Myogenic Differentiation of Muscular Dystrophy-Specific Induced Pluripotent Stem Cells for Use in Drug Discovery
Myogenic Differentiation of Muscular Dystrophy-Specific Induced Pluripotent Stem Cells for Use in Drug Discovery
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DOI：
10.5966/sctm.2013-0095
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发表时间：
2014-02-01
影响因子：
6
通讯作者：
Flynn, Peter
中科院分区：
文献类型：
--
作者：
Abujarour, Ramzey;Bennett, Monica;Flynn, Peter
人类诱导多能干细胞（iPSC）代表了用于疾病建模和治疗筛选的潜在任何细胞类型的可扩展来源。我们对各种遗传背景的骨骼肌建模特别感兴趣;然而，用于iPSCs肌源性分化的有效和可重复的方法以前尚未得到证实。异位肌源性分化1（MyoD）表达已显示在原代细胞类型中诱导肌生成，但在人类iPSC中复制相同的效果出人意料地具有挑战性。在这项研究中，我们报告了培养条件的优化使得直接MyoD介导的iPSCs分化为成肌细胞，而不需要中间步骤或细胞分选。MyoD诱导介导成熟肌细胞的有效细胞融合，产生多核肌球蛋白重链阳性肌管。我们将相同的方法应用于营养不良的iPSC，从四名Duchenne和Becker肌营养不良患者的成纤维细胞中产生了16个iPSC系。如来自健康供体的iPSC所示，在MyoD诱导后36小时内，培养物中的大多数iPSC（50%-70%）明确致力于肌原性。患者iPSC衍生的肌管成功地采用了骨骼肌程序，如通过全局基因表达谱所确定的，并且在功能上响应于用肥大蛋白胰岛素样生长因子1（IGF-1）和无翼型MMTV整合位点家族成员7A（Wnt 7a）的治疗，其分别在临床和临床前研究中被研究为肌营养不良症的潜在治疗。我们的研究结果表明，iPSCs没有内在的障碍，阻止MyoD诱导有效和快速的肌生成，从而提供了一个可扩展的正常和营养不良的成肌细胞来源，用于疾病建模和药物发现。
Human induced pluripotent stem cells (iPSCs) represent a scalable source of potentially any cell type for disease modeling and therapeutic screening. We have a particular interest in modeling skeletal muscle from various genetic backgrounds; however, efficient and reproducible methods for the myogenic differentiation of iPSCs have not previously been demonstrated. Ectopic myogenic differentiation 1 (MyoD) expression has been shown to induce myogenesis in primary cell types, but the same effect has been unexpectedly challenging to reproduce in human iPSCs. In this study, we report that optimization of culture conditions enabled direct MyoD-mediated differentiation of iPSCs into myoblasts without the need for an intermediate step or cell sorting. MyoD induction mediated efficient cell fusion of mature myocytes yielding multinucleated myosin heavy chain-positive myotubes. We applied the same approach to dystrophic iPSCs, generating 16 iPSC lines from fibroblasts of four patients with Duchenne and Becker muscular dystrophies. As seen with iPSCs from healthy donors, within 36 hours from MyoD induction there was a clear commitment toward the myogenic identity by the majority of iPSCs in culture (50%-70%). The patient iPSC-derived myotubes successfully adopted the skeletal muscle program, as determined by global gene expression profiling, and were functionally responsive to treatment with hypertrophic proteins insulin-like growth factor 1 (IGF-1) and wingless-type MMTV integration site family, member 7A (Wnt7a), which are being investigated as potential treatments for muscular dystrophy in clinical and preclinical studies, respectively. Our results demonstrate that iPSCs have no intrinsic barriers preventing MyoD from inducing efficient and rapid myogenesis and thus providing a scalable source of normal and dystrophic myoblasts for use in disease modeling and drug discovery.