| PrefaceAt present,stem cells transplantation has become a hot spots in the therapy of SCI. There are many suitable kind of cells for the transplantation,which contains the derived mesenchymal stem cells(MSCs),embryonic stem cells(ESCs),olfactory ensheathing cells(OECs),schwann cells(SCs)and so on.Transplanted cells can keep long-term survival in the damage zone,and they can secrete growth factors to replace the dead neurons,and to regulate the local micro-environment of the glial area.The transplanted stem cells can play their roles of cells replace,intercellular bridge and neurotrophic factors secretion.The MSCs show the much more the ascendancies during the transplant thearapy because they are large amount,easy to acquire,suitable for autologous transplantation and have no rejection compared with allografts.MSCs have a high degree of self-renew and multilineage differentiation capacity. They can be isolated from many tissues such as adult bone marrow,fat,umbilical cord blood and so on.Studies showed that,MSCs could be induced into neural cells under certain conditions in vitro.They could express many neural cell-specific markers,such asβ-tubulinâ…¢,microtubule-associated protein 2(MAP2),glial acidic protein(GFAP) and neuronal nuclear protein(NeuN) and so on.However,it is not clear whether MSCs from different sources including bone marrow stroma cells(BMSCs) and adipose-derived stroma cells(ADSCs) have the different ability of differentiation.In this study,therefore,MSCs from both BMSCs and ADSCs were choosed as the main study subject,to compare whether or not they possess the differences on inducing into neuron-like cells.This study aims to provide new experimental data for regenerative medicine.Partâ… Isolate and passage of BMSCs and ADSCsObjective To isolate the BMSCs and ADSCs from bone marrow and adipose of adult SD rat respectively,and then to culture the cells in vitro and to passage the cells until the 5th generation.The characteristics of them,as the stem cell were identified in order to carry out further study next step.Method 1.Derive and passage of ADSCs Abdominal adipose tissue from the adult SD rat was cut off in aseptic conditions,which was then cut into pieces with size about 1mm3,and then digested with 2.5%collagenase typeâ… for 60 minutes. Digestive juice was centrifuged at 1200 rpm for 8min,The cells were then re-suspended by DMEM/F12,centrifuged again at 1200 rpm for 8min,and inoculated in culture bottles.The medium was changed twice a week.Cells were then passaged according to 1:3 when they showed the 90%of fusioned growth.,The cells in passage 5 were used to induce into differentiation.2 derive and passage of BMSCs Femur and tibia bone from adult SD rat (with 200-250g of body weight) was cut off in aseptic conditions.The bone marrow cavity was then flushed with DMEM/F12 until no marrow in it,and then the cellular solution was centrifuged and inoculated in the culture bottles,two days later,the un-adherent cells were abandoned.Culture medium was changed twice a week.Cells wrer passaged according to 1:3 when they showed the 90%of fusioned growth.The cells in passage 5 were used to induce into differentiation.3.Stem cell characteristics detection Immunocytochemical stain was carried out by anti-CD44 to detect the primary cells of BMSCs and ADSCs.flow cytometry was use to Determine whether BMSCs and ADSCs have Cell surface markers included CD29,CD34,CD44,CD90.Results Most of primary BMSCs adherented to the bottom of the culture bottles 2 days later.The inoculated cells fusioned 7 days later in the primary.The incubation period of subcultured cells,however,showed the shorter significantly,only for 5 days to reach 90%fusion.Cells in passage 5 were homogeneousin the morphology,The primary of ADSCs adherented to the bottom of culture bottles 1 days later.The inoculated cells fusioned 9 days later in the primary.The incubation period of subcultured cells also showed the shorter significantly,only for 5 days to reach 90% fusion.ADSCs in passage 5 were more homogeneous than BMSCs. Immunocytochemistry and Flow Cytometry confirmed that both of cells possessed the characteristics of the stem cells.Discussion Both ADSCs and BMSCs in passage 5 were homogeneous,and showed the stem Cells characteristics,which might be induced further.Partâ…¡BMSCs and ADSCs induced into neuron-like cellsObjective To compare the ability of expressing positive neural markers between BMSCs and ADSCs when induced by both of epidermal growth factor(EGF) and basic fibroblast growth factor(bFGF) in vitro. Methods The cells were induced by EGF(10ng/ml) and bFGF(20ng/ml) at passage 5,which were added at 3 days after inducing