCs,具有与人胚胎干细胞相似的形态学及多向分化潜能;通过拟胚体和畸胎瘤实验证实iPSCs能在体内外分化成三胚层细胞;DNA印迹实验显示质粒载体序列未整合至iPSCs基因组中．因此,通过多顺反子质粒载体重编程技术成功建立的人iPSCs具有多向分化潜能,可减免发生插入突变和免疫排斥问题,为iPSCs在遗传性或退行性疾病的治疗奠定了实验基础．     

分化细胞经特定因子诱导重编程为多能干细胞

胚胎干细胞在再生医学领域有着十分诱人的应用前景。但是现有胚胎干细胞建系技术不能避开对卵细胞的操作, 成为ES细胞临床应用的障碍。通过反转录病毒载体系统, 在小鼠和人类高度分化细胞中表达干细胞因子Oct4, Sox2, Klf4和/或c-Myc等基因, 再经过干细胞标志因子Nanog或Oct4筛选, 可以获得与ES细胞特性十分近似的诱导多能干细胞系。这种不依赖于卵细胞的多能干细胞建系方法无疑是干细胞实验技术的重大进展, 也是对现有重编程理论假设的突破。综述了诱导多能干细胞系建系实验结果, 并对诱导重编程的机制和诱导多能干细胞系的临床应用前景进行了讨论。     

基于诱导性多潜能干细胞的亨廷顿舞蹈病发病机制研究

目的:基于诱导性多潜能干细胞(induced pluripotent stem cells,i PSC)多潜能性的特点将亨廷顿舞蹈病(Huntington disease,HD)患者和正常人特异性i PSC定向诱导分化成运动神经元,并在运动神经元的基础上探讨HD的发病机制。方法:将HD患者和正常人的i PS细胞在特定的生长因子和神经因子的作用下定向诱导分化成运动神经元。然后用免疫荧光染色检测运动神经元特异性标记物HB9和ISL1的表达。以DCFH-DA和JC-1为荧光探针,利用流式分析法分别对正常人和HD患者运动神经元细胞活性氧和线粒体膜电位进行检测。结果:经过25天诱导分化成功得到HD患者和正常人的运动神经元,并且免疫荧光染色显示,βIII-微管蛋白阳性的神经细胞同时表达运动神经元特异性的标志物HB9和ISL1。此外,经实验统计发现HD患者运动神经元细胞内代表活性氧水平的荧光强度(4704.33±390.50)较正常组(2840.33±166.20)有明显增强(P=0.002),而且代表线粒体膜电位红绿荧光强度比(2.74±0.13)较正常组(3.97±0.29)相比有明显降低(P=0.03)。结论:HD患者特异性i PSC能够诱导分化成运动神经元,为实验提供研究模型。HD的发病与运动神经元细胞线粒体功能障碍有关。     

Learning Biomarkers of Pluripotent Stem Cells in Mouse

Pluripotent stem cells are able to self-renew, and to differentiate into all adult cell types. Many studies report data describing these cells, and characterize them in molecular terms. Machine learning yields classifiers that can accurately identify pluripotent stem cells, but there is a lack of studies yielding minimal sets of best biomarkers (genes/features). We assembled gene expression data of pluripotent stem cells and non-pluripotent cells from the mouse. After normalization and filtering, we applied machine learning, classifying samples into pluripotent and non-pluripotent with high cross-validated accuracy. Furthermore, to identify minimal sets of best biomarkers, we used three methods: information gain, random forests and a wrapper of genetic algorithm and support vector machine (GA/SVM). We demonstrate that the GA/SVM biomarkers work best in combination with each other; pathway and enrichment analyses show that they cover the widest variety of processes implicated in pluripotency. The GA/SVM wrapper yields best biomarkers, no matter which classification method is used. The consensus best biomarker based on the three methods is Tet1, implicated in pluripotency just recently. The best biomarker based on the GA/SVM wrapper approach alone is Fam134b, possibly a missing link between pluripotency and some standard surface markers of unknown function processed by the Golgi apparatus.     

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.     

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

The laboratory rat was one of the earliest mammalian species for scientific research and used as animal disease models in physiology,toxicology,behavior,immunology,and tumor-biology for over 150 years (Jacob,1999).However,rat lags far behind mouse in generating human disease models and functional genomic studies because of the lack of authentic rat embryonic stem (ES) cells (Voigt and Serikawa,2009),whereas the first mouse ES cell line was established in 1981 (Evans and Kaufman,1981).By combining two or three kinase inhibitors which target GSK3,MEK and FGF signaling pathways in serum-free N2B27 medium,germline competent rat ES cells were first derived in 2008 (Buehr et al.,2008;Li et al.,2008).     

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。     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells

1. Download : Download high-res image (210KB)
2. Download : Download full-size image

     

小鼠ES细胞种系嵌合体的获得

种系嵌合体的获得是实现ＥＳ细胞介导的转基因途径的决定步骤,ＥＳ细胞种系分化能力的保持是决定种系嵌合的前提条件,而事体的主种系嵌合体的获得则是判定ＥＳ细胞系是否具有种系分化能力的唯一方法,为考察本室新近建立的３种小鼠ＥＳ细胞系ＭＥＳＰＵ２１．ＭＥＳＰＵ２２和ＭＥＳＰＵ２９的种系分化能力,选用近交系Ｃ５７ＢＬ／６Ｊ及远交系ＫＭＷ和ＩＣＲ为受体胚胎提供者,分别通过囊胚注射法和８细胞期桑椹胚注射法进行了嵌     

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.     

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD⁺-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.     

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate ^1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich''s ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged ^4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich''s ataxia patients and control individuals ⁶, human newborn fibroblasts, as well as human keratinocytes.     

Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics

Induced pluripotent stem cells (iPSCs) and their differentiated derivatives can potentially be applied to cell-based therapy for human diseases. The properties of iPSCs are being studied intensively both to understand the basic biology of pluripotency and cellular differentiation and to solve problems associated with therapeutic applications. Examples of specific preclinical applications summarized briefly in this minireview include the use of iPSCs to treat diseases of the liver, nervous system, eye, and heart and metabolic conditions such as diabetes. Early stage studies illustrate the potential of iPSC-derived cells and have identified several challenges that must be addressed before moving to clinical trials. These include rigorous quality control and efficient production of required cell populations, improvement of cell survival and engraftment, and development of technologies to monitor transplanted cell behavior for extended periods of time. Problems related to immune rejection, genetic instability, and tumorigenicity must be solved. Testing the efficacy of iPSC-based therapies requires further improvement of animal models precisely recapitulating human disease conditions.     

Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells

A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.