Fig. 4.

WJ-MSC secrete SHH with biological activity. A reporter assay shows osteogenic differentiation of murine mesenchymal C3H10T1/2 cells in response to WJ-MSC-secreted SHH. AP-positive cells were quantified along with total cells and were graphed. a DMEM 0.5% FBS was the CM vehicle. b, c Pur and recombinant N-Shh were used as positive controls. d The CM from three independent WJ-MSC samples were used. To block WJ-MSC-secreted SHH, C3H10T1/2 were preincubated with Cyc before treatment with WJ-MSC CM (e) or ethanol as vehicle (f). Additionally, the CM was preincubated with 5E1 (g) or its denaturated form as control (d5E1) (h). i Histogram quantification is representative for three independent WJ-MSC samples. *P < 0.05, two-way ANOVA. j SHH was detected in cells lysates by Western blot. It should be noted that we detected both the immature (N-SHH, 45 kDa) and processed forms of SHH (20 kDa) indicative of WJ-MSC as a source of this ligand (N-Shh corresponds to the commercial ligand and N.T to the embryonic chicken neural tube, both used as positive controls). Scale bar = 100 μm. AP alkaline phosphatase, CM conditioned medium, Cyc cyclopamine, FBS fetal bovine serum, Pur purmorphamine, SHH Sonic hedgehog, WJ-MSC Wharton’s jelly-derived mesenchymal stem cells