Focal Adhesion Kinase and ROCK Signaling Are Switch-Like Regulators of Human Adipose Stem Cell Differentiation towards Osteogenic and Adipogenic Lineages

Figure 3

Osteogenic differentiation of hASCs in BM, OM, and AM culture conditions supplemented with FAK, ERK, and ROCK inhibitors. (a) The cells were cultured in BM, OM, or AM supplemented with 2 μM FAK, 40 μM ERK, or 15 μM ROCK inhibitors in addition to medium controls. RUNX2A expression was analyzed with qRT-PCR at 7 d. FAK and ROCK: (independent experiments, 5 donors), ERK: (independent experiments, 3 donors). (b) ALP activity was analyzed with ALP assay at 7 d and 14 d. The ALP absorbance values were normalized with corresponding CyQUANT results, and the results are presented relative to the 7 d BM sample. Significance level 5%, designated with an asterisk (). FAK, ERK, ROCK: (independent biological replicates from 3 donors). (c) Matrix mineralization was analyzed with AR staining after 14 d and 21 d of culture. Quantitative results of AR staining are presented as graphs and corresponding representative images of the stained wells (21 d, area 1.9 cm²) are presented below; bright red dye represents mineral. Significance level 5%. FAK, ROCK: (independent biological replicates from 6 donors, control condition values of the graphs are the same since the experiments were conducted at the same time), ERK: (independent biological replicates from 5 donors). BM: basic medium; OM: osteogenic medium; AM: adipogenic medium; ALP: alkaline phosphatase; AR: Alizarin Red.

(a) RUNX2A expression

(b) ALP activity

(c) Mineralization