Background: Chondrogenic differentiation of human embryonic stem cells (hESCs) has
been investigated by maintenance of 3-dimensional cultures in the presence of various exogenous
growth factors added during defined stages of culture, or in cocultures with primary chondrocytes,
making the cultivation process rather complex. Thus, there is a need for easier and more handy expansion
and differentiation protocols.
Objective: The present study is aimed to investigate the potential of hESCs for chondrogenic differentiation
in simpler culture conditions.
Methods: The hESCs were directly cultured for 3 weeks on feeder-free gelatin-coated plates in chondrocyte
culture medium without any growth factor supplements after 6-day culture on feeder-free gelatin-
coated plate with conditioned medium.
Results: Immunocytochemical and gene expression analyses indicated that these human directly differentiated
cells (hDDCs), which derived from the hESCs, abundantly expressed Sox9, aggrecan, and procollagen
α1(II) mRNAs. Upon further passaging, the hDDCs behaved similarly to primary chondrocytes,
although the aggrecan mRNA expressions were maintained at a relatively constant level throughout
passaging. The procollagen α1(II) mRNAs expression was high in the beginning of the hDDC culture,
but declined upon further passaging, which is typical for the primary chondrocytes. The hDDCs
could be easily expanded in the monolayer culture using chondrocyte culture medium. Differentiation
assays showed that the hDDCs could be differentiated towards chondrocytes, but not adipocytes or
Conclusion: Our data suggests that the chondrogenic gene expression could be induced in the directly
differentiated hESCs without a need for chondrocyte coculture. In contrast, no osteogenic or adipogenic
differentiation was observed.