Expansion Of Bone Marrow Mesenchymal Stem Cells And Coculture With Neural Type Cells

The repair of central nervous system (CNS) disorders, such as Parkinson's disease (PD) and spinal cord injury, is difficult. Neural stem cells (NSCs) transplantation is believed to be effective in the treatment of neural diseases, however, NSCs are limited and difficult to be isolated from brain. Bone marrow mesenchymal stem cells (BMSCs) are a new cell source to CNS diseases because the cells possess the advantages of multi-differentiation potentials, weaker immunogenicity, easy obtaining and expansion, and secreting cytokines in in vivo microenvironment. Here, the expansion of BMSCs and the interaction between BMSCs and NSCs as well as the effect of expanded BMSCs on PD modeled cells in vitro were investigated.BMSCs were expanded in vitro firstly under three dimensional (3D) conditions by the combined use of collagen gel, hollow fiber membranes (HFMs) and air-lift loop bioreactor (ALB). During the expansion period, the cell growth curves and metabolism parameters were measured. And after7days, the cell viability and morphology were assayed microscopically. Moreover, the BMSC characters of expanded cells were identified by specific expressions of CD29, CD44, CD34and CD45and by multilineage differentiation into osteoblasts, chondrocytes and adipocytes. The results showed that the cultured BMSCs metabolized robustly and expanded about17-fold under dynamic condition,13-fold under static condition with the initial density of5×105cells·mL-1; after7days, the cells maintained high viability of about92%and spread with a spindle appearance and long cell protrusions in gel; the expanded cells were CD29and CD44positive, CD34and CD45negative, and they could differentiate into osteoblasts, chondrocytes and adipocytes; the cells expanded under dynamic condition spread more and showed stronger multi-differentiation potentials.The expanded BMSCs were then used to coculture with NSCs growing in scaffolds to investigate the interaction between BMSCs and NSCs. The NSCs encapsulated in calcium alginate beads (Ca-Alg-Bs) with appropriate density firstly grew to neurospheres before being cocultured with BMSCs. During the coculture period, the neurosphere structure and BMSC morphology were observed. And when the coculture ended, the NSC and BMSC expansion folds were measured; the phenotype and multi-differentiation potentials of NSCs and the differentiations of BMSCs into NSCs or neurocytes in the NSC proliferation medium were assayed by immunofluorescent staining; meanwhile the capacity of NSCs to differentiate into neurocytes in the differentiation medium was analyzed with flow cytometry, and the differentiations of BMSCs into NSCs or neurocytes were assayed by immunofluorescent staining. The results showed that2mm-diameter Ca-Alg-Bs prepared with1.5%sodium alginate and3.5%CaCl2solutions gelling for10min were suitable to encapsulate NSCs with cell density of0.8×105cells·mL-1; NSCs in beads could migrate out of neurospheres by contacting with BMSCs indirectly; BMSCs effected NSCs proliferation slightly, while influenced NSCs differentiation obviously so that the oligodendrocyte proportion increased3times and astrocyte proportion decreased1fold, but neuron proportion remained unchanged; part of the BMSCs could be induced into NSCs, astrocytes, oligodendrocytes and neurons by coculture with NSCs, and the proportions of these cells in the proliferation medium were8%,11%,6%and7%, respectively, while in the differentiation medium, the figures increased to18%,25%,15%and11%, respectively.Furthermore, PD cell model in vitro was constructed through PC12cell apoptosis induced by serum deprivation. The apoptotic PC12cells were then cocultured with NSCs alone or with NSCs and BMSCs together by means of Ca-Alg-Bs or transwell inserts. The repair of apoptotic PC12cells after the coculture was investigated and how BMSCs contributed to the repair of PD modeled PC12cells by NSCs was explored. During and at the end of the coculture, PC12cell morphology was observed and PC12cell survival, cell cycle and GDNF concentration in medium were detected. The results showed that NSCs inhibited PC12cell from apoptosis through secreting neurotrophic factor such as GDNF and the PC12cell apoptosis percentage decreased from44.0%to24.3%; the living PC12cells in G1phase reduced from79.9%to53.1%, while the cells in S and G2phases increased from15.3%and4.9%to35.5%and9.8%, respectively; these cell percentages of repaired PC12cells were close to those in normal cultured cell group, which meant that the process from G1phase to S and G2phases was nearly not obstructed. After BMSCs were introduced into the coculture system, GDNF concentration in the medium increased about33.4%comparing to the coculture system with NSCs alone. In the coculture system of BMSCs, NSCs and PC12cells, the ability of the cells to secret GDNF increased and the protection to the apoptotic PC12cells was enhanced additionally; the PC12cell apoptosis percentage further decreased from24.3%to15.1%, and the living cells in G1, S and G2phases were53.3%,36.7%and10.3%, respectively, which remained close to those in normal cultured cell group.