人胚胎干细胞建系，鉴定及分化为多能性间充质样前体细胞

发布时间：2018-03-25 19:34

本文选题：人胚胎干细胞　切入点：建系　出处：《浙江大学》2009年博士论文

【摘要】：人胚胎干细胞(hESC)是一类来源于植入前囊胚内细胞团(ICM),具有体外无限增殖潜能且能在特定条件下分化为多类成体组织或胚外组织的细胞群体。它作为体外研究人类胚胎发育的优势模型,进行药物筛选的良好平台,参与细胞治疗的无限资源,越来越受到全世界科研工作者的关注。目前,建立能广泛适用于临床的hES细胞系成为世界关注的焦点。然而大部分已经建立的hES细胞系,包括NIH认可的在内,均在建系之初以及后续的培养过程中直接或间接地接触到非人源化物质,这将限制其未来的临床应用前景。虽然人源饲养层培养体系,hESC自体来源的饲养层培养体系,以及多种无饲养层培养体系或许能安全有效地培养hESC,但是完全不接触任何异源物质的ICM分离过程也亟待建立。我们采用非接触性激光辅助孵出系统结合连续的培养来获得孵出囊胚,并以此为材料,不通过“免疫手术”等可能接触异源物质的操作,建立起一株属于中国族群的hES细胞系ZJUhES-1。该细胞系符合hESC的相关标准:具备典型的形态学特征;表达一系列hESC特异性标志;能长期增殖并维持稳定的二倍体核型;能在体内体外分化为多个胚层的细胞。同时,DNA指纹图谱鉴定证实ZJUhES-1具有特有的身份,不同于其它已报道细胞系。成功建立干细胞治疗需要具备多向分化潜能且免疫相容的干细胞群体,高效的定向分化技术,以及能安全有效地扩增干细胞群体的培养体系。除了所熟知的一些成体组织外,hESC逐渐成为又一类能分离获得间充质样细胞的组织来源。然而hESC单细胞消化后难以存活的特征,限制其目前的分化研究和未来的临床应用。因此我们采用ROCK抑制剂Y-27632提高hESC单细胞消化贴壁后的存活率,并以此单层系统为基础,利用无血清无血清替代物且成分明确的N2B27培养体系,将hESC分化为间充质样前体细胞(hESC-MP),整个过程不接触任何动物源性物质。分化所得的细胞呈现成纤维样细胞形态,符合MSC的评价标准,如自我更新潜能,表达多种MSC特异性标志,能分化为成骨细胞,脂肪细胞和软骨细胞等间质细胞。与其他文献报道的hESC-MSC相比,我们所获得hESC-MP还能分化为平滑肌细胞,心肌细胞,功能性肝细胞和表达不同神经递质的神经细胞,这使得它们更适合于未来的临床应用。此外,我们还通过Y-27632协助的单层诱导系统在含血清的培养条件下将hESC分化为另一类MSC。与N2827培养体系中获得的hESC-MP相比,这类hESC-MSC表达更高比例的MSC特异性分子标志,并呈现更强的增殖潜能。我们将其移植入四氯化碳(CCl_4)诱导的小鼠急性肝损伤模型中,初步发现这类细胞能改善肝功能,并促进肝组织自身修复。
[Abstract]:Human embryonic stem cells (ESCs) are a group of cells derived from pre-implantation blastocysts, which have unlimited proliferative potential in vitro and can differentiate into many kinds of adult or extraembryonic tissues under certain conditions. It is used to study human beings in vitro. The dominant model of embryonic development, More and more researchers all over the world pay more and more attention to the excellent platform for drug screening and unlimited resources for cell therapy. At present, the establishment of hES cell lines, which can be widely used in clinical applications, has become the focus of attention in the world. However, most of the established hES cell lines, including those recognized by NIH, Both of them were exposed directly or indirectly to non-humanized substances at the beginning of the establishment and the subsequent culturing process, which will limit their future clinical application prospects, although the human feeder layer culture system has autologous feeding layer culture system of hESC. And several culture systems without feeding layer may be able to culture hESCs safely and effectively, but the separation process of ICM without exposure to any heterogenous substance is urgent. We use non-contact laser-assisted hatching system combined with continuous culture. To get hatched blastocysts, Using this as material, a hES cell line ZJUhES-1, which belongs to Chinese population, was established without possible exposure to heterogenous substances such as "immune surgery". The cell line conforms to the relevant criteria of hESC: it has typical morphological characteristics; Expression of a series of hESC specific markers; long-term proliferation and maintenance of stable diploid karyotype; in vitro and in vivo differentiation into multiple embryonic layers of cells. At the same time, the identification of ZJUhES-1 fingerprinting confirmed that ZJUhES-1 has a unique identity. Different from other reported cell lines. The successful establishment of stem cell therapy requires a multi-directional differentiation potential and immune compatible stem cell population, efficient directional differentiation technology, In addition to some familiar adult tissues, hESC has gradually become the source of another kind of tissue that can isolate and obtain mesenchymal cells. However, hESC single cells are difficult to survive after digestion. Therefore, we use Y-27632, a ROCK inhibitor, to improve the survival rate of hESC after digesting and sticking, and base on this monolayer system. HESC was differentiated into mesenchymal precursor cells (hESC-MPN) by using serum-free and serum-free N2B27 culture system with clear components, and no contact with any animal-derived substances was observed in the whole process. The differentiated cells were fibroblast-like cells. According to the evaluation criteria of MSC, such as self-renewal potential, expression of many specific markers of MSC, differentiation into interstitial cells such as osteoblasts, adipocytes and chondrocytes. Our hESC-MP can also differentiate into smooth muscle cells, cardiomyocytes, functional hepatocytes and nerve cells expressing different neurotransmitters, which makes them more suitable for future clinical applications. In addition, we also differentiated hESC into another kind of MSCs in serum-containing culture by a monolayer induction system assisted by Y-27632. Compared with the hESC-MP obtained in N2827 culture system, these hESC-MSC expressed a higher proportion of MSC specific molecular markers. We transplanted it into CCl4)-induced acute liver injury model in mice and found that this kind of cells could improve liver function and promote liver self-repair.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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