Enrichment of osteogenic cell populations from rat bone marrow stroma.
The presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. The presence of these multiple subpopulations suggests a need for a method that can purify the osteogenic component, i.e. osteoprogenitors, from other components. The availabilities of monoclonal antibodies recognizing subpopulations of osteoblasts are providing means for antibody-based methods. The cell surface antigens STRO-1, ALP and HOP-26 were used for cell sorting experiments with fluorescence activated cell sorting (FACS). These cell populations were analyzed on differential gene expression, cell proliferation and differentiation into the osteoblastic lineage. The oligo-microarray results showed that only the ALP positive cell population expressed genes of the extracellular matrix; like different collagens, ECM-1 and matrix protease MMP-14. The real-time polymerase chain reaction (QPCR) results showed that STRO-1 and ALP positive cells had an upregulation in expression of lipoprotein lipase, osteocalcin, and collagen type I. Integrin beta-3 was only upregulated for ALP positive cells, while for these cells downregulation occurred for the genes myosine, alkaline phosphatase and integrin beta-1. HOP-26 positive cells showed an upregulation in collagen type I compared to control group. The DNA analysis revealed that the cells of the control group and the HOP-26 positive cells showed a 5 times higher cell growth compared to the STRO-1 and ALP positive cells. The alkaline phosphatase activity showed no activity for the control group. The STRO-1 and ALP positive cells had a higher activity compared to the HOP-26 positive. The calcium measurements revealed only for the control group calcium at day 24. Based on the results of our study, we conclude that the FACS method had no negative effect on the proliferating as well as differentiating response of the cells. Further, we conclude that by using an antibody-based cell selection method, different cell populations with different mRNA expression profiles and different osteogenic characteristics can be obtained.
van den Dolder
Juliette
J
Department of Periodontology and Biomaterials, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. j.vandendolder@dent.umcn.nl
Jansen
John A
JA
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D015519:Bone Density / Q000502:physiology; D001846:Bone Development / Q000502:physiology*; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D002454:Cell Differentiation / Q000276:immunology / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry; D010006:Osteoblasts / Q000166:cytology* / Q000276:immunology; D010012:Osteogenesis / Q000502:physiology*; D051381:Rats; D017154:Stromal Cells / Q000276:immunology
D000954:Antigens, Surface