culture.Medium was changes at 7 day following the culture.The cells were subcultured when they reached 90%of fusion. Some of neural markers such as Nestin,β-tubulin and GFAP were detected by immunocytochemistry and Western blot at 6h,12h,24h,72h,1w and 2w respectively after inducing.Morphological characteristics were observed by scanning electron microscopy(SEM) when the cells were induced 1 week later.Statistical analysis Immunocytochemistry photographs were obtained by the CCD imaging system.Counting method of positive cells by fluorescence staining as bellows.The cells with each kind of fluorescence stain were selected three samples. Every sample was then selected 10 non-overlapping field of vision.The positive cells from 100 cells in every vision were checked.Data was described by "mean±standard deviation(M±SD)",and analysised by Spss(Version 13.0).The respective data of induced ADSCs and BMSCs at same time point that expressed same fluorescence stain was analysed by the way of Independentsamples t-test.The change of fluorescence expressing accompanied by induced time was described follow One-Way ANOWA,with P＜0.05 for significant difference.Results Before differentiation,the scattered ADSCs and BMSCs expressed positive Nestin positive(Nestin+) staining.Following differentiation,much more of ADSCs differentiated into neuron-like cells compared with BMSCs upon the morphologies. The differentiated processes of neural-like cells from BMSCs showed the longer than those from ADSCs.At 6h after treatment with bFGF+EGF,some cells began to display morphological changes.Cytoplasm in BMSCs and ADSCs retracted towards the nucleus,forming a contracted cellular body with multipolar process-like extensions peripherally.At this time point,the Nestin+ ADSCs and BMSCs were 75.2±2.8 and 55.2±2.8,respectively.Thereafter,the Nestin+ percentage of both ADSCs and BMSCs began to decrease gradually.There were no detectable Nestin+ ADSCs and BMSCs at 72 h and 1w after inducement(ai),respectively.Both differentiated.ADSCs(dADSCs) and differentiated BMSCs(dBMSCs) began to express positiveβ-tubulinâ…¢(β-tubulinâ…¢+) at 24h ai.contacted with each other to form the network at 72 h ai,.The cells retained the stable ratio at 1w until 2 w ai. Positivity forβ-tubulinâ…¢+ was found in 62.7±2.2,87.0±2.5 and 83.2±5.1 at 72 h, 1w and 2w,respectively.Interesting,no matter BMSCs or ADSCs,very few of cells in which were induced into GFAP-positive(GFAP+) cells at all time points.As we could see in the results of the statistica analysis,Nestin-positive(F= 1877.764,P=0.000) andβ-tubulinâ…¢-positive(F=882.435,P=0.000) cells from each of the induce period of ADSCs were statistically significant(P＜0.05). Nestin-positive(F=2158.746,P=0.000) andβ-tubulinâ…¢positive(F=1800.764,P =0.000) cells from each of the induce period of BMSCs were also statistically significant(P＜0.05),which implied that the induction method used in this experiment could successfully induce ADSCs and BMSCs into the neuron-like cells. The comparison of positive could staining expression between ADSCs and BMSCs at every different time points was also as the significant difference(P＜0.05).In short,there were more Nestin+ andβ-tubulinâ…¢+ in ADSCs than those in BMSCs following induction.Western blot analysis showed the equal results with the immunocytochemical findings as described above.Discussion In this study,we have demonstrated that ADSCs and BMSCs could be readily induced into neural-like cells by undergoing morphological and phenotypic changes,following the inducement by using cytokines.The low levels of Nestin and GFAP expression in undifferentiated ADSCs and BMSCs as well as the high percentage of cells undergoing neural differentiation in dADSCs and dBMSCs suggested that ADSCs and BMSCs might retain a native potential for neural differentiation.Interesting,few of MSCs were differentiated into GFAP-positive cells (GFAP+) by using bFGF and EGF.which indicated that the MSCs passessed the plastic potentials,and the microenvironment might play an important role in their differentiation.ADSCs and BMSCs in the same induced conditions appeared the different results significantly,which suggested that the ADSCs differentiated much more efficiently into Nestin+ andβ-tubulinâ…¢+ cells than BMSCs did.Compared with BMSCs,ADSCs had a stronger capacity to differentiation into the neuro-like cells with positive Nestin.In conclusions,this study showed that the dADSCs underwent more efficient neural differentiation,and expressed more obvious neural markers than the dBMSCs. ADSCs,as the alternative sources of BMSCs,may be considered as a new kind of seed cells for CNS repairing following the injury. |
