Assessment of a preclinical model for studying the survival and engraftment of human stem cell derived osteogenic cell populations following orthotopic implantation.

INTRODUCTION: Preclinical studies with osteoprogenitor cells derived from human embryonic stem cells (hESC) do not lead to substantial bone regeneration in vivo. The degree of survival following implantation might play a role in their long term efficiency. We investigated the initial engraftment of hESCs-derived cells during two weeks post-implantation and compared it to such response for adult bone marrow stromal cells (hBMSC)-derived osteoprogenitor cells. METHODS: hBMSC and H9-hES cells pre-treated with osteogenic factors were implanted into a calvarial defect in both adult WT and nude rats. At days 7 and 14 post-implantation, samples were analysed for persistence of implanted cells, initiation of regeneration of host bone, angiogenesis and apoptosis. RESULTS: At day 7, hESC and hBMSC were detected within defects in both rat strains. By day 14 human cells were only detected in immune-deficient rats whilst still maintaining an osteoblastic phenotype and engendered a significant increase in bone formation. In WT animals, the participation of implanted cells was very limited due to their poor survival. CONCLUSION: This study demonstrates the ability of hESC and hBMSC derived osteoprogenitor cells to survive transplantation, to engraft and to develop an osteogenic phenotype during the early stage following implantation, validating the appropriate preclinical model.

Bone tissue formation from human embryonic stem cells in vivo.

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.

The hyalectan degrading ADAMTS-1 enzyme is expressed by osteoblasts and up-regulated at regions of new bone formation.

During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.