| Bone marrow mesenchymal stem cells ( MSCs) , also referred to as bone marrow stromal cells, are stem cells from adult bone marrow. They can not only differentiate into various cell lineages that derive from mesoderm during development, but into glias and neuron - like cells that usually come from ectoderm.MSCs are easily available, capable of rapid expansion in culture, able to cross damaged blood - brain barrier, capable of long - term survival and integration in host brain and immunologically inert, which makes MSCs one of best donor cells for neurotransplantation.However, studies on MSCs application in neurosystem are still rare in china. Here, we aim to establish reliable methods for adult mice MSCs culture and differentiation towards neuron -like cells.Methods1. MSCs were obtained from femurs of Kunming adult mice (6 -8w) aseptically under anesthesia. Through discarding non - adherent cells, MSCs were purified and expanded. Invert phase - contrast microscopy was used to observe morphology change of MSCs each day. HE stain was used to observe morphology more clearly.2. For neurogenic differentiation, MSCs were incubated in prein-duction media consisting of DMEM/10% FCS/1mmol/L β - Mercap-toethanol( BME) 24h. Then, preduction media were removed and cells were transferred into neuronal induction media ( DMEM/8 mmol/ L β- Mercaptoethanol). Cells were fixed for NSE and GFAP immu-nocytochemistry at 6h. Cells exhibiting increasing NSE staining were counted and compared to total cells, the mean and deviation were computed. To assay multilineage differentiation potential, MSCs were cultured in adipogenic media (1 X 10-6 mol/L dexamethason/10% FCS/DMEM) for 12 days. Oil red 0 stain was used to identify adipo-cyte.Results1. The morphologies of the first 5 passages of MSCs are stable, most of which appear fibroblast - like. The first 5 passages can reach confluence after 7-9 days.2. After induced by β- Mercaptoethanol, cell bodies of most MSCs contract and exhibit spherical or pyramidal, with increasing thin processes. Most of MSCs before inducement express low but detectable NSE, while the others show negative stains. After inducement, NSE expressions raise apparently. Cells showing strong or moderately NSE expression are71.4±11.1% .No GFAP expression is detected both before and after inducement.3. After exposure to adipogenic media 12 days, fat droplets in the cells are stained with Oil Red O. Undifferentiated MSCs can 't be stained with Oil Red O.DiscussionAlthough cultures of MSCs have been studied extensively for many years, specific markers for characterizing the cells have not been developed. The general criteria used now are as followings: ①, cells are obtained by their plastic - adherent nature ②. fibroblast - like cells are prominent in cultures ③. cells show multilineage differentiation potentials. The cells we prepared fulfill these criteria, so we can conclude that they are MSCs.In our experiment, we diluted mice bone marrow directly into culture dishes. By this way, we not only increased cell number obtained from bone marrow, but also reduced contamination and destroy or damage to cells during complex procedures. Thus , survival rate of primary culture is increased.There are three morphologically distinct cell types in our cultures; spindle - shaped cells, large fat cells and very small round cells, which is consistent with the observations of Colter et al. According to the analysis of flow cytometer, Colter et al inferred that this very small round cell formed mature MSCs during log phase. Moreover, samples enriched for the small cells had a greater potential for multilineage differentiation than those enriched for the large cells.NSE and GFAP are specific marker of neuron and astrocyte respectively. Increased NSE expression and negative GFAP stain after inducement indicate that our protocols induce neuronal ,but not astro-cytic differentiation. This is consistent with the report of Woodbury et al. But whether these differentiated cells pos...
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