| The found of adult stem cells and the study of induced them into neurons have opened up a new way for curing nerve system diseases and damages of human and animal. In order to explore a kind of adult stem cell, which can be used to induce into neuron expediently,safely and effectively,to become a ideal substitute material for transplantaton,the present study has established the culture system of fetal mouse neural stem cell and epidermal stem cell in vitro, and probed the condition of directed differentiated into dopaminergic neuron from neural stem cell and epidermal stem cell into neurons. 1．Neural stem cells were dissociated and cultured from fetal mouse cerebral. The growth curve revealed that neural stem cell grew continually when cultured in vitro. Single cell could proliferate to become neurosphere,and the cells derived from the obtained neurosphere had proliferation and differentiation capacity when passaged, the clone form rate was 93%. Immunocytochemistry study indicated that the neurosphere cells were nestin positive.2．It was found that the following methods was favorite for obtaining pure and normal neural stem cells, pipetted firstly, then filtrated, and after stay for a while collected supernatant liquid to centrifuge. When culture media were refreshed by just adding new media and no discarding. The neural stem cells could sustain proliferation status and until to 12 passages when cultured with stem cell culture media supplement bFGF. Removed bFGF from culture media, replaced with 2-5% FBS, neural stem cells could differentiate to three kinds cells,which express positive staining of NF,GAFP, Galc-C respectively, they are neurons,oligodendrocytes,astrocytes, their percentage were 21.1%, 59.7%, 19.2% respectively.3．NSCs were cryopreserved using 10% BSA and 8%DMSO NSCs cryopreservation media by following protocol, 30 min at 4℃→2 hr at -20℃→3 falls in nitrogen steam up liquid nitrogen, each 30min→put into liquid nitrogen. Thawed NSCs using standard method and cultured, the results showed that there was no significant effect of cryopreservation duration on NSCs viability, and the viability were not different significantly within passages.Cerebral NSCs and diencephalons NSCs were successfully induced to be dopaminergic neuron by DMEM/F12 supplement 2% B27+100pg/mL Il-1 + 0.05g/L Vc + 5 u/mL Erythropoietin (inducer group).Induced cells had typical neural cell morphology character, immunocytochemistry check with TH antibody revealed that plasma was positive stain to brown-yellow, nucleus was not stained. The percentage of TH positive cells increased along with the increase of induction times. TH positive cell rate derived from diencephalons (22.49%) was significantly higher than that of cerebral derivation (10.65%). When corpus striatum extract + DMEM/F12 (1:1) used as induction media (corpus striatum group), 14.49% diencephalons NSCs was differentiated into dopaminergic neuron, significantly lower than inducer group.Goat epidermal stem cells were round or elliptical at beginning of in vitro culture with big obvious nucleus, then became colony-like, could grow 14～20 d. They grew well in Ca2+ -free keratinocyte conditioned media and self-made stem cell culture media. The two culture media had no significant effect on epidermal stem cell clone form rate, but had significant effect on clone sustaining time. The clone sustaining time in conditioned media was higher significantly than that in self-made stem cell culture media. Epidermal stem cells could grow on mouse fetal fibroblast feeder layer or goat fibroblasts feeder layer. The two feeder layer had no significant effect on the beginning time of epidermal stem cell clone form, but had significant effect on clone sustaining period(P=0.049<0.05). The clone sustaining time on mouse fetal fibroblast feeder layer was higher significantly than that on goat fibroblasts feeder layer. Epidermal stem cells and stem cell clone all express integrin-β1. The plasma of those cells that had round shape and same size, stained to be brow...
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