GSE121154
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).