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STEM_625_sm_suppinfofigures.pdf1541KFigure S1 - Myogenic gene expression induced by Pax3 and Pax7. Real time RTPCR expression analysis for Pax3, Pax7, Myf5, MyoD, and myogenin in monolayer outgrowths from the PDGFαR+Flk-1- cell fraction, sorted from uninduced and induced day 5 iPax3 and iPax7 EBs. Transcripts are normalized to GAPDH. Error bars indicate standard errors from 3 independent experiments. ** p<0.01 compared to iPax3-induced cells, *** p<0.001 compared to iPax3-induced cells, and +++ p<0.001 compared to iPax7-induced cells. Figure S2 - Engraftment of Pax7-induced ES-derived myogenic progenitors into mdx mice following intra-arterial transplantation. Pax7-induced PDGFαR+Flk-1-- derived cells were injected into the femoral artery, and 4 weeks later muscles were sectioned and stained for dystrophin and GFP (n=8 mice). Immunostaining for dystrophin (red) and GFP (green) reveals the presence of GFP+Dystrophin+ myofibers in transplanted mice. Scale bar is 100 μm. Figure S3 - FACS characterization of the GFP+ mononuclear fraction in transplanted muscles. (AB) Left panels show FACS analyses for GFP in control and transplanted muscles (iPax3 and iPax7, respectively) which had been dissociated with collagenase. GFP+ cells were then counterstained with antibodies to relevant surface markers (right panels). Plots show isotype or secondary control staining profile (gray line) versus specific antibody staining profile (red line). Percentages represent the fraction of cells that express a given surface antigen. These analyses were performed in 2 mice per group (iPax3 and iPax7), which gave similar results. Figure S4 - Presence of donor-derived satellite cells following the transplantation of ES-derived myogenic progenitors into Pax7−/− mice. Immunofluorescence analyses of Pax7−/− mice transplanted with (A) Pax3- or (B) Pax7-induced ES-derived myogenic progenitors (a total of 4 and 2 transplanted mice, respectively). The upper panel shows single myofibers stained with GFP (Alexa 488), and M-cadherin (Alexa 555) or Pax7 (Alexa 555) antibodies, in addition to DAPI. Scale bar is 100 μm. Figure S5 - FACS profile of ex vivo expanded GFP+ cells purified from iPax3 and iPax7 transplanted muscles. GFP+ donor derived cells obtained from mdx mice that had been transplanted with Pax3- (A) or Pax7-induced (B) ES-derived myogenic progenitors were expanded in proliferation medium with and without dox induction and evaluated by FACS analyses. Plots show isotype or secondary control staining profile (gray line) versus specific antibody staining profile (red line). Percentages represent the fraction of cells that express a given surface antigen.

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