Cultivation of human embryonic stem cells without the embryoid body step enhances osteogenesis in vitro

¹ Massachusetts Institute of Technology, Cambridge, Massachusetts

^* To whom correspondence should be addressed. E-mail: rlanger{at}mit.edu.

	Abstract

Osteogenic cultures of embryonic stem cells (ESC) are predominately derived from 3-dimensional cell spheroids called embryoid bodies (EB). An alternative method that has been attempted, and merits further attention, avoids EB through the immediate separation of ESC colonies into single cells. However, this method has not been well characterized and the effect of omitting the embryoid body step is unknown. Herein we found that culturing hESC without EB leads to over a 7 fold increase (P=0.018) in the number of osteogenic cells and to spontaneous bone nodule formation after 10-12 days. In contrast, when hESC were differentiated as EB for 5 days followed by plating of single cells, bone nodules formed after 4 weeks only in the presence of dexamethasone. Furthermore, regardless of the inclusion of EB, bone matrix formed including cement line matrix and mineralized collagen which displayed apatitc mineral (PO₄) with calcium to phosphorous ratios similar to hydroxyapatite and human bone. Together these results demonstrate that culturing hESC without an embryoid body step can be used to derive large quantities of functional osteogenic cells for bone tissue engineering.

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