A Myogenic Double-Reporter Human Pluripotent Stem Cell Line Allows Prospective Isolation of Skeletal Muscle Progenitors.
A Myogenic Double-Reporter Human Pluripotent Stem Cell Line Allows Prospective Isolation of Skeletal Muscle Progenitors.
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DOI：
10.1016/j.celrep.2018.10.067
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发表时间：
2018-11-13
期刊：
影响因子：
8.8
通讯作者：
Darabi R
中科院分区：
文献类型：
--
作者：
Wu J;Matthias N;Lo J;Ortiz-Vitali JL;Shieh AW;Wang SH;Darabi R
人类多能干细胞(HPSCs)的成肌分化是通过基因过度表达或定向分化来实现的。然而，病毒整合、长期培养以及多余细胞的存在是主要障碍。利用CRISPR/Cas9n构建了PAX7/MYF5双报告基因的人胚胎干细胞(HESC)系。这一策略使得通路筛选能够确定hPSCs中有效的生肌诱导。接下来，表面标记筛选可以鉴定CD10和CD24，以纯化肌源性祖细胞和排除非肌源性细胞。CD10在人类卫星细胞和骨骼肌祖细胞上也有表达。使用转基因和/或无报告的hPSCs的体外和体内研究进一步证实了该细胞的肌肉生成潜力，方法是在NSG-mdx4Cv小鼠中形成表达人肌营养不良蛋白的新纤维以及供体来源的卫星细胞。本研究利用双报告细胞来源为hPSCs的成肌分化提供了生物学见解，并确定了一种改进的成肌分化和纯化策略。Wu等人。研究表明，通过为骨骼肌谱系生成双报告的人类胚胎干细胞系，可以筛选控制通路和表面标记的细胞，定义成肌诱导和纯化方案，并利用肌肉营养不良小鼠模型的体内研究评估其再生潜力。
Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/ MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy. Wu et al. demonstrate that, by generation of a double-reporter human embryonic stem cell line for skeletal muscle lineage, cells can be screened for governing pathways and surface markers, defining myogenic induction and purification protocol and evaluation of their regenerative potential using in vivo studies in a mouse model for muscular dystrophy.
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DOI：
10.1073/pnas.1610605114
发表时间：
2017-06-06
影响因子：
11.1
作者：
Buckingham, Margaret
通讯作者：
Buckingham, Margaret
DOI：
10.1007/7651_2014_134
发表时间：
2016-01-01
期刊：
INDUCED PLURIPOTENT STEM (IPS) CELLS
影响因子：
--
作者：
Darabi, Radbod;Perlingeiro, Rita C. R.
通讯作者：
Perlingeiro, Rita C. R.
影响因子：
5.2
作者：
Arpke, Robert W.;Darabi, Radbod;Kyba, Michael
通讯作者：
Kyba, Michael
影响因子：
82.9
作者：
通讯作者：
--
影响因子：
--
作者：
Hagos, Engda G.;Dougan, Scott T.
通讯作者：
Dougan, Scott T.