Figure 7.

FAPs enhance myogenic differentiation. (a) Immunohistological analysis of co-cultures revealed increased MP differentiation in the presence of FAPs. MPs (5,000) were isolated from GFP⁺ mice and co-cultivated with 5,000 GFP⁻ FAPs or GFP⁻ MPs. After ten days, cultures were fixed and stained for MyHC. Data are expressed as the ratio of nuclei in GFP⁺MyHC⁺ cells (myonuclei) to total nuclei in GFP⁺ cells. (b) FAPs induced MP differentiation in a dose-dependent manner. GFP⁺ MPs were co-cultivated with increasing numbers of GFP⁻ FAPs and re-isolated by FACS after ten days. The expressions of markers of myogenic differentiation were analysed by qRT-PCR using Taqman probe and primer sets spanning exon–exon boundaries. TBP, TATA-binding protein. P values were determined using ANOVA. (c) FAPs increase MP commitment to terminal differentiation. GFP⁺ MPs were co-cultivated with equal numbers of GFP⁻ FAPs or GFP⁻ MPs and re-isolated by FACS after seven days. qRT-PCR analysis was performed as described in b. Gene expression is presented in arbitrary units. (d) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay revealed little difference in proliferation between MP-only or MP-FAP co-cultures. Assays were performed on freshly sorted cell cultures containing either 5,000 MPs, or 2,500 MPs co-cultured with 2,500 FAPs for seven days. Data are expressed as absorbance at 570 nm (A570). (e) No difference in BrdU incorporation in MPs exposed to equal numbers of GFP⁺ MPs or GFP⁺ FAPs. GFP⁻ MPs (5,000) were co-cultivated with either 5,000 GFP⁺ MPs or 5,000 GFP⁺ FAPs for a total of seven days. BrdU (10 μM) was applied during the last 24 h of co-culture. GFP⁺ cells were removed using FACS and immunostaining for BrdU was performed on the remaining GFP⁻ MPs. Error bars in a–e represent the mean ± s.e.m., n = 3 (b, c and e), 4 (d) or 6 (a). *P < 0.05, ***P < 0.001 (Student’s t-test).