In vitro direct chondrogenesis of murine embryonic stem cells by bypassing embryoid body formation

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.