Fig 2: PMSCs cultured under muscle differentiation conditions increased levels of muscle markers (Pax3/7, MyoD, MyoG, and MHC) and decreased OCT4 and SOX2 levels using Western blots. Compared to PMSCs cultured under control conditions, (a) OCT4 was decreased at 1 and 14 days under muscle differentiation conditions. (b) SOX2 levels were decreased at each time point under muscle differentiation conditions. (c) Pax3/7 was increased at day 7 and decreased by day 14 under muscle differentiation conditions. (d) MyoD, (e) MyoG, and (f) MHC were increased at each time point under muscle differentiation conditions. Protein levels were quantified by densitometry and normalized to ß-actin. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni's multiple comparison test was performed to determine *P < 0.05, **P < 0.01, and ***P < 0.001 comparing control to muscle differentiation conditions, or #P < 0.05, ##P < 0.01, and ###P < 0.001 comparing the same treatment over time.

Fig 3: Model of IGFBP-6 functions on PMSC differentiation into skeletal muscle. PMSCs under normal growth conditions (10% FBS) express high levels of pluripotency-associated markers OCT4 and SOX2. As these cells commit towards the skeletal muscle lineage, increased IGFBP-6 correlated with increased Pax3/7 that decreased as differentiation markers (MyoG, MyoD, and MHC) were increased. Both committed and differentiated muscle cells continued to express and secrete IGFBP-6. As IGFBP-6 increased, there was an increase in multipotency markers, as well as, an earlier commitment and differentiation towards the muscle lineage. Thus, IGFBP-6 was required for maintaining multipotency and enhancing muscle commitment and differentiation.

Fig 4: PMSCs treated with IGFBP-6 increased pluripotency-associated and muscle differentiation markers. IGFBP-6, OCT4, SOX2, Pax3/7, MyoD, MyoG, and MHC protein levels were quantified within PMSCs grown in muscle differentiation media with or without IGFBP-6 (375 ng/mL) supplementation using Western blots. (a) IGFBP-6 treatment increased IGFBP-6 levels as compared to PMSCs grown in muscle differentiation media only. IGFBP-6 treatment also increased pluripotency-associated markers (b) OCT4 and (c) SOX2 levels. (d) IGFBP-6 treatment increased muscle lineage commitment marker Pax3/7 at each time point. Muscle differentiation markers (e) MyoD, (f) MyoG, and (g) MHC levels increased with IGFBP-6 treatment at early time points (1–3 days) but showed reduced levels at later time points (7–14 days). Protein levels were quantified by densitometry and normalized to ß-actin. Data is presented as the mean ± SEM of 3 independent experiments. Two-way ANOVA with Bonferroni's multiple comparison test was performed to determine *P < 0.05, **P < 0.01, and ***P < 0.001 comparing treatments, or #P < 0.05, ##P < 0.01, and ###P < 0.001 comparing the same treatment over time.