IN VIVO / EX VIVO OSTEOGENESIS OF HUMAN EMBRYONIC STEM CELLS

Abstract

[Aim] This study was aimed at comparing the in vivo osteogenic differentiation potential of human embryonic stem cells (hESC) and human somatic osteoblast cell line.

[Method] HESCs were propagated on mouse embryonic feeder cells. They were shown to be pluripotent by expression oct4, sox2 and nanog molecular markers. Human fetal osteoblasts (hFOB) cell lines were cultured in DMEM media without phenol red. Osteogenic differentiation was initiated by supplementing the culture medium with ?-glycerophosphate, ascorbic acid, dexamethasone and vitamin D3. After 21 days of in vitro culture, osteogenesis in both cell types was confirmed by expression of osteocalcin and bone sialprotein molecular markers and calcium deposition by alizarin red staining. For in vivo assessment of osteogenesis, hESCs were propagated on feeder free culture system for 2 weeks. Both hESCs and hFOB cells were then seeded into PLGA scaffolds with cell carrier, Extracel, and left in culture media containing the same osteogenic supplements used for in vitro osteogenic differentiation. Before going in vivo the cells were stained with CFDA, a cell tracing reagent. After 14 days of in vitro differentiation, the cell-scaffold constructs were placed subcutaneous into the dorsum of the mice. Sacrifices were made at the end of 2nd, 4thand 7th weeks after implantation. The removed samples were processed and stained to assess the capacity of both hESCs and hFOB to form mineralized tissue. The tissue sections were stained with H&E, von Kossa followed by immunostaining for osteonectin and alkaline phosphatase to confirm osteogenic differentiation.

[Results] In hESC differentiated samples harvested after 7 weeks of implantation, discrete areas of mineralized tissue could be observed within the scaffolds. This was confirmed by von Kossa staining, H&E staining and immunostaining with osteonectin and alkaline phosphatase (ALP). There was no evidence of teratoma formation. In hFOB samples harvested 7 weeks after implantation, immunostaining with osteonectin and ALP confirmed the progression to osteogenic differentiation within the scaffolds. However, the samples did not stain positive for von Kossa staining. A possible explanation for this could be that the hFOB cells were at an early stage in differentiation. In samples harvested after 4 weeks of implantation, the scaffolds were found to be empty and devoid of the implanted cells. This was observed in samples of both cell types. Mineralization could not be detected in samples of both cell types harvested after 2 weeks of implantation.

[Conclusion] This study demonstrates the possibility of generating osteoblasts from direct differentiation of hESCs under our osteogenic conditions. These osteoblasts when implanted subcutaneous into SCID mice, where able to form mineralized tissue. A comparison of in vivo osteogenesis in hFOB and hESCs was done. This comparison helped us to confirm the efficacy of in vivo osteogenesis in hESC.