Cultivation Of H-MSCs Cells And Differentiation Into Neuron-like Cells In Vitro

Objactive To isolate and cultivate h-MSCs in vitro,preinduce by basic fibroblast growth factor(bFGF), then induce by different concentration of CNTF, observe the variations of cell appearances and expressions of CD34, CD44, CD54, NSE and Nestin by immunohistochemistry.To approach establishment a more convenient and effective cultivation method of human bone marrow mesenchymal stem cells (h-MSCs) in vitro,and test the effect of induction h-MSCs differentiation into neuron- like cells by ciliary neurotrophic factor (CNTF) and the eligible concentration of CNTF.Methods MSCs were cellected from 68 patients in the blood department, the second and the first auxiliary hospital of Nan Hua University,who did not have cancer, infection or hematopathy,in18～55 years old,including 39 males and 29 females. h-MSCs were isolated by density centrifugation method and whole bone marrow adherence method with DMEM/F12 containing serum and then amplified, cryopreser- vation and resuscitation.Phase-contrast inverted microscope was used to observe mor- phology change of h-MSCs and draw the growth curve of the first, third and sixth passages h-MSCs.The third passage h-MSCs were divided into 4 groups:experimental group,purely preinduction group, purely induction group and control group. In experi- mental group, h-MSCs were preinduced by bFGF in 24 hours, then induced by diffe- rent concentration CNTF: 5ng/mL,10ng/mL and 20ng/mL in 12 hours.In purely prein- duction group, h-MSCs were only preinduced by bFGF. In purely induction group, h-MSCs were induced by 5ng/mL, 10ng/mL and 20ng/mL CNTF. In control group, h-MSCs were not induced by CNTF or preinduced by bFGF. Immunohistochemistry was used to identify the cultivated for CD34,CD44,CD54 and differentiated cells for NSE and Nestin. Results1.h-MSCs were fusiform and fibroblast-like cells, and had stable proliferative capa- city, cryopreservation, resuscitation in vitro. The growth characters of h-MSCs after resuscitation and before cryopreservation were identical;2.The growth curves of h-MSCs were like"S"; There were not significant differences between the cell concentrations of the first passage and the third passag(eP=0.704). There were not significant differences between the cell concentrations of the first passage and the sixth passage(P=0.132), and also between the third and sixth(P=0.256);3.The results of immunohistochemistry disclosed the CD44,CD54 were all positive expression,and the CD34 were nafetive expression;4. After the preinduction 24 hours by bFGF, the cell appearances did not exhibite variations. After the induction 5 hours by CNTF, a few cellls diminished and contract- ed. The cells were taper and had protubrance. After the induction 12 hours by CNTF, the cells exhibited obvious variations and had 2 or more protubrances like neuron cells in 10ng/mL group and 20ng/mL group of experimnet group;Few cell appearances exhibited variations in 5ng/mL experiment group. The cell appearances did not exhibite variations and were fusiform in control group, purely preinduction groupand purely induction group;5.The results of immunohistochemistry disclosed NSE and Nestin were all positive expression after inducing. There were significant differences between all groups(F=98.822,F=105.395,P<0.01); There were significant differences between the experiment group and control group, purely preinduction group,purely induction group(P<0.01),but not btween the three groups.There were not significant difference between the positive cells rates of 20ng/mL group and 10ng/mL group(P=0.413,P=0.496),but they were all higher than 5ng/mL group(P<0.01)in experiment group. In 10ng/mL of experiment group, the rates of NSE positive cells were (46.38±4.99)% and Nestin positive cells were (45.76±4.46)%.They were the most of all groups. Conclusions1.The combination method of density centrifugation method and whole bone marrow adherence method, was a more convenient and effective system of cultivation and purification h-MSCs in vitro;2.h-MSCs could be cultivated, proliferated,cryopreservation and resuscitation in vitro. The growth characters of h-MSCs after resuscitation and before cryopreservation were identical;3.In this experiment, h-MSCs could be induced and differentiated into neuron-like cells by CNTF; and 10ng/mL CNTF may be the more eligible concentration.