The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12 days using defined growth factors and small molecules. This is followed by 28 days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages28
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Chondrogenesis
  • Human iPSCs
  • Stem cells
  • Tissue-engineered cartilage


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