Current approaches on the chondrogenic differentiation of embryonic stem cells (ESCs) involve embryoid body (EB) formation, resulting in a fragmented process where control of differentiation, integration, and scalability are difficult to achieve, thus hampering any potential application to cartilage tissue engineering and regenerative medicine. Our study aimed at developing a simplified two-step process which avoids EB formation and results in enhanced chondrogenic differentiation of murine ESCs. Specifically, mESCs were cultured in HepG2 conditioned medium for 3 days and then directed into chondrogenic differentiation for 15 days without prior EB formation. Analysis of chondrogenic differentiation demonstrated well-developed Alcian blue-stained cartilage nodules, production of sulfated glycosaminoglycan and collagen matrix, the presence of structured type II collagen and sox-9 molecules, as well as distinct gene expression of type II collagen, aggrecan, link-protein, scleraxis and sox-9 transcripts. To our knowledge, this represents one of the first reports demonstrating the enhanced derivation of chondrogenic cells from mESCs without EB formation in a simplified and easily integrateable and scalable bioprocess with potential applications in cartilage tissue engineering.
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