Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).
Adult human bone marrow contains a minority population of MSCs that contribute to the regeneration of tissues such as bone, cartilage, muscle, ligaments, tendons, fat, and stroma. Evidence that these MSCs are pluripotent, rather than being a mixture of committed progenitor cells each with a restricted potential, includes their rapid proliferation in culture, a characteristic morphology, the presence of typical marker proteins, and their consistent differentiation into various mesenchymal lineages. These attributes are maintained through multiple passages and are identifiable in individual stem cells.
Since stem cells are present in both the bone marrow and other tissues, we thought it possible that cells with a similar appearance and pluripotent mesenchymal potential would be present in the blood. We applied techniques used successfully with marrow MSCs to identify similar cells in elutriation fractions of normal human blood.
BMPCs were elutriated by diluting the buffy coats from 500 ml of anticoagulant-treated, platelet-depleted blood 1:4 in RPMI-1640 medium (RPMI) and layering 25-ml portions over 20 ml of Lymphoprep™. These samples were centrifuged at 2000 rpm for 20 min. The leukocyte-rich interface cells were collected, made up to 20 ml in RPMI, and separated by density-gradient centrifugation. The interface cells, now depleted of red blood cells, were collected, resuspended in 50 ml of sterile RMPI and 5% heat-inactivated FCS, and introduced into the sample line of the flow system of a Beckman JE-50 cell elutriator charged with elutriation buffer. The chamber was centrifuged at 25 000 rpm at 10°C and the flow rate adjusted to 12 ml/min. After about 150 ml had been collected, the flow rate was increased by 1 ml/min. Fractions nos. 1-6 (flow rates of 12-16 ml/min) contained most of the lymphocytes. Monocytes usually appeared in fractions 6 or 7 (as determined by flow cytometric analysis in a fluorescence-activated cell sorter (FACS). BMPCs were concentrated in fractions 7 and 8, along with monocytes and lymphocytes. Elutriation fractions with more than 50% and less than 75% monocytes were collected and concentrated by centrifugation at 1200 rpm for 5 min, and the cell pellets were combined, reconstituted in DMEM plus 20% sterile heat-inactivated FCS, counted, washed in medium, repelleted, and then resuspended in DMEM to 5 × 106/ml and dispensed into either tissue-culture plastic slides or glass chamber slides. Cells thus obtained were observed in time-lapse cinematography, assayed for proliferation, and examined immunohistologically and histochemically, and their ability to become fibroblasts, osteoclasts, osteoblasts, and adipocytes was documented.
BMPCs were found in elutriation fractions containing less than 30% T cells and more than 60% monocytes from the blood of more than 100 normal persons. BMPCs adhered to plastic and glass and proliferated logarithmically in DMEM-20% FCS without added growth factors. The initial elutriate had only small, round, mononuclear cells; upon culture, these were replaced by fibroblast-like cells and large, round, stromal cells. The formation of cells with fibroblast-like and stromal morphology was not affected by eliminating CD34, CD3, or CD14 cells from the elutriation fraction. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycero-phosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed AP and its production was doubled by the addition of BMP2 (1 ng) (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures contained two other types of cell: sudanophlic adipocytes and multinucleated giant cells, which stain for TRAP and vitronectin receptors (attributes of osteoclasts). Cultured BMPCs were immunostained by antibodies to vimentin, type I collagen, and BMP receptors (heterodimeric structures expressed on mesenchymal lineage cells). The cultured cells also stained strongly for the SH-2 (endoglin) antigen, a putative marker for marrow MSCs. BMPCs express the gene for SDF-1, a potent stroma-derived CXCα chemokine.
In the circulation of normal individuals is a small population of CD34- mononuclear cells that proliferate rapidly in culture as an adherent population with a variable morphology, display cytoskeletal, cytoplasmic, and surface markers of mesenchymal precursors, and differentiate into several lineages (fibroblasts, osteoblasts, and adipocytes). These are all features found in bone-marrow-derived MSCs. Therefore, autologous blood could provide cells useful for tissue engineering and gene therapy. In addition, the demonstration of similar cells in the inflammatory joint fluids and synovium of patients with rheumatoid arthritis (RA) suggests that these cells may play a role in the pathogenesis of RA.