In VitroDifferentiation andIn VivoMineralization of Osteogenic Cells Derived from Human Embryonic Stem Cells

Abstract

The first report of the derivation of embryonic stem (ES) cell lines from human blastocysts had major implications for research into developmental biology and regenerative medicine. Finding efficient and reproducible methods to derive therapeutically useful cells from an ES cell source is a key feature of many regenerative medicine strategies. We have previously demonstrated that it is possible to induce osteogenic differentiation of murine ES cells by supplementing the culture medium with ascorbic acid, beta-glycerophosphate, and dexamethasone. This study investigated whether methods for driving osteogenic differentiation developed with murine ES cells could be applied successfully to human ES cells. The H1 line was propagated in vitro on murine feeder layers and shown to be pluripotent by expression of the markers Oct-4 and SSEA-4. Subsequently, differentiation was initiated via embryoid body (EB) formation and, after 5 days in suspension culture, cells harvested from EBs were replated in a medium containing osteogenic supplements. We found that the treatment regimen previously identified as optimal for murine ES cells, and in particular the addition of dexamethasone at specific time points, also induced the greatest osteogenic response from human ES cells. We identified mineralizing cells in vitro that immunostained positively for osteocalcin and found an increase in expression of an essential bone transcription factor, Runx2. When implanted into SCID mice on a poly-D, L-lactide (PDLLA) scaffold, the cells had the capacity to give rise to mineralized tissue in vivo. After 35 days of implantation, regions of mineralized tissue could be identified within the scaffold by von Kossa staining and immunoexpression of the human form of osteocalcin. We did not see any evidence of teratoma formation. These data therefore demonstrate the derivation of osteoblasts from pluripotent human ES cells with the capacity to form mineralized tissue both in vitro and in vivo. We have also shown that a culture methodology established for differentiation of murine ES cells was entirely transferable to human ES cells. Further development of this technology will result in the capacity to generate sufficient yields of osteogenic cells for use in skeletal tissue repair.

Taking stem cells to the clinic: Major challenges

“…It is therefore tempting to suggest that the brain may be a tumor-privileged site. When hESC-derived osteocytes or cardiomyocytes were transplanted into the bone or heart of SCID mice, there was also no teratoma production within 1 month after injection [Bielby et al, 2004;Laflamme et al, 2007]. The longer hESCs are differentiated in vitro, the risk of teratoma formation seems to be reduced.…”

Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-β/activin/nodal signaling using SB-431542

“…(13) Recent publications have demonstrated the development of clinically suitable ex vivo differentiation protocols for obtaining insulin-producing b cells (14,15) and neuronal cells (16,17) from hESCs. Several protocols have been reported in which hESCs were directed to differentiate into musculoskeletal tissue: osteoblastic cells, (18)(19)(20)(21)(22) chondroyctic cells, (23) cardiomyocytes, (24)(25)(26)(27)(28) and skeletal myoblasts. (29) However, most of these protocols relied on using coculture with differentiated cells or complex culture conditions to induce differentiation.…”

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…The presence of all three germ layers demonstrates that EB culture is a crude but effective means of recapitulating early stages of embryogenesis in vitro Itskovitz-Eldor et al 2000). Theoretically, embryonic stem cells can differentiate to any cell type and many examples of in vitro differentiation exist including generation of skeletal and brain tissue (Bielby et al 2004;Parekkadan et al 2008). Consequently, ES cells provide a renewable source of cellular material for tissue engineering and regenerative medicine (Ungrin et al 2008).…”