Freshly isolated Lin-SP cells express FAPs surface markers but are capable of myogenic differentiation.

<p><b>A</b>: RT-PCR analysis of freshly isolated Lin- SP cells from wild type (WT) and <i>mdx^5cv</i> (MDX) muscle for myogenic markers (Pax7 and Myf5) and FAPs markers (PDGFRα and Sca1). Positive controls (PC) are sorted Sca1-positive cells for Sca1 and Lin- MP cells for Pax7, Myf5 and PDGFRα. Negative controls (NC) are sorted Sca1-negative cells for Sca1 and CD45-positive MP cells for Pax7, Myf5 and PDGFRα. <b>B, C</b>: FACS analysis of PDGFRα and Sca1 protein expression in Lin- SP cells (<b>B</b>) and Lin- MP cells (<b>C</b>) from wild type (WT) and <i>mdx^5cv</i> (MDX) muscle. Percentages of cells double positive (red) and double negative (green) for PDGFRα and Sca1 are shown. <b>D</b>: Confirmation by RT-PCR for PDGFRα expression in Lin- SP and Lin- MP cells sorted into PDGFRα-positive (Pα+) and PDGFRα-negative (Pα−) sub-fractions. <b>E</b>: <i>In vitro</i> myogenic differentiation of Lin- SP and MP cells sorted based on PDGFRα (Pα) expression. Cells were fixed after 14 days in culture and immunolabelled for α-actinin (green) to reveal myotubes. Cultures were counterstained with DAPI (blue) to visualize nuclei. Lin- MP Pα+ cells correspond to the previously characterized FAPs. Lin- MP Pα− cells are enriched in myogenic cells and also contain fibroblasts. Cultured wild type Lin- MP Pα− cells had 2,261 myotubes while cultured <i>mdx^5cv</i> Lin- MP Pα− cells had only 541 myotubes. Lin- SP Pα− cells did not survive in culture and are not shown. Scale bar = 400 μm. <b>E</b>: Cultures of Lin- SP Pα− cells were double labeled with antibodies to α-actinin (green) and collagen I (red) to visualize myotubes and fibroblasts, respectively. Cultures of Lin- SP Pα− cells from dystrophic muscle (MDX) do not contain myotubes but give rise to fibroblasts. Scale bar = 100 μm. <b>G</b>. Quantitative RT-PCR for Pax7 expression in Pdgfrα+ and Pdgfrα− Lin-SP cells, and Pdgfrα− Lin-MP cells. Data is presented as means +/− s.d. from 3 technical replicates.