Fig. 1.

Directed iPSC differentiation to skeletal cells. (A) Schematic showing the differentiation stages from iPSCs to articular and hypertrophic chondrocytes and then toward osteoblasts. Differentiation to sclerotome in the first 6 d includes aggregating monolayer cells into pellets at day 4. Culture medium and conditions are shown below and above the timeline and culture formats are illustrated at the bottom. (B) Chondronoids spontaneously mature toward hypertrophy, and hypertrophy can be enhanced with T3. Toluidine blue-stained sections (MCRIi019-A) at days 48 and 69. Some chondronoids were treated with T3 for 3 wk before harvesting at day 69. Scale bar is 500 µm. Immunostaining shows that the chondronoids contain an extensive collagen II-rich ECM throughout the time course. Collagen X is not apparent at day 48 but has been deposited into the ECM by day 69. (Scale bar is 200 µm.) (C) TEM at day 52 showing a typical chondrocyte and an extensive network of collagen II fibrils in the ECM. (D) Changes in mRNA abundance of selected cartilage, hypertrophic cartilage, and bone proteins. N = 3 independent differentiation experiments. (E) Most highly expressed core matrisome genes. Those not reaching the statistical threshold for differential expression (adj.P.value < 0.05) in at least one of the comparisons are indicated with a red asterisk in D and E.