Figure 5. Adipocytic conversion of human blood-derived adher- ent cells in vitro. Immunohistochemistry of human blood-derived adherent cells revealed expression of the adipocytic transcription factor, CEBP (A). The ability of blood-derived cells to form ad- ipocyte-like cells was further investigated by assessing lipid accu- mulation as demonstrated by Oil Red O staining after culture with rabbit serum. As has been seen in marrow stromal cells and in most blood-derived cell types, small lipid-containing structures are present in cultures grown in 20% FBS (B). In cultures grown in 20% rabbit serum, there is a dramatic increase in the number and size of lipid vacuoles (C), which begin to coalesce in vitro (D). panded 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 adipo- cytes, 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 PPAR 2, the adipogenic transcription factor. However, CEBP was detected by immunohis- tochemistry 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 (Fig. 5) (Diascro et al., 1998). These data show that clonogenic adherent cells of fibro- blastic 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 as- say 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 myelosup- portive 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 (os- teoblasts, adipocytes, and reticular cells). This is, to the best of our knowledge, the first indication that cells with true osteogenic potential, directly demon- strated 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 pro- vided (Ferrari et al., 1998; Hou et al., 1999). Our data pro- vide the complementary notion that stem cells of extravas- cular 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 exist- ence provides a significant opportunity for further charac- terization of their differentiation potential, a novel and accessible source of progenitor cells of solid-phase meso- dermal tissues, and an interesting glimpse of a hitherto un- recognized level of circulation-mediated, systemic integra- tion of skeletal physiology. The authors acknowledge Prof. J. Spencer, Dr. J. Fredenburgh, Dr. Larry W. Fisher, and Zimmer, Inc. This work was supported by the Telethon Fondazione Onlus (grant E1029 to P. Bianco). Submitted: 28 March 2001 Revised: 28 March 2001 Accepted: 19 April 2001 References Beresford, J.N., J.H. Bennett, C. Devlin, P.S. Leboy, and M.E. Owen. 1992. Ev- idence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J. Cell Sci. 102:341– 351. Bianco, P., and G. Cossu. 1999. Uno, nessuno e centomila: searching for the identity of mesodermal progenitors. Exp. Cell Res. 251:257–263. Bianco, P., M. Costantini, L.C. Dearden, and E. Bonucci. 1988. Alkaline phos- phatase positive precursors of adipocytes in the human bone marrow. Br. J. Haematol. 68:401–403. Bianco, P., and M. Riminucci. 1998. The bone marrow stroma in vivo: ontog- eny, structure, cellular composition and changes in disease. In Marrow Stro- mal Cell Cultures. J.N. Beresford and M. Owen, editors. 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