Prospective In Vivo Identification of Osteogenic Stem/Progenitor Cells from Bone Marrow-Derived Lin⁻/Sca-1⁺/CD45⁻ Cells.

We previously developed an in vivo prospective assay for identification of non-cultured cells with MSC potential. Using this assay we identified a population of cells that were slowly cycling and of low density that were capable of multilineage differentiation both in vitro and in vivo (Z. Wang et al, Stem Cells. 2006 24(6):1573). Further characterization of these cells suggested that they resemble a homogenous population of rare Lin⁻/Sca-1⁺/CD45⁻ cells that have the morphology and express several markers of undifferentiated embryonic-like stem cells. In vitro the Lin⁻/Sca-1⁺/CD45⁻ cells may differentiate into cells from all three germ-layers (M. Kucia et al, Leukemia. 2007 21(2):297). To determine the in vivo fate of this population, we transplanted 500 or 5,000 Lin⁻/Sca-1⁺/CD45⁻ cells from a GFP mouse into SCID mice in each group (n=3) immediately after cell sorting to evaluate tissue generation in vivo. At 4 weeks the regenerative potential of these populations was evaluated by micro-CT and histology, and cells were tracked by gross examination of the harvested tissues by fluorescent microscopy. The results showed that a large number of GFP+ cells are located in the implants, indicating that the transplanted cells maintain the ability to contribute to the generation of new tissue. Bone-like tissue was observed in the Lin⁻/Sca-1⁺/CD45⁻ group with as low as 500-cells/implant, while 5,000 Lin⁻/Sca-1⁺/CD45⁻ cells generated significantly larger mineralized tissue volume, which was confirmed by micro-CT. Lin⁻/Sca-1⁺/CD45⁺ cell only implantation did not form any mineralized tissue, however, while mixed with 2x10⁶ whole bone morrow cells, positive mineralized tissue occurred. Whole bone marrow mixture also improve bone formation in Lin⁻/Sca-1⁺/CD45⁻ cell implants compared the actual bone volumes measured by micro-CT. This study demonstrates that non-cultured BM-derived Lin⁻/Sca-1⁺/CD45⁻ cells exhibit the capacity to form bone in vivo with as low as 500 cells/implant. Whole bone marrow mixtures can enhance the bone formation, presumably through the interaction of other populations cells. Based on these findings, it is proposed that non-cultured BM-derived Lin⁻/Sca-1⁺/CD45⁻ cells are enriched osteogenic cells that can be applied to bone regeneration in vivo.