Clonogenic adherent cells were observed in cultures of peripheral blood from all species investigated, albeit with significant variation in colony forming efficiency (number of colonies per 106
nucleated cells) across animal species, and from one individual donor to another (). In all species except human (in which a total of two colonies were obtained), two distinct types of cell morphology were observed. The majority of the colonies were composed of cells with fibroblastic morphology, but a number of colonies consisted of cells that exhibited a distinctive polygonal shape (). However, upon characterization of cloned strains of both types with a broad panel of markers, the pattern was virtually identical ( and ). This was noted for the lack of expression of hematopoietic (CD45, CD14) and endothelial (endoglin, CD34, Factor VIII–related antigen, Muc-18, PAL-E, EN4) markers, variable expression of osteogenic makers (osteopontin and bone sialoprotein), and the consistent expression of collagen types I and III, fibronectin, osteonectin, α-smooth muscle actin, CD44, VCAM-1, and the β1 integrin subunit. The phenotypic profile of blood-borne adherent cells overall was nearly identical between species (rabbit, guinea pig, and human) and quite similar to that of marrow stromal cells derived from each species. However, the human blood-derived adherent cells were negative for the human marrow stromal marker, Stro-1, as well as for endoglin and Muc-18, all of which are expressed in human marrow stromal 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.
Figure 2 Morphological features of blood-derived adherent clonogenic cells. The majority of clones that developed from all animal species, including the two human colonies (A and B) consisted of cells with a fibroblastic morphology. However, between 5 and 13% (more ...)
Figure 3 Representative immunophenotype of blood-derived adherent cells. Although two morphologically distinct types of cells were detected in three of the four animal species, their immunophenotype was identical, and this did not vary significantly from one species (more ...)
To investigate whether the blood-borne adherent cells had an osteogenic differentiation potential, polyclonal strains from guinea pig and clonal strains from mouse, rabbit, and human cultures were transplanted with a ceramic-based carrier into the subcutis of immunocompromised mice ( and ). Histology-proven bone was demonstrated in 12–50% of the strains transplanted, depending on the animal species ( and ). 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 murine 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).
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 particles (c) and (more ...)
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 sequences as probes (). This demonstrated that genuine osteoblasts and osteocytes were of donor origin, thus they originated from clonogenic cells that had been expanded in cultures of blood cells. The hematopoietic tissue (particularly prominent in transplants generated by guinea pig cells) was of recipient origin, as expected.
It has been previously documented that osteogenic cells also form marrow adipocytes upon in vivo transplantation. Because of the difficulty in visualizing the nuclei of adipocytes, which are rarely intercepted in tissue sections, we sought to determine the adipogenic potential of the cell strains in vitro. All strains, including marrow stromal cells, were negative for PPARγ2, the adipogenic transcription factor. However, CEBPα was detected by immunohistochemistry in both human clonal strains, and adipogenesis was observed in cells from all species upon culture with rabbit serum, a known inducer of adipogenesis in marrow stromal osteoprogenitor cells () (Diascro et al. 1998
Figure 5 Adipocytic conversion of human blood-derived adherent cells in vitro. Immunohistochemistry of human blood-derived adherent cells revealed expression of the adipocytic transcription factor, CEBPα (A). The ability of blood-derived cells to form (more ...)
These data show that clonogenic adherent cells of fibroblastic growth habit can be isolated in culture from the adult peripheral blood in a variety of species. A subset of these cells proved to be osteogenic using the best in vivo assay available to date for probing the osteogenic capacity of putative skeletal stem cells. These cells are also capable of accumulating lipid, turning into adipocyte-like cells, upon defined culture conditions in vitro, as do marrow stromal cells in vitro and in vivo (Bianco et al. 1988
; Beresford et al. 1992
). The formation of a hematopoietic marrow in some transplants in vivo also indicated that a myelosupportive reticular stroma was formed. Thus, blood-borne adherent cells share with marrow stromal stem cells some critical functional features, such as the ability to generate at least three phenotypes of the so-called stromal system (osteoblasts, adipocytes, and reticular cells).
This is, to the best of our knowledge, the first indication that cells with true osteogenic potential, directly demonstrated by the formation of histology-proven bone in vivo, can circulate. Our data clearly demonstrate that cells with multiple differentiation potential similar to that of post-natal marrow stromal stem cells (osteo-adipo-fibrogenic) can negotiate the circulation. Indication of the ability of stromal or other extravascular tissue progenitor cells (e.g., myogenic) to home to their respective peripheral tissues when infused into the circulation has recently been provided (Ferrari et al. 1998
; Hou et al. 1999
). Our data provide the complementary notion that stem cells of extravascular mesodermal tissues exist (physiologically) in the circulating peripheral blood. The origin of these cells, the mode by which they gain access to the blood stream, and their physiological role are all issues that remain to be properly addressed. However, establishment of their existence provides a significant opportunity for further characterization of their differentiation potential, a novel and accessible source of progenitor cells of solid-phase mesodermal tissues, and an interesting glimpse of a hitherto unrecognized level of circulation-mediated, systemic integration of skeletal physiology.