In vitro direct osteogenesis of murine embryonic stem cells without embryoid body formation

Embryonic stem cells (ESCs) posses the ability to self-renew and differentiate into a multitude of lineages, including the osteogenic lineage in vitro. Currently, most approaches have focused on embryonic body (EB)-mediated osteogenic differentiation, which relies on formation of all three germ layers resulting in limited yields and labour-intensive culture processes. Our study aimed at developing an efficient culture strategy resulting in the upregulated in vitro osteogenic differentiation of murine ESCs (mESCs), which completely avoided EB formation. Specifically, mESCs were cultured in HepG2 conditioned medium for 3 days and then directed into osteogenic differentiation for 21 days without prior EB formation. The mineralised bone nodules generated were characterized by Alizarin red S-staining, phenotypic alkaline phosphatase expression, time-course analysis of ALPase activity, the presence of type I collagen and osteopontin, and osteocalcin, cbfa-1/runx-2, and osterix gene expression. Our method of direct osteogenic differentiation of mESCs represents a novel and efficient approach that results in enhanced yields and could have significant applications in bone tissue engineering.