In vivo functional assessment of human ESC-derived skeletal myocytes

描述(由申请人提供)： 人类胚胎干细胞(HES)可能为退行性疾病和癌症的治疗应用提供无限的细胞来源。一个挑战是利用HES细胞的巨大分化潜力选择性地衍生不同类型的感兴趣细胞。我们从与小鼠OP9基质细胞共培养的分化中的HES细胞中分离出间充质前体细胞，通过FACS分选人类间充质标志物CD73。通过应用标准间充质干细胞分化方案，我们成功地从这些前体细胞获得成骨细胞、脂肪细胞、软骨细胞和骨骼肌细胞。然而，为了确保这些细胞适合于未来的治疗应用，我们开发了一种无基质分化方法，并证明HES细胞可以再次分化为CD73+间充质祖细胞。此外，我们还为从CD73+前体细胞高效培养成熟骨骼肌细胞建立了更为特异的培养条件。基于我们的初步结果，表明纯化的HES来源的骨骼肌细胞可以融合形成肌管，本方案旨在通过移植到免疫缺陷小鼠和肌肉营养不良小鼠模型中研究HES来源的骨骼肌细胞在体内的功能。与HES来源的骨骼肌细胞相关的优势是其高增殖能力，表达骨骼肌承诺/干细胞标记Pax3和Pax7，以及潜在增强的迁移能力。本项目的具体目标是：1)体外研究人胚胎干细胞向骨骼肌细胞分化的特征，优化CD73+前体细胞向骨骼肌细胞选择性分化的培养条件，并鉴定CD73-细胞产生的促进骨骼肌分化的可能的可溶性因子(S)，以及2)在小鼠模型中评估人胚胎干细胞来源的骨骼肌成肌细胞后代在体内的功能能力。首先，我们将探索心脏毒素诱导免疫缺陷小鼠(SCID Beige)损伤后的肌肉再生，然后将结果转化为肌营养不良症小鼠模型(MDX小鼠)。值得注意的是，从这个系统中分离肌肉分化所必需的因子(S)可以帮助从HES获得更好的可移植心肌细胞，以及成人干细胞，因此具有重要的临床意义。

DESCRIPTION (provided by applicant): Human embryonic stem (hES) cells may provide an unlimited source of cells for therapeutic applications in degenerative diseases and cancer. One challenge is to harness the huge differentiation potential of hES cells towards selective derivation of different cell types of interest. We isolated mesenchymal precursors from differentiating hES cells cocultured with mouse OP9 stromal cells, by FACS sorting for the human mesenchymal marker CD73. By applying standard mesenchymal stem cell differentiation protocols, we succeeded in obtaining osteoblasts, adipocytes, chondrocytes, and skeletal myocytes from these precursors. Nevertheless, to ensure that these cells are suitable for future therapeutic applications, we developed a stroma-free differentiation method and demonstrated that hES cells could again be differentiated into CD73+ mesenchymal progenitors. In addition, we set up more specific culture conditions for the efficient generation of mature skeletal myocytes from the CD73+ precursors. Based on our preliminary results, showing that purified hES-derived skeletal myocytes can fuse to form myotubes, this proposal aims to study the function of hES-derived skeletal myocytes in vivo by transplantation into immunodeficient mice and in a mouse model of muscular dystrophy. The advantages associated with hES-derived skeletal muscle cells are their high proliferative capacity, expression of skeletal muscle commitment/stem cell markers Pax3 and Pax7, and the potentially enhanced migratory ability. The specific aims of this project are: 1) to characterize the in vitro differentiation of hES cells into skeletal myocytes, by optimizing the culture conditions for the selective differentiation of CD73+ precursors into skeletal myocytes, and by identifying putative soluble factor(s) produced by CD73- cells that lead to the enhancement of skeletal muscle differentiation, and 2) to assess the functional capacity of hES-derived skeletal myoblast progeny in vivo after artificial injury in a mouse model. First, we will explore muscle regeneration after cardiotoxin-induced lesions in immunodeficient mice (SCID Beige), and then translate the results into a mouse model of muscular dystrophy (mdx mice). Of note, isolation of factor(s) essential for muscle differentiation from this system could help to obtain better transplantable myocytes from hES, as well as adult stem cells, with consequently important clinical relevance.