Effect Of Peripheral Nerve Cells Suspension On Differentiation Of Bone Marrow Stromal Cells In Vitro

Background and purpose: Parkinson's disease (PD) is one of the most common chronic neurodegenerative diseases, and its etiopathogenisis and pathogenesis are unknown. The strategy now is to control the clinical symptoms, but can not halt the progression of the disease. Cell transplantation can delay the progression even cure the disease. And the cells for transplantation have been changing, from dopamine adrenergdrenic chromaffin cells,embryo substantia nigra cells to nerve stem cells and marrow stromal cells (MSCs) nowadays, and the latter one is outstanding in all candidates. MSCs exist in bone marrow which can differentiate into not only hematopoetic cells, but also into non-hematopoietic cells; the properties of easy isolation, culture, proliferation and being able to auto-transplant make MSCs the ideal cells for cell transplantation. Now researchers usually control the differentiation of MSCs using cytokines and chemicals in vitro, transplanting when cells are enough.Microenvironment is the most advantageous condition for cell differentiation. Peripheral nerves include Schwann cells (SCs) and Fibroblast, except for axons. SCs are glial cells of peripheral nerve system, which can secrete growth factors and support axons. It is said that SCs could secrete more than 20 sorts of neurotrophic factors, extracellular matrix, and cell adhesion factors that are used for peripheral nerve damage. There is an investigation suggesting that SCs can induce nerve stem cells differentiate into tyrosine hydroxylase positive neurons.It is hard to get SCs because of the complicate technique,expensive reagent,not adequate purity and so on.There are a few reports about Fibroblasts. Fibroblast can secretetransforming growth factor,basic fibroblast growth factor,insulin-like growth factor,epidermic cell growth cell and so on.So far investigators can induce MSCs into neuron-like cells, which express the characteristic markers of neuron. Our experiment use peripheral nerve cells suspension and mesencephalic conditioned media to induce MSCs differentiate into dopaminergic (DA) neurons. The purpose is to approach a way that can induce MSCs differentiate into DA neurons in vitro, establishing the groundwork for cell transplantation therapy of PD. Methods:(1)Isolation, culture, and purification of MSCs The rat marrow was isolated from femurs and tibias of SD male rats, and then maintained in L-DMEM supplemented with 10% fetal bovine serum. Kept it at the condition of 37℃,5%CO2,and renewed the culture medium firstly after 24 hours. The nonadherent cells were cleared by replacing the culture medium. When the cultures reached confluency, the cells were gone down to posterity, using trypsin, and MSCs were purified by this way. We used the 3rd to 5th passages of MSCs for inducing.(2)Peripheral nerve cells suspension A SD rat of 300~350g weight was anesthetized by 2.5% ethypropymal sodium, getting bilateral sciatic nerve. Then made the nerve fiber from nerve track smashed, and put the fragment into centrifuge tube with 0.5% trypsin and 0.06% collagenase under water bath of 37℃for 90 minutes. Stopped the enzyme reaction with culture medium, attenuated the cells into 3×105/L after centrifuged, and kept the cells suspension in incubator.(3) Control group: MSCs were just preinduced by bFGF for 24h. Experimental groups:①MSCs with peripheral nerve cells suspension;②MSCs with peripheral nerve cells suspension and mesencephalic conditioned media. MSCs in both experimental groups were preinduced by bFGF for 24h before being induced by peripheral nerve cells suspension or mesencephalic conditioned media.Results:(1)The rat MSCs could be cultured and proliferated in vitro, and the number were twice for 3~4 days.(2)The number of cells increased noticeably after being preinduced bybFGF. Cell bodies contracted and many of the cells had the typical morphologic features of neuron.(3)The experiment groups had more NSE-positive and TH-positive cells than that of control group.(4)There were no TH-positive cells in the control group. The experimental group of MSCs with peripheral nerve cells suspension and mesencephalic conditioned media had more TH-positive cells than two other groups.Conclusion:(1)The rat MSCs could be successfully cultured and proliferated in vitro.(2)bFGF could promote MSCs to differentiate into neuron.(3)bFGF and peripheral nerve cells suspension could induce MSCs to differentiate into neuron-like cells, which expressed NSE.(4) peripheral nerve cells suspension and mesencephalic conditioned media could induce to MSCs differentiate into dopaminergic cells, which expressed TH.