JCB Report Circulating Skeletal Stem Cells Sergei A. Kuznetsov,* Mahesh H. Mankani,* Stan Gronthos,* Kazuhito Satomura,‡ Paolo Bianco,§ and Pamela Gehron Robey* *Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892; ‡First Department of Oral and Maxillofacial Surgery, School of Dentistry, University of Tokushima, Tokushima 770-8504, Japan; and §Dipartmento di Medicina Sperimental e Patologia, Universita “La Sapienza,” Rome 00161, Italy Abstract. We report the isolation of adherent, clono- genic, ﬁbroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguish- able from skeletal stem cells found in bone marrow (stromal stem cells, “mesenchymal stem cells”). The os- teogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either poly- clonal or single colony–derived strains were trans- Introduction The post-natal bone marrow of mammals harbors two pop- ulations of stem/progenitor cells that are thought of as part of separate lineages and exhibit distinct fundamental biolog- ical properties: (a) hematopoietic stem cells, which are non- adherent and circulating, and (b) stromal stem cells, which are adherent and fibroblastic in habit, and noncirculating (Friedenstein et al., 1968; Bianco and Riminucci, 1998). Stromal stem cells can give rise to all major skeletal tissues, such as cartilage, bone, myelosupportive stroma, and associ- ated adipocytes, and fibrous connective tissue (Friedenstein et al., 1966, 1968; Owen, 1988). Throughout development, these particular tissues are thought to be formed entirely by extravascular mesodermal progenitors that associate with developing skeletal rudiments via migratory processes that do not rely upon their ability to circulate in blood (Bianco and Riminucci, 1998). Other solid-phase mesodermal tissues such as skeletal muscle also fall into the same general cate- gory (Cossu et al., 1996; Bianco and Cossu, 1999). Recent data have shown that both muscle and skeletal cells of donor origin differentiate within muscle and bone when marrow-derived cell populations are infused into the systemic circulation under some experimental conditions Address correspondence to Sergei A. Kuznetso, Building 30, Room 228 30 Convent Drive MSC 4320 Bethesda, MD 20892. Tel.: (301) 402-2476. Fax: (301) 402-0824. E-mail: skuznets@mail.nih.gov planted into the subcutis of immunocompromised mice, and the donor origin of the fully differentiated bone cells was proven using species-speciﬁc probes. This is the ﬁrst deﬁnitive proof of the existence of circulating skeletal stem cells in mammals. Key words: blood cells • tissue culture • adherent col- onies • transplantation • osteogenesis (Ferrari et al., 1998; Hou et al., 1999). These data provide evidence for at least some degree of systemic transplant- ability of progenitors of solid-phase mesodermal tissues. Complete reconstitution of the hematopoietic system after the systemic infusion of progenitor/stem cells illustrates the natural properties of the hematopoietic stem cell; hemato- poietic stem cells are capable of negotiating the circulation and homing to the bone marrow in normal physiology. Whether any physiological event relevant to extravascular mesodermal tissues calls for the existence of a naturally circulating progenitor cell remains to be determined. Criti- cal to addressing this question would be the demonstration of circulating progenitors for solid-phase mesodermal tis- sues in the intact organism. In this study, we isolated adherent and clonogenic cells from whole blood of adult animals of four different species (mouse, rabbit, guinea pig, and human). We demonstrated that some polyclonal strains and several single colony– derived strains formed bone upon in vivo transplantation, the gold standard assay of osteogenic potential. Further- more, they were capable of forming adipocytes in vitro. These studies prove for the first time that some circulating cells that can form adherent colonies on plastic are compe- tent to form genuine bone tissue in vivo, and establish for the first time the existence of circulating osteogenic stem cells.  The Rockefeller University Press, 0021-9525/2001/05/1133/7 $5.00 The Journal of Cell Biology, Volume 153, Number 5, May 28, 2001 1133–1139 http://www.jcb.org/cgi/content/full/153/5/1133 1133