Experimental Study On Optimal Culture In Vitro Of MPC And Directed Differentiation Of MPC Into Neuron-like Cells In Mice

Objective: To sudy the method of optimal culture in vitro of mesenchymal progenitor cells (MPC) from compact bone in C57 mice to establish the suitable amplifying condition for MPC; to study the experimental research on inducing directed differentiation of MPC from compact bone of mice into neuron-like cells in vitro.Method:Sampling methods: Female healthy C57 mice weighing 18±2g, of clean degree and of 3 weeks old were selected in this study. Femur and tibia fragments after digested by collagenase (typeⅡ) were the source of MPC.Grouping: The digested bone fragments were cultured in different match with DMEM/F12 culture medium, DMEM/F12 culture medium andα-MEM culture medium and fetal bovine serum (FCS) produced in TBD Company and FCS produced in GIBCO Company, which were usually used in stem cells culture. They were divided randomly into 6 groups with 6 portions in each group. They were DMEM/F12+10%TBD FCS group, DMEM/F12+10%GIBCO FCS group, IMDM+10% TBD FCS group,IMDM+10%GIBCO FCS group ,α-MEM +10%TBD FCS group andα-MEM+10%GIBCO FCS group.Culture method: We took the compact bone fragments into culture dishes; added corresponding culture fluid based on different groups and put them into 37℃incubator with 5% CO2. The culture medium was changed after 48 hours at the first time. From then on, the culture medium was changed once every 2-3 days. The cell growth condition was observed everyday. When the area of the cell attachment of primary cells in best growth group was more than 80% of the area of the bottom of culture plate, the culture medium was sucked out. After digested by 0.25% trypsin contained 0.04% EDTA, the cells were gathered and put into culture bottle with fixed cell concentration. After passaged, the culture medium was changed once every 2-3 days. When the area of cell attachment was more than 70% -80% of the total area of bottom of the culture bottle in the best growing group, we passaged the cells with cell concentration 1×10~3/cm~2.Purity identification of MPC: MPC of P3 was examined with single fluorescence labeling of flow cytometry with the marker, FITC labeled rabbit anti mouse CD29, CD31, CD44, CD90 and PE labeled CD45, CD106. The parallel control group was MPC with homotype control antibody. Considering the experimental needs and limited time in the stage of the graduate student study, we only selected MPC cultured with optimum scheme for the purity identification.Identification of multipotential differentiation of MPC: The result of induction of committed differentiation of MPC into osteocyte or adipocyte was examined with alizarin red and oil red O staining, which was used to verify MPC had the multipotential differentiation potential of stem cells. For the same reason, we only selected MPC cultured with optimum scheme for the identification of multipotential differentiation.Method of induction of committed differentiation of MPC into neuron-like cells: MPC of P4 cultured with optimum scheme was selected to be induced with bodyfluid of microenviroment (the supernatant cultured with primary neuron). After 24 h, MPC after induction were selected as the experiment specimens, which were for morphological observation; for immunocytochemistry detection of the expression by neuronal specific markers neuron specific enolase (NSE) and neurofilament triplet (NF) in and meanwhile, for immunofluorescence assay.Result:Optimized culture in vitro of MPC in C57 mice:After 5-day primary culture, cell attachment to the bottom of culture dishes could be observed. The cells were mainly round and oblate. After 10 days, becoming spindle and polygonal, attachment cells spread gradually. They were in plump shape and with high refraction. There was no overlapping among cells, but contact inhibition. After passage, the cell morphology tended to coincide, mostly spindle, in dense arragement and vigorous growth. When the area of cell attachment was more than 70% -80% of the total area of bottom of the culture bottle in the best growing group, all the cells were passaged and counted. Based on the observation of 3 passages, the result showed that the cell quantity of DMEM/F12+10%GIBCO fetal bovine serum group was more than that of the other groups in the cell count ofevery passage. The statistical test verified that compared with the cell quantity of the other groups, that of DMEM/F12+10%GIBCO fetal bovine serum group had significant difference (P﹤0.05). It indicated that DMEM/F12+10%GIBCO fetal bovine serum were more beneficial to the culture and reproduce in vitro of MPC.Purity identification of MPC:MPC cultured with optimized scheme expressed the mensenchymal cells surface protein CD29, CD44, CD90 and CD106, while they did not express hematopoietic stem cell surface protein CD31 and CD45. It indicated that MPC that we obtained were with good homology, high purity and without the influence of hematopoietic stem cell.Identification of multi-directional differentiation capability of MPC:In the experiment of directed induction of MPC cultured with opmized scheme into osteocytes, a plenty of calcium deposition were found in extracellular matrix through alizarin red staining after induction. In the experiment of directed induction of MPC cultured with opmized scheme into adipocytes, lipid droplet in the cells was found punctuate red through oil red O staining. The two experiments indicated that MPC which we obtained could differentiate to osteocytes or adipocytes and they were with good activity and multi-directional differentiation capability.Method of induction of committed differentiation of MPC into neuron-like cells:At the time point of 24 hours after induction, the cell morphology of MPC has changed. There were processes growing from the cell bodies, different in length, which were similar to neurons. The result of immunocytochemistry detection showed that the expression of both NSE (73.73%±9.88%) and NF (60.26%±7.19%) of MPC after induction was positive, which was verified by immunofluorescence assay.Conclusion:1. The experiment verified that it's the most beneficial to the quantitative proliferation that MPC cultured with DMEM/F12+10%GIBCO fetal bovine serum from compact bone of mice and the MPC group that we obtained was with good homology, high purity, strong proliferation activity and multiple differentiation potential.2. The method of induction of the supernatant cultured with primary neuron could make MPC from compact bone of mice differentiated into neuron-like cells directly.