核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。     

High-efficient generation of induced pluripotent stem cells from human astrocytes

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.     

Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.     

疾病特异诱导多能干细胞研究进展

通过病毒或非病毒转导体系,在小鼠和人的体细胞中人为表达几个与细胞多能性相关的转录因子,从而使细胞达到类似于胚胎干细胞（embryonic stem cells,ESCs）状态,是近年来新发展起来的体细胞重编程技术。这些被重编程的细胞称为诱导多能干细胞（induced pluripotent stem cells,iPS细胞）。这项技术为获得患者和疾病特异的多能干细胞提供了新的途径。患者和疾病特异的iPS细胞的获得,不仅在避免免疫排斥的宿主特异的细胞移植治疗上有广泛前景,并对了解疾病发生机理、药物筛选和毒性研究有着重要的意义。该文综述从iPS细胞技术的发明入手,着重讨论疾病iPS细胞的研究进展及其在应用于治疗时亟需解决的问题。     

Natural and artificial routes to pluripotency

Pluripotent cells of the blastocyst inner cell mass (ICM) and their in vitro derivatives, embryonic stem (ES) cells, contain genomes in an epigenetic state that are poised for subsequent differentiation. Their chromatin is hyperdynamic in nature and relatively uncondensed. In addition, a large number of genes are expressed at low levels in both ICM and ES cells. Also, the chromatin of naturally pluripotent cells contains specialized histone modification patterns such as bivalent domains, which mark genes destined for later developmentally-regulated expression states. Female pluripotent cells contain X chromosomes that have yet to undergo the process of X chromosome inactivation. Collectively, these features of very early embyronic chromatin are required for the successful specification and production of differentiated cell lineages. Artificial reprogramming methods such as somatic nuclear transfer (SCNT), ES cell fusion-mediated reprogramming (FMR), and induced pluripotency (iPS) yield pluripotent cells that recapitulate many features of naturally pluripotent cells, including many of their epigenetic features. However, the route to pluripotent epigenomic states in artificial pluripotent cells differs drastically from that of their natural counterparts. Here, we compare and contrast the differing routes to pluripotency under natural and artificial conditions. In addition, we discuss the intrinsically metastable nature of the pluripotent epigenome and consider epigenetic aspects of reprogramming that may lead to incomplete or inaccurate reprogrammed states. Artificial methods of reprogramming hold immense promise for the development of autologous cell graft sources and for the development of cell culture models for human genetic disorders. However, the utility of artificially reprogrammed cells is highly dependent on the fidelity of the reprogramming process and it is therefore critically important to assess the epigenetic similarities between embryonic and induced pluripotent stem cells.     

Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.     

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.     

A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.     

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。