Correspondence to: Sergei A. Kuznetsov, Building 30, Room 228 30 Convent Drive MSC 4320 Bethesda, MD 20892. Tel:(301) 402-2476 Fax:(301) 402-0824 E-mail:skuznets{at}mail.nih.gov.
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Abstract |
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We report the isolation of adherent, clonogenic, fibroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguishable from skeletal stem cells found in bone marrow (stromal stem cells, "mesenchymal stem cells"). The osteogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either polyclonal or single colonyderived strains were transplanted 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 colonies, transplantation, osteogenesis
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Introduction |
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The post-natal bone marrow of mammals harbors two populations of stem/progenitor cells that are thought of as part of separate lineages and exhibit distinct fundamental biological properties: (a) hematopoietic stem cells, which are nonadherent and circulating, and (b) stromal stem cells, which are adherent and fibroblastic in habit, and noncirculating (
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 (
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 colonyderived strains formed bone upon in vivo transplantation, the gold standard assay of osteogenic potential. Furthermore, 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 competent to form genuine bone tissue in vivo, and establish for the first time the existence of circulating osteogenic stem cells.
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Materials and Methods |
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Blood Collection
All procedures were performed in accordance to specifications of institutionally 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 protocols, 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 harvested from donors of varying ages as indicated in Table 1.
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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 1). In most instances, standard plastic culture vessels were used (Becton Dickinson). Some human samples were plated in either fibronectin-coated flasks (Becton Dickinson) or collagen type Icoated dishes (Flexcell International); however, this was not found to increase colony formation. Culture media of several compositions were used based on MEM (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/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 dexamethasone (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. 13 d later, the cells were released by trypsin-EDTA (Life Technologies). Colonies were detached either simultaneously to generate polyclonal strains (guinea pig), or individually using cloning cylinders to generate single colonyderived strains (mouse, rabbit, and human). Cells were then plated at 0.10.2 x 105 cells/cm2. Subsequent passages were performed when cells were approaching confluence. Adipogenic conversion of passaged cells was determined by using medium with 20% rabbit serum (Life Technologies), as previously described (
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 (
Phenotypic Characterization
Polycolonal and single colonyderived strains of blood-derived adherent cells and marrow stromal cells from each species were plated in eight-chamber slides (VWR Scientific) at 2 x 104 cells/cm2 for immunohistochemical analysis with a panel of antibodies, as described previously (-naphthyl acetate esterase, acid phosphatase, 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 Photoshop 5.0 software (Adobe Systems Inc.).
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In Vivo Transplantation Assay
An in vivo transplantation assay was employed to study the osteogenic potential 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 (
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In Situ Hybridization for Species-specific Repetitive DNA Sequences
In situ hybridization for species-specific DNA sequences was used to determine the origin of bone formed in the transplants of guinea pig and human bloodderived adherent cells. A 3,878-bp long guinea pig repetitive 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 primers were created, and one was chosen based on its performance in PCR and, subsequently, in in situ hybridization. This set was sense: 5'-CTCCTGTCCTGCATCCACT-3' and antisense: 5'-GGATATGAGAGACAGTGGTG-3'. Two consecutive PCR amplifications were used to generate digoxigenin-labeled guinea pigspecific probes. In the first PCR, Platinum Taq DNA Polymerase kit (Life Technologies) was used with 1 µg of specific primers and 1 µg 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 purified using the Qiaex II Gel Extraction Kit (QIAGEN) according to the manufacturer's recommendations. The product was reamplified in the second 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 (
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Results and Discussion |
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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 (Table 1). 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 (Fig 2). However, upon characterization of cloned strains of both types with a broad panel of markers, the pattern was virtually identical (Table 2 and Fig 3). This was noted for the lack of expression of hematopoietic (CD45, CD14) and endothelial (endoglin, CD34, Factor VIIIrelated 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, -smooth muscle actin, CD44, VCAM-1, and the ß1 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 (
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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 (Fig 1 and Fig 4). Histology-proven bone was demonstrated in 1250% of the strains transplanted, depending on the animal species (Table 1 and Fig 4). 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 (
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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 (Fig 4). 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 PPAR2, the adipogenic transcription factor. However, CEBP
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 (Fig 5) (
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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 (
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 (
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Acknowledgements |
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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
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References |
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