Engineering embryonic stem-cell aggregation allows an enhanced osteogenic differentiation in vitro.

Abstract

Pluripotent embryonic stem (ES) cells hold great promise for the field of tissue engineering, with numerous studies investigating differentiation into various cell types including cardiomyocytes, chondrocytes, and osteoblasts. Previous studies have detailed osteogenic differentiation via dissociated embryoid body (EB) culture in osteoinductive media comprising of ascorbic acid, beta-glycerophosphate, and dexamethasone. It is hoped that these osteogenic cultures will have clinical application in bone tissue repair and regeneration and pharmacological testing. However, differentiation remains highly inefficient and generates heterogeneous populations. We have previously reported an engineered three-dimensional culture system for controlled ES cell-ES cell interaction via the avidin-biotin binding complex. Here we investigate the effect of such engineering on ES cell differentiation. Engineered EBs exhibit enhanced osteogenic differentiation assessed by cadherin-11, Runx2, and osteopontin expression, alkaline phosphatase activity, and bone nodule formation. Results show that cultures produced from intact EBs aggregated for 3 days generated the greatest levels of osteogenic differentiation when cultured in osteoinductive media. However, when cultured in control media, only engineered samples appeared to exhibit bone nodule formation. In addition, polymerase chain reaction analysis revealed a decrease in endoderm and ectoderm expression within engineered samples. This suggests that engineered ES cell aggregation has increased mesoderm homogeneity, contributing to enhanced osteogenic differentiation.