Fig. 3

Effect of GDF6 treatment on mesenchymal lineage commitment of H9 hESCs. a Flow cytometry analysis of CD73, CD146, and CD271 expression in vehicle- and GDF6-treated H9 cells. b ALP staining and quantitative ALP activity assay after 14 days of OI for vehicle- and GDF6-treated H9 cells. c ARS staining and quantification after 14 days of OI for vehicle- or GDF6-treated H9 cells. d Alcian blue staining and quantification after 21 days of CI for vehicle- or GDF6-treated H9 cells. qRT-PCR gene expression analysis of osteogenic (ALPL, RUNX2, IBSP, OCN) (e) and chondrogenic markers (SOX9 and COL2a1) (f) after lineage-specific differentiation in H9 cells cultured in the presence or absence of GDF6. All in vitro experiments were performed three times independently. **P < 0.01, ***P < 0.001. For b–d, Student’s t test; for e and f, two-way ANOVA with Tukey’s post hoc test