Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems.

The objective of this study was to compare the osteogenic potential of human embryonic stem cells (hESCs) within two- and three-dimensional (2D and 3D) culture systems. hESCs of the H1 line (Wicell Inc., Madison, Wisc., USA) were induced to form embryoid bodies (EBs) through 5 days of suspension culture within non-adherent culture dishes. Following enzymatic dissociation, the EB-derived single cells were seeded on either novel 3D porous PLGA scaffolds or 2D culture dishes with the same total cell number. Osteogenic differentiation was induced through culture media supplemented with dexamethasone, L-ascorbic acid and beta-glycerophosphate. After 3 weeks of in vitro culture, quantitative and qualitative assays of osteogenic differentiation were conducted. Osteocalcin secretion and alkaline phosphatase (AP) activities were detected at significantly higher levels within 3D culture compared with the 2D system. Subsequently, the cell-scaffold constructs were implanted in iliac crest defects of immunosuppressed rabbits. After 4 weeks, the constructs were subsequently explanted and characterized by histology and X-ray analysis. Formation of new bone was detected within and around the implanted scaffolds. The results demonstrate that the osteogenic differentiation of human embryonic stem cells is enhanced in a 3D culture system compared to a 2D culture environment. Upon implantation in situ, the differentiating human embryonic stem cells can contribute positively to the repair and regeneration of bone defects.