Study On Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neural Cells And Osteoblasts

Bone-marrow mesenchymal stem cells (BMSCs) have generated a great deal ofexcitement and expectation as a potential source of cells for cell-based therapeuticstrategies in regenerative medicine, primarily because of their intrinsic ability to self-renewand differentiate into functional cells of multiple tissues including fat, bone, muscle, neuraland cartilage. They are easily isolated and expanded with high efficiency and induced todifferentiate under well defined culture conditions.1. Isolation, in vitro expansion, and characterization of bone marrowmesenchymal stem cellsTo establish a method of in vitro isolation, purification and amplification of bonemarrow mesenchymal stem cells of adult rat, three isolation methods were evaluated andthe results indicated the high purification of bone marrow mesenchymal stem cells can beobtained by incontinuous density Percoll gradient centrifugation technique. The purity wasexamined by immunochemical staining and Flow Cytometry. The growth curve was testedby MTT assays. Over 90% cells were harvested by discontinuous density Percoll gradientcentrifugation and morphological observation accorded with mesenchymal stem cells. Thegrowth curve indicated that the cells growed in a good status and Flow cytometry analysisusing 8 surface markers showed that the 5 and 20 passages of BMSCs were positive forCD29, CD90, CD44, CD106, CD71 and negative for CD34 and CD45.2. Differentiation of bone marrow mesenchymal stem cells into cells witha neural phenotype In order to study factors that are involved in the neural differentiation of BMSCs, cellswere treated with: group (1) bFGF, DMSO, butyl hydroxy anisol (BHA); (2) bFGF,DMSO; (3) DMSO, BHA; (4) DMSO. Treatment (1) induced differentiation of 90% cellsinto neural cells within 29 hours (h), while a longer treatment was required when usingtrestment (2) (3) (4). Morphological changes were more dramatic after treatment (1)compared with treatment (2) (3) (4). Treatment (1) resulted in 80% positive cells forneuron-specific enolase (NSE) after 29 h while almost all cells were negative in othergroups. After 32 h, NSE expressions were observed in all treatments. Moreover, bonemarrow mesenchymal stem cells were cultured in serum-free L-DMEM media for 24 hafter treatments, cells were treated with (3) (4) tended to senescence.To investigate the neural markers' expression in the differentiation of BMSCs, cellswere first treated for 24 h with preinduction media L-DMEM consisting of fetal calf serum(FCS) and bFGF, then induced with induction media L-DMEM consisting of DMSO andbutyl hydroxy anisol (BHA). Nestin, a neuroectodermal stem cell marker was expressedearlier than NSE andβ-tubulin.BMSCs and the neural cells derived from BMSCs were prelabeled with BrdU andtransplanted into the injured spinal cord rats. Then, the rats behaviors were assessed byBBB score 1-8 w after transplantation respectively. Treated rats generally showed betterfunctional recovery than control rats after operation. Immunohistochemistry identificationshowed that BrdU positive cells could be found in the spinal cord injury site.3. Differentiation of bone marrow mesenchymal stem cells intoosteoblastsTo evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) at different times during subculturing period, BMSCs were induced byconditioned medium (1) at the seeding time, (2) at 50% confluence, and (3) at 85-90%confluence. Proliferation and differentiation of BMSCs were observed under invertedphase-contrast microscope. Alkaline phosphatase (ALP) in BMSCs was stained by Gomori,the calcified nodules were stained by von kossa method, and ALP activity was measured. BMSCs at different times responded to the osteogenic supplements by changing theirmorphology, showing the specific property of osteoblasts. Positive staining for ALP andcalcified nodules were observed. Cells induced at 85-90% confluence had stronger reactionfor ALP and more calcified nodules than the others and the alkaline phosphatase (ALP)activity of BMSCs was increased more significantly.To investigate the osteogenic potential of BMSCs at different passages, cells atpassage 5, 10, 15 and 20 were induced to differentiate into osteoblasts and results showedno difference among these passages.Our results contribute to the understanding of mechanisms involved in thedifferentiation of mesenchymal stem cells.