Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds

• Published in: Stem cell reports 2019-09, Vol.13 (3), p.530-544
• Main Authors: Kawata, Manabu, Mori, Daisuke, Kanke, Kosuke, Hojo, Hironori, Ohba, Shinsuke, Chung, Ung-il, Yano, Fumiko, Masaki, Hideki, Otsu, Makoto, Nakauchi, Hiromitsu, Tanaka, Sakae, Saito, Taku
• Format: Article
• Language: English
• Subjects: cartilage; chondrocyte; chondrocyte differentiation; pluripotent stem cells; regenerative medicine; small-molecule compound
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2019.07.012

A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5–9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation. [Display omitted] •Combination of a GSK3 inhibitor and an RAR agonist induces chondrocytes from hPSCs•The present protocol shows excellent differentiation efficiency•The potential to form hyaline cartilaginous tissues in vivo is demonstrated•RA and Wnt/β-catenin signals collaboratively regulate key genes for chondrogenesis Saito and colleagues show that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, a GSK3 inhibitor and an RAR agonist, within 5–9 days. Genome-wide analysis of RAR and β-catenin association with DNA for sequential samples at each protocol stage demonstrates that RA and Wnt/β-catenin signaling is collaboratively involved in direct regulation of chondrocyte differentiation.