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Stem Cell Reports. 2019 Sep 10;13(3):530-544. doi: 10.1016/j.stemcr.2019.07.012. Epub 2019 Aug 8.

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

Author information

1
Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
2
Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
3
Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
4
Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
5
Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
6
Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
7
Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Bone and Cartilage Regenerative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan. Electronic address: tasaitou-tky@umin.ac.jp.

Abstract

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.

KEYWORDS:

cartilage; chondrocyte; chondrocyte differentiation; pluripotent stem cells; regenerative medicine; small-molecule compound

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