Muc-18, all of which are expressed in human marrow stro- mal cells (Gronthos et al., 2000). In addition, blood- derived cells from all species were negative for alkaline phosphatase, an enzyme commonly expressed in marrow stromal cells from mouse, guinea pig, and humans. To investigate whether the blood-borne adherent cells had an osteogenic differentiation potential, polyclonal strains from guinea pig and clonal strains from mouse, rab- bit, and human cultures were transplanted with a ceramic- based carrier into the subcutis of immunocompromised mice (Figs. 1 and 4). Histology-proven bone was demon- strated in 12–50% of the strains transplanted, depending on the animal species (Table I and Fig. 4). All transplants that contained bone were generated by cells that displayed a fibroblastic morphology in vitro. However, it was not possible to completely rule out an osteogenic potential for the polygonal cells due to their overall lower frequency. A variable proportion of the bone-containing transplants also demonstrated the formation of a complete hematopoietic marrow, including adipocytes. Control transplants of mu- rine and human skin fibroblasts were consistently devoid of bone, as has been reported previously (Krebsbach et al., 1997; Kuznetsov et al., 1997; Satomura et al., 2000) (data not shown). Positive controls using marrow stromal cells from all species consistently generated an ossicle complete with bone and hematopoietic marrow and adipocytes, as has been described (Krebsbach et al., 1997; Kuznetsov et al., 1997) (data not shown). The donor origin of tissues formed in the transplants was documented for guinea pig and human by in situ hybridization using species-specific DNA-repetitive se- quences as probes (Fig. 4). This demonstrated that genu- ine osteoblasts and osteocytes were of donor origin, thus they originated from clonogenic cells that had been ex- Figure 4. Bone formation by in vivo transplantation of blood- derived adherent cells. Strains of blood-derived adherent cells from guinea pig (GP, in A, B, and E), mouse (M, in C), rabbit (R, in D), and human (H, in F) were attached to ceramic parti- cles (c) and transplanted into the subcutis of immunocompromised mice. After 8 wk, the transplants were harvested and sections were stained with Goldner’s to distinguish between bone (b, green in A and B) and osteoid (open arrow, red in B). In all spe- cies, bone was deposited on the surfaces of the ceramic particles by mature osteoblasts (white ar- row in B, arrows in C, D, and F). In transplants in which there was a significant amount of bone formed, a fully functional he- matopoietic marrow (hp) was also established (A and B), com- plete with megakaryocytes (ar- row in B) and adipocytes (a). Sections of paraffin-embedded transplants generated by guinea pig (G) and human (J) blood- derived adherent cells were used for in situ hybridization using a guinea pig–specific sequence (G, H, and I) and a human-specific alu sequence (J, K, and L) as probes. Osteocytes completely embedded in bone were easily identifiable in both guinea pig and human transplants, indicat- ing the donor nature of the cells that formed the bone. Specificity of the probes was demonstrated by the positive reaction of the guinea pig probe with guinea pig bone (H) and negative reaction with mouse bone (I), and by pos itive re- action of the human probe with human bone (K) and negative reaction with mouse bone (L). Kuznetsov et al. Circulating Skeletal Stem Cells 1137