Osteoblast formation from MesenchymalStem Cells Derived from mouse Bone Marrow in vitro

This study was designed to evaluate osteogenic potential of mesenchymal stem cells (MSCs) isolated from mouse bone marrow in vitro. MSCs were isolated by collecting the thigh bone of 50 male albino mice, both the femur and tibia were collected and cells were flushed from bones and MSCs isolated based on the ability of adherence to plastic surfaces. Reactivity MSCs to CD105 and CD34 were tested by immunocytochemistry. Isolated MSCs exhibited positive reactivity to CD105 and negative for the haematopoietic surface marker CD34.Differentiation of isolated MSCs into osteoblast was induced by osteogenic
medium consisting of high glucose- DMEM supplemented with 50 µg /ml Ascorbic acid, 1nM dexamethasone and10 Mm of  beta glycerophosphate disodium salt hydrate after 21 days.Osteoblast activity was monitored by evaluation alkaline phosphatase (ALP) activity in osteogenic medium by Reflotron at (0, 7, 14 and 21) days of differentiation. Results recorded that a significant (P≤ 0.05) increase in ALP activity in osteogenic medium at 7 and 14 day culture in comparison with zero day (32.13±0.46 and 23.33±0.88 vs. 5.33±1.76 IU/L) and decreased at 21 day (15.33±1.76 IU/L).The presence of beta-actin and osteocalcin genes was confirmed in differentiated cells by Reverse transcriptase PCR (RT-PCR) on day 14. Two bands were present with molecular size (200) bp for beta actin and (169) bp for osteocalcin.