Fig. 4.

Characterization of hiPSC-derived Chondrogenic Cells. (a) Relative gene expression of hiPSC-derived hiPSC-MSCs at day 21 (equal to day 0 of chondrogenic differentiation) and chondrogenic cell pellets at day 28 (equal to day 7 of chondrogenic differentiation) and 35 (equal to day 14 of chondrogenic differentiation), as determined by qPCR. Data are displayed as means and standard errors of triplicate experiments per sample. Cells at day 14 of chondrogenic differentiation show significantly increased gene expression of the hyaline chondrogenic markers COL2A1, COL9A1, COL11A1, SOX9, and aggrecan (ACAN) compared to hiPSC-MSCs. They also show an increased expression of COL1A2 and COL10A1 representative of fibro- and hypertrophic cartilage respectively. (*** indicates p < 0.001). (b) Histological evaluation of hiPSC-derived chondrogenic cell pellets at day 42 (equal to day 21 of chondrogenic differentiation); H&E stain shows chondrocytes and formation of a chondrogenic matrix. Alcian blue stain demonstrates positive glycosaminoglycan production, and immunohistochemistry shows positive stains for Collagen type II. (left 100 μm, right 50 μm). (c) Histological evaluation of hiPSC-MSCs osteogenic (upper panel) and adipogenic (lower panel) differentiation. Alizarin Red S staining used for osteogenic differentiation evaluation and Oil Red O staining used to assess the adipogenic differentiation of hiPSC-MSCs after 3 weeks of differentiation