A myogenic double reporter human pluripotent stem cell line allows prospective isolation of skeletal muscle progenitors

Myogenic differentiation of iPSCs 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 novel double reporter hESC line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hESC/iPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors. CD10 expression was also confirmed on human satellite cells and muscle progenitors. In vivo studies using transgene/reporter-free hESC/iPSCs further validated myogenic potential of the cells by dystrophin restoration and satellite cell engraftment in NSG-mdx4cv mice. In addition, side-by-side in vitro and in vivo comparison proved superior specificity of CD10/CD24 compared to recently reported markers (ERBB3/NGFR). This study provides new biological insights for myogenic differentiation using a new cell resource and identifies CD10 as a potential myogenic marker. Overall design: hESCs were analyzed at 5 differentiation time-points (Day0, Day4, Day10-sorted for MYF5 EGFP positive or negative- and day20 myotubes after myogenic differentiation of MYF5 EGFP positive fraction). For each time-point, 3 independent biological replicates were harvested for further analysis. 1 x 106 cells were harvested / sorted at each time- point and RNA extracted using QIAGEN miRNeasy RNA isolation kit (217004, QIAGEN) according to manufacturer's protocol. Each RNA sample was converted to cDNA using oligo-dT primers and was further converted into indexed sequencing libraries using Illumina kit. Indexed libraries were subsequently pooled together and then sequenced on an Illumina HiSeq 4000 for a 50 cycle single end run. To compare transcriptome profile of PAX7/MYF5 double reporter hESC derived myogenic cells to human skeletal myoblasts, we enlisted ENCODE polyA RNA-Seq data of the human skeletal muscle myoblasts from the Wold lab (GEO: GSM958744).

诱导多能干细胞（induced pluripotent stem cells, iPSCs）的肌源性分化目前多通过基因过表达或定向分化策略完成，但病毒整合、长期培养以及非目标细胞残留均为该过程的主要障碍。本研究借助CRISPR/Cas9n系统构建了PAX7/MYF5双报告基因人类胚胎干细胞（human embryonic stem cells, hESCs）系，可实现前瞻性的细胞状态检测。该策略可用于通路筛选，以明确人类胚胎干细胞/诱导多能干细胞的高效肌源性诱导方案。后续通过表面标志物筛选，成功鉴定出可用于纯化肌源性祖细胞的CD10与CD24标志物，且证实CD10在人类卫星细胞与肌肉祖细胞中均有表达。借助无转基因/无报告基因的人类胚胎干细胞/诱导多能干细胞开展的体内实验，通过在NSG-mdx4cv小鼠中实现肌营养不良蛋白恢复与卫星细胞定植，进一步验证了该类细胞的肌源性潜能。此外，体外与体内平行对照实验证实，相较于近期报道的ERBB3与NGFR标志物，CD10/CD24具备更优异的特异性。本研究为肌源性分化提供了全新的细胞资源与生物学视角，并鉴定出CD10作为潜在的肌源性标志物。

整体实验设计：对人类胚胎干细胞在肌源性分化过程中的5个时间节点进行分析：第0天、第4天、第10天（分选MYF5 EGFP阳性与阴性细胞群），以及MYF5 EGFP阳性细胞群完成肌源性分化后的第20天（此时为肌管）。每个时间节点均设置3次独立生物学重复用于后续分析。每个时间节点收集/分选1×10^6个细胞，并按照试剂盒说明书，使用QIAGEN miRNeasy RNA提取试剂盒（货号217004，QIAGEN）提取总RNA。以寡聚dT引物将每份RNA样品反转录为cDNA，随后使用Illumina试剂盒构建带索引的测序文库。将所有带索引的文库混合后，在Illumina HiSeq 4000平台上进行50个循环的单端测序。为比较PAX7/MYF5双报告基因人类胚胎干细胞来源的肌源性细胞与人类骨骼肌成肌细胞的转录组谱，我们调取了Wold实验室发布的人类骨骼肌成肌细胞的ENCODE polyA RNA测序数据（GEO登录号：GSM958744）。