In vivo functional assessment of human ESC-derived skeletal myocytes

描述（由申请人提供）： 人胚胎干细胞（hES）可以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来选择性衍生不同的感兴趣的细胞类型。我们通过FACS分选人间充质标志物CD 73，从与小鼠OP 9基质细胞共培养的分化hES细胞中分离间充质前体。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中获得成骨细胞、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用于未来的治疗应用，我们开发了无基质分化方法，并证明了hES细胞可以再次分化为CD 73+间充质祖细胞。此外，我们建立了更具体的培养条件，从CD 73+前体细胞的成熟骨骼肌细胞的有效一代。基于我们的初步结果，显示纯化的人胚胎干细胞衍生的骨骼肌细胞可以融合形成肌管，该建议旨在研究人胚胎干细胞衍生的骨骼肌细胞在体内的功能，通过移植到免疫缺陷小鼠和小鼠模型的肌营养不良症。与hES衍生的骨骼肌细胞相关的优点是它们的高增殖能力、骨骼肌定型/干细胞标志物Pax 3和Pax 7的表达以及潜在增强的迁移能力。该项目的具体目标是：1）通过优化CD 73+前体选择性分化为骨骼肌细胞的培养条件，并通过鉴定由CD 73-细胞产生的导致骨骼肌分化增强的推定可溶性因子，表征hES细胞体外分化为骨骼肌细胞，和2）在小鼠模型中评估人工损伤后hES衍生的骨骼成肌细胞后代的体内功能能力。首先，我们将探索免疫缺陷小鼠（SCID Beige）心脏毒素诱导损伤后的肌肉再生，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，从该系统中分离肌肉分化所必需的因子可以帮助从hES获得更好的可移植肌细胞以及成体干细胞，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.