Objective
The main objective is to differentiate mesenchymal stem cells from human bone marrow into osteoblast using osteoblast differentiation media.
Method
Mononuclear cells isolated from bone marrow using Ficoll-paque were cultured in DMEM, 10% FBS and 1% antibiotics pen strep (Penicillin and streptomycin). Cell viability was checked by Trypan blue method and 95% cell viability was achieved at every consecutive passage. Further, cells were grown in Osteoblast differentiation media (0.1% dexamethasone, 10mM beta Glycerophosphate, 0.2mM ascorbic acid with 10% FBS and 1% Penicillin (10000U/ml) and streptomycin(10000µg/ml) GIBCO, Invitrogen, NY USA) . Differentiated osteoblast cells were stained with von-kossa for calcium phosphate deposits. MSC cells at each passage P1 and P2 were checked for FACS analysis using CD29, CD90, CD105, CD34, CD45, CD133 markers compared with controls.
Results
Mesenchymal stem cell markers, CD29+ positive cells are highly expressed in hMNC (97.2%) when compared to CD90+ (36.5%) and CD 105+ (56.6%). With subsequent passages CD29+, CD90+ and CD105+ expression increased which almost was 99% in P3. CD133+ is expressed in very high fraction of cells in the fresh sample (77%) and decreases in subsequent passages. This is an expected phenomenon as CD133+ is a pure HSC marker. Therefore CD29+, CD90, CD90+, correlates with pure hMSC cells from HSC cells and can be used for differentiation.
Conclusion
Bone marrow is the major source of hMSCs contributing circulating bone marrow mono-nuclear cells, Therefore differentiation of MSC from HSC can be of great importance in regenerative medicine.
