Figure 7
Characterization of hiPSC chondrogenic differentiation in 3D by DMMB assay for sulfated glycosaminoglycans. Chondrogenic differentiation of hiPSCs was assessed by culturing hiPSCs in fibrin gels followed by culture in APEL2/GSKi and subsequent culture in chondrogenic differentiation medium for a total of 21 days in culture after encapsulation. The influence of the duration of LPM differentiation or of chemical treatment during LPM differentiation (for 2 days) on chondrogenic differentiation was assessed by sulfated glycosaminoglycan assay on day 21. (A) Mean ± SEM sGAG/DNA, normalized to the mTeSR1 control for each experiment, representing 3 independent experiments (mean of each experiment plotted as a separate point) comparing the hiPSCs that were cultured in APEL2/GSKi for 1, 2 or 3d followed by 19, 18, or 17 d of chondrogenic differentiation, respectively. Asterisks denote statistically significant increase in normalized sGAG/DNA relative to the mTeSR1 undifferentiated control via one-way ANOVA and Tukey’s post-hoc test at α = 0.05. (B) Mean ± SEM sGAG content per DNA content from each condition from 3 independent experiments (mean of each experiment plotted as a separate point) wherein the hiPSCs were cultured in APEL2/GSKi for 2 d in the presence of either 0.1% DMSO, 20 µM thalidomide, or 3 µM atRA and subsequently cultured in chondrogenic differentiation medium for 18 d with no chemical treatments. Asterisks denote a statistically significant decrease relative to the APEL2/GSKi DMSO control via one-way ANOVA and Tukey’s post-hoc test at α = 0.05.
