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Simple Two Small-Molecule Compound Approach to the Chondrogenic Differentiation of iPSCs

Review of “Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds” from Stem Cell Reports by Stuart P. Atkinson

The generation of chondrocytes from human pluripotent stem cells (PSCs) via the exposure to a potent cocktail of small molecules compounds represents a promising strategy both for those studying chondrocyte differentiation and those looking to employ resulting cells to cartilage regeneration strategies. A previous study from the laboratory of Taku Saito (The University of Tokyo, Tokyo, Japan) used CHIR99021 to activate canonical Wnt signaling (by suppressing β-catenin degradation via the inhibition of glycogen synthase kinase (GSK) 3β activity) as a means to promote the osteoblastic differentiation of mouse embryonic stem cells [1]. CHIR99021 treatment of human PSCs may also be useful for the chondrogenic differentiation, given the mesendodermal characteristics of both osteoblasts and chondrocytes. Additionally, the team knew that retinoic acid (RA) and retinoids played essential roles in limb bud formation and subsequent chondrogenesis during skeletal development [2, 3], and so may be of use in the chondrogenic differentiation of PSCs. 

Now, researchers from the Saito laboratory describe how the treatment of human induced pluripotent stem cells (hiPSCs) with CHIR99021 and an RA receptor agonist (TTNPB) robustly induced the formation of large amounts of chondrocytes that form hyaline cartilaginous tissues in vivo [4]. Furthermore, this new study also delves into the molecular mechanisms controlling differentiation in the hope of understanding the process at a more profound level.

Kawata et al. discovered that hiPSCs exposed to CHIR99021 and TTNPB passed through mesendoderm and mesoderm stages before chondrocyte formation within five to nine days. Encouragingly, transplantation of chondrogenically differentiated hiPSCs into articular cartilage defects in model mice led to the robust generation of hyaline cartilaginous tissues in vivo with a lack of teratoma formation or other tumor formation observed around the transplanted joints or in other organs.

Their subsequent analyses delved into the programs controlling the differentiation process. First, an initial microarray analysis of sequential samples of differentiating hiPSCs found evidence for a step-by-step differentiation to chondrocytes; a finding confirmed via Genomic Regions Enrichment of Annotations Tool (GREAT) gene ontology analysis of transposase-accessible chromatin using sequencing (ATAC-seq) data that describes the sequential opening and closing of chromatin elements. Chromatin immunoprecipitation sequencing (ChIP-seq) with an anti-human RARα antibody combined with ATAC-seq then demonstrated that RA receptor alpha bound to enhancers around mesendodermal, mesodermal, and chondrogenic genes at various differentiation stages of chondrocyte differentiation, while β-catenin ChIP-seq suggested that Wnt/β-catenin signaling collaboratively regulates crucial chondrogenesis-associated gene expression with RA signaling at multiple stages of differentiation.

The authors of this new study suggest that their simple method, the exposure of hiPSCs to a Wnt activator and an RA receptor agonist, will provide the large numbers of cells required for the repair/regeneration of cartilage defects and for in vitro modeling purposes to explore of the molecular mechanisms underlying chondrocyte differentiation.

For more on how the directed differentiation of human induced pluripotent stem cells may provide a means to generate cells for cartilage regeneration and modeling efforts, stay tuned to the Stem Cells Portal!


  1. Kanke K, Masaki H, Saito T, et al., Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions. Stem Cell Reports 2014;2:751-760.
  2. Niederreither K, Subbarayan V, Dollé P, et al., Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nature Genetics 1999;21:444-448.
  3. Niederreither K, Vermot J, Schuhbaur B, et al., Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse. Development 2002;129:3563.
  4. Kawata M, Mori D, Kanke K, et al., Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds. Stem Cell Reports 2019;13:530-544.