Prospective identification and skeletal localization of cells capable of multilineage differentiation in vivo

A prospective in vivo assay was used to identify cells with potential for multiple lineage differentiation. With this assay, it was first determined that the 5-fluorouracil resistant cells capable of osseous tissue formation in vivo also migrated toward stromal derived factor-1 (SDF-1) in vitro. In parallel, an isolation method based on fluorescence-activated cell sorting was employed to identify a very small cell embryonic-like Lin-/Sca-1+CD45- cell that with as few as 500 cells was capable of forming bone-like structures in vivo. Differential marrow fractionation studies determined that the majority of the Lin-Sca-1+CD45- cells reside in the subendosteal regions of marrow. To determine whether these cells were capable of differentiating into multiple lineages, stromal cells harvested from Col2.3 Delta TK mice were implanted with a gelatin sponge into SCID mice to generate thymidine kinase sensitive ossicles. At 1.5 months, 2,000 green fluorescent protein (GFP)+ Lin-Sca-1+CD45- cells were injected into the ossicles. At harvest, colocalization of GFP-expressing cells with antibodies to the osteoblast-specific marker Runx-2 and the adipocyte marker PPAP gamma were observed. Based on the ability of the noncultured cells to differentiate into multiple mesenchymal lineages in vivo and the ability to generate osseous tissues at low density, we propose that this population fulfills many of the characteristics of mesenchymal stem cells.