Differentiation Of Mesenchymal Stem Cells Derived From Bone Marrow Of Rat And Human Placenta Into Cholinergic Neuron-like Cells In Vitro

Objectives:To culture and amplify mesenchymal stem cells from bone marrow of rat and human placenta in vitro;To explore the differentiation of rat marrow mesenchymal stem cells(MSCs) into cholinergic neuron-like cells by inducing reagents.Methods and materials:1.Disassociation and cultivation of mesenchymal stem cells1.1 Nucleated cells were separated from the whole bone marrow and then were subcultured into culture flask contained 10%FBs and DMEM/F12,they were cultured in incubator filled with five percent of carbon dioxide and in condition of saturation humidity.Culture fluid were replaced every 72 hours.1.2 Disassociation and cultivation of human placenta stem cells:pieces of placenta digested by collagenaseⅣand trypsinase scatter into monoplast cell suspension,mononuclearcells centrifugated and filtered by sieve were collected, then replaced in medium contained DMEM/F12.After that,they were cultured in incubator filled with five percent of carbon dioxide and in condition of saturation humidity.Culture fluid were replaced every 72 hours.Lamellate fibroblast-like cells were observed to adhere to bottom of culture flask after 3-4 weeks.2.Induction Mesenchymal stem cells to differentiate into into cholinergic neuron-likecells2.1 Induction Mesenchymal stem cells derived form rat bone marrow to differentiate into into cholinergic neuron-like cells:with 2-4 passage of MSCs by inducing reagents.The protocol was as follows:groupⅠ,MSCs were incubated with 10%FBS for 2 days and then medium was replaced by DMEM/F12 complemented with 10ug/L of bFGF,0.1mmol/L of 2-ME,1mmol/L of RA, 10ug/L of NGF for 8 days.groupⅡ,on base ofⅠ,And then 10ug/L of EGF and 1250u/mL of heparin were added into the culture sysytem for culture for 8 days. Except of DMEM/F12,Nothing treated for control group.2.2 Induction Mesenchymal stem cells form human placenta to differentiate into into cholinergic neuron-like cells:with 4 passage of hPMSCs by inducing reagents. The protocol was as follows:groupⅠ,MSCs were incubated with 10%FBS for 2 days and then medium was replaced by DMEM/F12 complemented with 10ug/L of bFGF,0.1mmol/L of 2-ME,1mmol/L of RA,10ug/L of NGF for 19 days. groupⅡ,on base ofⅠ,And then 10ug/L of EGF and 1250u/mL of heparin were added into the culture sysytem for culture for 19 days.Except of DMEM/F12 for culture for 21 days,Nothing treated for control group.3.Morphologic changes of mesenchymal stem cells(MSCs) induced for different days were observed under inverted microscope.All of the data was tested with SPSS13.0 software.4.Usedβ- actin as intra- control,RT-PCR and gel electrophoresis were used to detected gene expression of nestin,nurr-1,chat for induced MSCs5.Immunological fluorescent staining was used to Nestin,CHAT,NeuN,Ache positive cells6.Flow cytometry(fcm) was not only used to identify the expression of protein related to cell surface mark of hPMSCs and the number of induced hPMSCs postived for Nestin,CHAT,NeuN,Ache in different time but also used to calculate the rate of induced cell differentiation into cholinergic neuron-like cells7.By means of ELISA,the contains of production,ie acetylcholine in supemate was detected for induced hPMSCs in different time.Results:1.The Morphologic changes of induced MSCs:for 1 day,the shape of these induced cells gradually grow into round or ellipse from long fusiform shape,stretched out ecptoma;Parts of them connected with each other by ecptoma,others conjunct to periplasm to shape neuro-bulb.Moreover,intracytoplasm secretor granules of induced MSCs rise than before.The morphous rate in experiment group is higher than control group,The difference is obviously in statistics in this groups.The respective morphous rate in 1,3,5,10 day in control group is less than 1%. Induced cells higher express nestin,nurr1,chat than control,the expression strengthen of mRNA was difference obviously in statistics(p＜0.01).Indirect immunological fluorescent staining show:It is a higher transformation efficiency (positive for nestin,chat,NeuN,AchE;50-80%) in experiment group than control (about 1%cells positive for chat)2.Morphologic change of hPMSCs is similar to BMSCs.MSCs couldn't express CD34(4.8%),CD45(0.9%),CD106(2.3%),HLA-DR(1.0%)and were strongly positive for Adhesion molecules CD29(99.8%),CD44(99.7%).Induced for 14 days,RT-PCR showed that the induced neuron-like cells expressed special neurocyte genes of nestin,nurr-1,chat.Indirect immunological fluorescent staining showed:positive cells with Nestin,CHAT,Ache for groupⅠ,Ⅱreached 29.7%, 85.8%,24.5%;10.4%,76.3%,98.2%respectively.ELISA detected that the contained acetylcholine for 1,2,3 week in supernate fluid was 638.9±7.9 nmol/l,393.6±10.3nmol/l,905.1±10.1nmol/l in groupⅠ,and was 39.2±26.6nmol/l, 82.1±23.2nmol/l,234.9±19.2nmol/l in groupⅡ.While was only 0±4.6 nmol/l, 0±4.7nmol/l,20±9.4nmol/l observed in MSCs cultured in regular medium.(P＜0.01)Conclusions:1.MSCs from marrow and placeta can be cultured and expanded in vitro and differentiate into cholinergic neuron-like cells which can be able to synthesis,release and disintegrate acetylcholine by inducing reagents.GroupⅠshowed a higher transformation efficiency than groupⅡ2.MSCs strongly expressed CD29,CD44,but no CD34,CD45,CD106,HLA-DR. was found.