Mouse Mesenchymal Stem Cells Isolation、Culture and Differentiation into Neuron-like Cells

Abstract: PrefacePluripotent stem cells have been detected in multiple tissues in the adult, participating in normal replacing and repairing, while undergoing self – renewal. Although bone marrow is the major source of adult hematopoietic stem cells (HSCs). It also contains another kind of stem cells-Mesenchymal stem cells ( MSCs ). Mesenchymal stem cells are thought to be multipotent cells, which have the potent to differentiate into lineages of mesenchymal tissues, including muscle, cartilage and fat. In recent studies, mesenchymal stem cells are used clinically to treat osteogenesis imperfection. Here we induced the mouse mesenchymal stem cells to neuron – like cells in vitro, which provided the possibilities using the mesenchymal stem cells to treat a variety of neurological diseases technically and theoritically.Materials and MethodsMaterials1. Source of bone marrow; femurs of mice four weeks old (18 -20g).2. Reagents used in cell cultureDMEM – LG, FBS ( Fetal Bovine Serum) , ATRA ( All – trans -retinoid acid) , Recombinant human bFGF , BME ( (3 – mercaptoeth-anol).3. AntibodiesPolycolonal human, mouse, rat anti – neuron specific enolase, Polycolonal mouse anti – glial fibrillary acidic protein.MethodsIsolation,, culture and purification of mouse MSCsmMSCs were isolated from the femurs of adult mice four weeks old and propagated in vitro. mMSCs were originally cultured in DMEM -LG with 10% FBS,100U/ml penicillion,100mg/ml streptomycin. At the second day in the cell culture the unadherent cells were carried out. After that we replaced the media with fresh complete media every two or three days. For each passage the cells were plated at about 3 x 10 /ml and grew to confluency.Neuronal InductionSubconfluent cultures of mMSCs were maintained in DMEM – LG / 10% FBS. 24h prior to neuronal induction, media were replaced with preinduction media consisting of DMEM – LG / 20% FBS / 1mM BME. To initiate neuronal differentiation, the preinduction media were removed, and the cells were washed with PBS then transferred to neuronal induction media composed of DMEM – LG / 1 – 10 mM BME. In the later experiments DMEM – LG /20% FBS / bFGF (50ng/ml) was used as the neuronal preinduction media and DMEM – LG / ATRA ( 150mg/ml) was used as the neuronal induction media. During induced differentiation we observed the changes of the cell morphology, the number of the cells and the expressions of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) in the cells.ImmunocytochemistryThe BME – induced mMSCs were fixed with 4% paraformalde-hyde, incubated with primary antibodies of NSE and GFAP overnight at 4C , incubated with secondary antibody for 30min. DAB served as chromagen.Western BlotThirty micrograms of protein extracted from untreated and bFGF, ATRA – induced mMSCs cultures were separated on a 8% gradient acrylamide gel and eletrophoretically transferred to a nitrocellulose membrane. The blot was probed for NSE expression. Secondary antibody was alkaline phosphatase conjugated.ResultsIsolation , culture and Purification of the mMSCsTypically, mMSCs were isolated from bone marrow by their adherence to plastic and grew as fibroblastic cells that developed into visible symmetric colonies. By the method of passage, we further purified the mMSCs cultures.Neuronal DifferentiationTo induce the neuronal phenotype, mMSCs were maintained in media containing neuronal induction reagents. Within the 5h of exposure to neuronal induction, changes in morphology of some of the mMSCs were apparent. Initially, cytoplasma in the mMSCs retracted towards the nucleus, forming a contracted multipolar, cell body, leaving membranous, process – like extensions peripherally. Over the induction process cell body became increasing spherical and retractile, exhibiting a typical neuronal perikaryal appearance. Western Blot a-nalysis indicated that cells exhibited increased expression of the neuronal marker NSE after 5h of bFGF, ATRA treatment. Immunocyto-chemistry analysis indi…
Key words: Mesenchymal stem cells; neuronal differentiation

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