Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro.

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.

Frank O ，Heim M ，Jakob M ，Barbero A ，Schäfer D ，Bendik I ，Dick W ，Heberer M ，Martin I ... -  《JOURNAL OF CELLULAR BIOCHEMISTRY》

Is 1, 25-dihydroxyvitamin D3 an ideal substitute for dexamethasone for inducing osteogenic differentiation of human adipose tissue-derived stromal cells in vitro?

Zhou YS ，Liu YS ，Tan JG 《CHINESE MEDICAL JOURNAL》

Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2.

In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex) and recombinant human bone morphogenic protein-2 (rhBMP-2). Cellular proliferation was determined by 3H-thymidine incorporation into DNA at both Days 2 and 7 when BMSc was cultivated with HY at concentrations of 0, 0.5, 1.0, 2.0 and 4.0 mg/ml. HY accelerated cellular proliferation when compared with cultures in the absence of HY at both Days 2 and 7. BMSc proliferation under the high HY concentration of 4 mg/ml was significantly higher than under the other, lower HY concentrations of 0.5, 1.0 and 2.0 mg/ml. When BMSc were cultivated under HY at concentrations of 0, 1.0 and 4.0 mg/ml and its 12 combinations with rhBMP-2 at concentrations of 0 and 10 ng/ml and Dex (+, -) at both Days 2 and 7, cellular responses were examined by 3H-thymidine incorporation into DNA, cellular alkaline phosphatase (ALP) activity, and pro-collagen type I C-terminal propeptide production. HY accelerated cellular proliferation irrespective of the presence of Dex and rhBMP-2. HY increased expression of ALP activity at Day 7, whereas had inhibitory effect at Day 2. HY and Dex showed an interaction on expression of ALP acitivity irrespective of the HY dose by Day 7. Collagen synthesis was inhibited by HY irrespective of the presence of other factors at both Days 2 and 7. When BMSc were cultivated with HY of 4.0 mg/ml alone, its combinations with Dex (+) and 10 ng/ml rhBMP-2, and with DMEM/FBS alone, expression of bone-related marker genes was evaluated by real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) analysis. Osteocalcin was up-regulated under both rhBMP-2 and HY-Dex-rhBMP-2 at Day 2, as also under 4 mg/ml HY, Dex, HY-Dex, Dex-rhBMP-2, and HY-Dex-rhBMP-2 by Day 7. Type 1alpha1 collagen was induced by rhBMP-2 on Day 2, and by Dex-rhBMP-2 on Day 7. Osteonectin and type X collagen was only marginally induced by HY at Day 2. Type 1alpha1 collagen and type X collagen were down-regulated in the presence of 4 mg/ml HY by Day 7. These results suggest that HY stimulates BMSc proliferation, osteocalcin gene expression, and a secretion of enzymes such as that of ALP activity in vitro. More importantly, HY can interact with Dex and rhBMP-2 to generate direct and specific cellular effects, which could be of major importance in bone tissue engineering.

Zou X ，Li H ，Chen L ，Baatrup A ，Bünger C ，Lind M ... -  《BIOMATERIALS》

Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.

Rickard DJ ，Sullivan TA ，Shenker BJ ，Leboy PS ，Kazhdan I ... -  《DEVELOPMENTAL BIOLOGY》

Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells.

von Knoch F ，Jaquiery C ，Kowalsky M ，Schaeren S ，Alabre C ，Martin I ，Rubash HE ，Shanbhag AS ... -  《BIOMATERIALS》