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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoates / pharmacology*
  • Cartilage / metabolism
  • Cartilage / pathology
  • Cell Differentiation / drug effects*
  • Chondrocytes / cytology
  • Chondrocytes / metabolism*
  • Chondrocytes / transplantation
  • Chondrogenesis
  • Chromatin / metabolism
  • Collagen Type X / genetics
  • Collagen Type X / metabolism
  • Gene Expression
  • Humans
  • Mice
  • Mice, Inbred NOD
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • Pyridines / pharmacology*
  • Pyrimidines / pharmacology*
  • Receptors, Retinoic Acid / genetics
  • Receptors, Retinoic Acid / metabolism
  • Retinoids / pharmacology*
  • Wnt Signaling Pathway
  • beta Catenin / genetics
  • beta Catenin / metabolism


  • Benzoates
  • Chir 99021
  • Chromatin
  • Collagen Type X
  • Pyridines
  • Pyrimidines
  • Receptors, Retinoic Acid
  • Retinoids
  • beta Catenin
  • 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid