| The goal of this research is to investigate the effects of the microenvironment on the osteogenic differentiation of human embryonic stem cells (hESCs). Human embryonic stem cells present a potentially unlimited supply of cells that may be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. Current techniques used for bone tissue repair employ the use of auto- and allografting methods, however, these methods have inherent limitations that restrict their universal application. The limitations of these reparative strategies suggest that an alternative approach is required, and hESCs may provide a repository of cells for such an approach. One of the major gaps in the knowledge regarding hESCs is the lack of understanding the biological cues from the microenvironment that control and direct differentiation. Therefore, we hypothesized that controlling the local in vitro and in vivo microenvironments can direct osteoblastic differentiation of hESCs.;We showed the importance of cell culture conditions in the in vitro microenvironment, and the importance of implantation site and scaffold design in the in vivo microenvironment. First, we developed a transwell co-culture system consisting of hESCs with human bone marrow stromal cells (hBMSCs). This demonstrated that pro-osteogenic soluble signaling factors secreted by hBMSCs into the cellular microenvironment directed differentiation of hESCs into osteoblasts. Secondly, we reproducibly derived mesenchymal progenitors from hESCs (hES-MSCs) that possess the characteristic hBMSC immunophentoype, are capable of multilineage differentiation along, and genetically modified them with bone specific transgenes to track osteoblastic differentiation. Distinct osteoprogenitor cells were identified and when implanted in an orthotopic calvarial defect microenvironment, participated in the bone regeneration process. Lastly, we delivered osteoprogenitor hES-MSCs in vivo within hydroxyapatite/tri-calcium phosphate (HA/TCP) scaffolds to investigate the effects that porosity and permeability design have on cell differentiation and overall bone tissue formation. We demonstrated osteoprogenitors derived from hESCs survive and play a role in overall bone tissue formation within HA/TCP scaffolds with high porosity and permeability. |
