title: Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro.
abstract: We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.
authors.affiliation: Department of Surgery, Research Division, University of Basel, Switzerland.
authors.forename: Oliver
authors.initials: O
authors.lastname: Frank
authors.forename: Manuel
authors.initials: M
authors.lastname: Heim
authors.forename: Marcel
authors.initials: M
authors.lastname: Jakob
authors.forename: Andrea
authors.initials: A
authors.lastname: Barbero
authors.forename: Dirk
authors.initials: D
authors.lastname: Schäfer
authors.forename: Igor
authors.initials: I
authors.lastname: Bendik
authors.forename: Walter
authors.initials: W
authors.lastname: Dick
authors.forename: Michael
authors.initials: M
authors.lastname: Heberer
authors.forename: Ivan
authors.initials: I
authors.lastname: Martin
mesh_terms: D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002454:Cell Differentiation; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D058574:Integrin-Binding Sialoprotein; D009363:Neoplasm Proteins; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics*; D053495:Osteopontin; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D016212:Transforming Growth Factor beta
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D017931:DNA Primers; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D053495:Osteopontin; D003907:Dexamethasone