Human embryonic stem cells (hESCs) 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. An area of particular interest is stem cell transplantation for bone tissue regeneration where hESCs may be used to repair skeletal defects. One of the major gaps in the knowledge regarding hESCs is the lack of understanding of the growth factors and three-dimensional signals that control differentiation. 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. Previous work has shown that exposing hESCs to exogenous factors such as dexamethasone, ascorbic acid and P-glycerophosphate can induce osteogenesis in vitro [1-3]. However, the specific factors that regulate and influence the commitment of hESCs along the osteoblast lineage have not yet been identified. It is possible that soluble factors secreted by human bone marrow stromal cells (hBMSCs) may provide the necessary signaling molecules to direct osteogenic differentiation of hESCs.

When bone marrow is cultured in vitro, adherent non-hematopoietic cells proliferate and exhibit characteristics of bone marrow stroma in vivo. Within this diverse population of hBMSCs there exist early progenitor mesenchymal stem cells that are capable of self-renewal and have multi-lineage differentiation potential into cell types such as osteoblasts, chondrocytes, and adipocytes. In the presence of dexamethasone, ascorbic acid and P-glycerophosphate, it has been demonstrated that hBMSCs can be differentiated readily into mineralizing osteoblasts both in vitro and in vivo [4-7]. The in vitro equivalent of bone formation is characterized by the formation of mineralized nodules, increased alkaline phosphatase activity, and up regulation of osteoblastic genes such as runx2, osteocalcin, bone sialoprotein, and collagen type I [5, 8-10]. The use of this well-defined in vitro model allows the control of the differentiation state of hBMSCs at varying time points within their lineage progression towards functional osteoblasts. Subsequently, soluble factors derived from hBMSCs may be controlled, thus enabling the establishment of a co-culture system that stimulates hESC differentiation.

Therefore, the goal of this study was to determine if the lineage progression of hESCs toward osteoblasts could be directed by soluble signaling factors derived from differentiated hBMSCs. Because the differentiation of hBMSCs in vitro is so well characterized, the ability to manipulate and control hBMSCs along with the osteogenic factors derived from them will be integral to controlling the osteoblastic differentiation of hESCs. In this study, we demonstrate that osteogenic growth factors secreted by hBMSCs into the local microenvironment can promote osteoblastic differentiation of hESCs, and the secretion of these factors was dependent on the state of cell differentiation.

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