Fig. 4: CLEC14A is a marker for a human myogenic cell in satellite cell position.

a CLEC14A- staining of PAX7null, PAX7neg, PAX7pos cells. PAX7null cells are strongly positive for CLEC14A. Some PAX7neg cell populations from dornors with intact PAX7 gene are also CLEC14A-positive. Reconstitution of PAX7null cells with a lentiviral PAX7 vector changed the expression profile of CLEC14A and NCAM (bottom line). Red, PAX7; green, CLEC14A; blue, Hoechst. Bar 20 µm. b PAX7 and CLEC14A expression in PAX7neg cell colonies were mutual exclusive. Fifty-six cell populations from donors with intact PAX7 genes were analyzed for expression of PAX7 (green) and CLEC14A (red). Cell colonies are either positive for PAX7 or for CLEC14A. Some colonies contained both, cells that were either PAX7 or CLEC14A-positive. Staining for PAX7 and CLEC14A in “mixed” cell populations depicted mutually exclusive expression. Scale bar: 20 µm. c Gene expression in PAX7null cells selected by FACS-sorting (see also Supplementary Fig. 9) after lentiviral PAX7 transduction determined by qPCR 3, 6, and 12 days after DOX induction. All data were normalized to two reference genes and relativized to the mean of PAX7pos cell colonies. The dotted line at twofold and 0.5-fold represents the threshold for differential expression. d TA muscle section from a NOG mouse 3 weeks after transplantation of CLEC14Apos cells shows newly built human muscle fibers. Quantification reveals similar results as in Fig. 3b. Quantification as in 3c, but without PAX7 analysis. Each dot represents one mouse grafted with PAX7neg-CLEC14Apos cells (n = 8). Scale bar: 20 µm. e Staining for huCLEC14A, PAX3 and Syndecan-4 indicates cells in satellite cell position (arrows) in the muscle tissue of the PAX7null patient. Nuclei are stained with Hoechst (blue). About 8% of nuclei were positive for CLEC14A and localized in a satellite cell position (133 nuclei analyzed). Scale bars: 10 µm. f SmFISH for huCLEC14A in a frozen muscle section of a normal human donor. The red arrow indicates a nucleus in satellite cell position and the white one a endothelial cell nucleus. Scale bar: 20 µm.