Figure 4. Osteogenic differentiation in RIV9 and RIV4 hiPSCs.
(A) Photomicrographs of unstained and stained osteogenic cultures from H9 hESCs and RIv9 and RIV4 hiPSCs on day 30 of differentiation. Matrix mineralization detected with OCN, Aliz S, and Von Kossa stains.
(B) 20-day-old osteogenic hPSCs deposit significant levels of calcium ions over spontaneously differentiating cultures (n=5 independent samples ± SD). *P<0.05 One-Way ANOVA over spontaneous differentiations.
(C) Levels of inorganic phosphate levels measured with the PiPer assay on day 20 content, n=5 independent samples ± SD, *P<0.05 One-Way ANOVA over H9. Bottom graph depicts calcium:phosphate ratios determined from Ca2+ and PiPer assays, day 20, n=3 independent replicates ± SD. *P<0.05 One-Way ANOVA versus H9.
(D) Morphological time course of hPSC osteogenic differentiation reveals differences in calcification yield between different hPSC lines.
(E) Calcification time course analysis shows heterogeneity between osteogenesis in different hPSC lines. Levels of inorganic phosphate also vary depending on differentiation day; n=3 independent replicates ± SD. #P<0.05 RIV9-H9; *P<0.05 RIV4-H9, One-way ANOVA.
(F, G) RIV9 and RIV4 cultures begin their calcification process earlier than H9 cells as determined by video bioinformatics analysis, n=10 technical replicates from three biological replicates±SEM.
OCN, osteocalcin; Aliz S, Alizarin Red S.