In Vivo Transplantation Assay An in vivo transplantation assay was employed to study the osteogenic po- tential of the blood-derived adherent cells (see Fig. 1) along with marrow stromal cells (as a positive control) and murine and human fibroblasts (as negative controls), as previously reported (Krebsbach et al., 1997). Cells were trypsinized, pelleted, resuspended in 1 ml of culture medium, and 1.5–3.0 106 cells were mixed with 40 mg of hydroxyapatite/tricalcium phosphate ceramic powder (particle size 0.5–1.0 mm, generously provided by Zimmer, Inc.). After a 90-min incubation at 37 C with rotation, the par- ticles with attached cells were collected by a brief centrifugation. A sec- ondary matrix was formed by mixing 15 l of mouse fibrinogen (3.2 mg/ml in PBS) and 15 l of mouse thrombin (25 U/ml in 2% CaCl2; Sigma- Aldrich) such that a solid fibrin gel was formed. The transplants were placed in subcutaneous pockets in the backs of 8–15-wk-old immunocom- promised female mice (bg-nu/nu-xid; Harlan Sprague Dawley). The trans- plants were recovered 8 wk later, fixed in 4% phosphate-buffered formalin (Sigma-Aldrich), decalcified in 10% EDTA, pH.8.0 (Quality Biological, Inc.), and embedded in paraffin or glycol methacrylate. 5- or 7- m sections were prepared from each block and either stained with hematoxylin and eosin (paraffin sections) or Goldner’s modified trichrome stain (glycol methacrylate), or used for in situ hybridization (paraffin). In Situ Hybridization for Species-specific Repetitive DNA Sequences In situ hybridization for species-specific DNA sequences was used to de- termine the origin of bone formed in the transplants of guinea pig– and human blood–derived adherent cells. A 3,878-bp long guinea pig repeti- tive DNA sequence (GenBank accession no. AF318129; provided by Prof. J. Spencer, Queen’s University, Kingston, Ontario, Canada, and Dr. J. Fredenburgh, Hamilton Civic Hospitals Research Center, Hamilton, Ontario, Canada) was evaluated, and revealed no homology with either mouse or human genomic DNA in a BLAST search. Several sets of prim- ers were created, and one was chosen based on its performance in PCR and, subsequently, in in situ hybridization. This set was sense: 5 -CTC- CTGTCCTGCATCCACT-3 and antisense: 5 -GGATATGAGAGA- CAGTGGTG-3 . Two consecutive PCR amplifications were used to gen- erate digoxigenin-labeled guinea pig–specific probes. In the first PCR, Platinum Taq DNA Polymerase kit (Life Technologies) was used with 1 g of specific primers and 1 g of either guinea pig or mouse genomic DNA. Using this primer set, one intense band (345 bp) was formed with guinea pig but not mouse genomic DNA. The band was excised and puri- fied using the Qiaex II Gel Extraction Kit (QIAGEN) according to the manufacturer’s recommendations. The product was reamplified in the sec- ond PCR using the same Platinum Taq DNA Polymerase kit except that 0.1 mM dNTP was substituted with 0.1 mM dATP, 0.1 mM dCTP, 0.1 mM dGTP, 0.065 mM dTTP (PerkinElmer), and 0.035 mM digoxigenin-11- dUTP (Boehringer). The digoxigenin-labeled probe specific for human repetitive alu sequence was created using primers and PCR conditions reported previously (Kuznetsov et al., 1997). The guinea pig– and human- specific digoxigenin-labeled probes were used for in situ hybridization according to a technique specifically modified for decalcified sections of hydroxyapatite-containing transplants (Kuznetsov et al., 1997). Digoxige- nin-labeled DNA was detected by immunohistochemistry using antidigox- igenin alkaline phosphatase-conjugated Fab fragments (Boehringer). Results and Discussion Clonogenic adherent cells were observed in cultures of pe- ripheral blood from all species investigated, albeit with sig- nificant variation in colony forming efficiency (number of colonies per 106 nucleated cells) across animal species, and from one individual donor to another (Table I). In all spe- cies except human (in which a total of two colonies were obtained), two distinct types of cell morphology were ob- served. The majority of the colonies were composed of cells with fibroblastic morphology, but a number of colo- nies consisted of cells that exhibited a distinctive polygonal shape (Fig. 2). However, upon characterization of cloned strains of both types with a broad panel of markers, the pattern was virtually identical (Table II and Fig. 3). This was noted for the lack of expression of hematopoietic Kuznetsov et al. Circulating Skeletal Stem Cells 1135 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) con- sisted of cells with a fibroblastic morphology. However, between 5 and 13% of the colonies, depending on the species, had a dis- tinctly polygonal morphology in mouse (C), rabbit (D), and guinea pig (not shown). (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 col- 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 immu- nophenotype was identical, and this did not vary significantly from one species to another. Their similarity to marrow stromal cells is demonstrated by their expression of (A) type I collagen, (B) fi- bronectin, (C) osteonectin, and (D) -smooth muscle actin, and variable expression of (E) osteopontin and (F) bone sialoprotein.