Materials and Methods Blood Collection All procedures were performed in accordance to specifications of institution- ally approved animal protocols. Blood from mice (BALB/cj and Swiss NIH- SW-CR), rabbits (New Zealand), and guinea pigs (Hartley) was collected by cardiac puncture in syringes containing sodium heparin (Fisher Scientific) at a final concentration of 100 U/ml. Human venous blood was collected from healthy volunteers under National Institutes of Health (NIH)-approved pro- tocols, and was mixed with sodium heparin at a final concentration of 100 U/ml. Alternatively, human buffy coat concentrates with citrate (CDPA-1), byproducts of blood donated for transfusion, were also used. Blood was har- vested from donors of varying ages as indicated in Table I. Cell Culture Whole blood from mice, rabbits, and guinea pigs, and whole blood, total buffy coat cells, and mononuclear buffy coat cells from humans, were plated in a range of cell densities (Table I). In most instances, standard plastic culture vessels were used (Becton Dickinson). Some human sam- ples were plated in either fibronectin-coated flasks (Becton Dickinson) or collagen type I–coated dishes (Flexcell International); however, this was not found to increase colony formation. Culture media of several compo- sitions were used based on MEM (Life Technologies), 2 mM L-gluta- mine, 100 U/ml penicillin, 100 g/ml streptomycin sulfate (Biofluids), with 20% serum (FBS; Equitech-Bio) or human serum blood type AB (Pel- Freez), or a combination of the two), with or without 10 8 M dexametha- sone (Sigma-Aldrich) and 10 4 M ascorbic acid phosphate (Wako), or 100 ng/ml of mouse or human leukemia inhibitory factor (Life Technologies, R&D Systems). However, medium composition did not influence the number of colonies formed within an experiment, and consequently the data were pooled. Cells were cultured at 37 C in an atmosphere of 100% humidity and 5% CO2. Complete medium replacement was performed 1 d after plating and twice a week thereafter. Colonies consisting of 50 or more cells were counted between 10 and 41 d, depending on the animal species. 1–3 d later, the cells were released by trypsin-EDTA (Life Tech- nologies). Colonies were detached either simultaneously to generate poly- clonal strains (guinea pig), or individually using cloning cylinders to gen- erate single colony–derived strains (mouse, rabbit, and human). Cells were then plated at 0.1–0.2 105 cells/cm2. Subsequent passages were per- formed when cells were approaching confluence. Adipogenic conversion of passaged cells was determined by using medium with 20% rabbit serum (Life Technologies), as previously described (Diascro et al., 1998). Cultures of Bone Marrow Stromal Cells and Skin Fibroblasts Long bones of mice, rabbits, and guinea pigs were obtained according to specifications of an approved animal protocol. Fragments of human bone were obtained from patients undergoing corrective surgery in accordance with NIH regulations governing the use of human subjects in research. Single cell suspensions of bone marrow were prepared and cultures of bone marrow stromal cells were established as described previously (Kuz- netsov and Gehron Robey, 1996). Murine skin fibroblasts and human foreskin fibroblasts were cultured as described previously (Kuznetsov and Gehron Robey, 1996; Krebsbach et al., 1997; Satomura et al., 2000). Phenotypic Characterization Polycolonal and single colony–derived strains of blood-derived adherent cells and marrow stromal cells from each species were plated in eight- Figure 1. Identification of skeletal stem cells in peripheral blood. Blood obtained from mice, rabbits, guinea pigs, and hu- mans was plated in vitro. In all four species, adherent clonogenic colonies were formed. After ex vivo expansion, they were at- tached to ceramic particles and transplanted in the subcutis of immunocompromised mice. After 8 wk, the transplants were ex- amined for bone formation. The donor origin of bone in guinea pig and human transplants was determined using species-specific repetitive DNA sequences. chamber slides (VWR Scientific) at 2 104 cells/cm2 for immunohis- tochemical analysis with a panel of antibodies, as described previously (Gronthos et al., 2000) (see Table II). Bound antibody was localized using either peroxidase-conjugated Ig (ABC kit; Vector Laboratories) or FITC- conjugated Ig (Wako), serotyped according to the species of primary anti- body. In other cases, histochemical staining was performed by standard protocols using diagnostic kits for -naphthyl acetate esterase, acid phos- phatase, alkaline phosphatase (kits 91-A, 387-A, and 86-C, respectively; Sigma-Aldrich), and Oil Red O for visualization of lipid accumulation. Slides were viewed using an Axioplan 2 (Carl Zeiss, Inc.), and images were acquired uisng a DMC-1 digital camera (Polaroid Corp.) and Photo- shop 5.0 software (Adobe Systems Inc.). Table I. Donor and Cell Culture Characteristics in the Determination of Colony-forming Efficiency of Blood-derived Adherent Cells Aminal species Mouse Rabbit Guinea pig Human No. of donors 37 5 5 10 Age range 3 wk–2 yr 9–10 mo 5–8 mo 22–60 yr Plating density 105/cm2 0.2–1.4 1.4–3.2 0.5–2.2 1–37.3 *Guinea pig cultures were derived as polyclonal rather than single colony–derived strains. Colony-forming efficiency/106 cells 0.93 0.18 2.7 Rare Range between donors 0–3.8 0–0.58 1.1–3.9 0–0.025 No. of polygonal/ fibroblast-like colonies 5/33 6/41 15/319 0/2 Percent polygonal 13 13 5 0 The Journal of Cell Biology, Volume 153, 2001 1134 No. of osteogenic clones/clones transplanted 1/8 3/19 2/4* 1/2