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The study of human embryonic stem cells (hESCs) can provide invaluable insights into the development of numerous human cell and tissue types in vitro. In this study, we addressed the potential of hESCs to undergo chondrogenesis and demonstrated the potential of hESC‐derived embryoid bodies (EBs) to undergo a well‐defined full‐span chondrogenesis from chondrogenic induction to hypertrophic maturation. We compared chondrogenic differentiation of hESCs through EB direct‐plating outgrowth system and EB‐derived high‐density micromass systems under defined serumfree chondrogenic conditions and demonstrated that cell‐tocell contact and bone morphogenetic protein 2 (BMP2) treatment enhanced chondrocyte differentiation, resulting in the formation of cartilaginous matrix rich in collagens and proteoglycans. Provision of a high‐density three‐dimensional (3D) microenvironment at the beginning of differentiation is critical in driving chondrogenesis because increasing EB seeding numbers in the EB‐outgrowth system was unable to enhance chondrogenesis. Temporal order of chondrogenic differentiation and hypertrophic maturation indicated by the gene expression profiles of  Col 1, Col 2 , and  Col 10 , and the deposition of extracellular matrix (ECM) proteins, proteoglycans, and collagen II and X demonstrated that the in vivo progression of chondrocyte maturation is recapitulated in the hESC‐derived EB model system established in this study. Furthermore, we also showed that BMP2 can influence EB differentiation to multiple cell fates, including that of extraembryonic endodermal and mesenchymal lineages in the EB‐outgrowth system, but was more committed to driving the chondrogenic cell fate in the EB micromass system. Overall, our findings provide a potential 3D model system using hESCs to delineate gene function in lineage commitment and restriction of chondrogenesis during embryonic cartilage development.  Disclosure of potential conflicts of interest is found at the end of this article.

Effects of Culture Conditions and Bone Morphogenetic Protein 2 on Extent of Chondrogenesis from Human Embryonic Stem Cells