The Research Of VEGF On Neural Differentiation Of Human Embryonic Stem Cells In Vitro

In recent years,the differentiation from human embryonic stem cells (hESCs) toneuronal cells provides an ideal vector for cell transplantation therapy of neurodegenerativediseases.The generation of neural stem cells (NSCs) is a key step in the induction fromembryonic stem cells to specific neurons or glial cells.There are broadly three kinds ofmethods used in hESCs differentiated into NSCs:First is the method with embryonic body(EB) culture period.It is the most widely used one.However,this method is too long andrequires a large amount of hESCs.Second one is co-culture with the special-induced stromalcells.But it has not been explained whether the factors secreted from stromal cells have othereffect on the neural cells.So this hinders the application to some extent.Third is the methodof using specific factors to direct differentiate embryonic stem cells.Studies found that whena variety of signal transduction in cells and cell interaction were removal,ESCsspontaneously differentiated into the neuronal cells.The inductions of NSCs from hESCsusing this method have been reported,but there does not form a unified and effective method.Researches of neurogenesis in the vertebrate have shown that vascular endothelial cellspromote adult neural stem cell proliferation through vascular niche In vitro,endothelial cellscan induce rat neural stem cells into neurons.Vascular endothelial growth factor (VEGF)secreted by endothelial cells,play an essential role in these process in vitro and in vivo.In the hippocampus,VEGF promot neural stem cells growth and survival through VEGFR2.Studiesfound that stern cells could produce signals to regulate the proliferation and differentiation ofthemselves.Preliminary studies have revealed Notch signal transduction pathway has animportant effect in the proliferation of multipotent neural stem cells.In addition,mitogenactivated protein kinase (MAPK) signal transduction pathway is widely expressed in thecentral nervous system.A variety of extracellular signals through this pathway may affect thesynaptic transmission and neurons survival.In this study,we use the EB method and thedirect differentiation to induce human embryonic stem cells into neural stem cells in order toexplore the suitable induction method.Then we will explore whether VEGF play a role in theneural differentiation of human embryonic stem cells in vitro and the effect of VEGF isrelated to Notch and p38 MAPK signal transduction pathway or not.PartⅠDifferentiation of Human Embryonic Stem Cells intoNeural Stem Cells in vitroObjective To find an efficient and economical method to differentiate humanembryonic stem cells into neural stem cells in vitroMethods hESCs were matured and free from the feeder layer,digested for thesingle-cell.The cells with 1×10~5 density cultured in dish and induced by Noggin and ITSFndirectly without EB culturing.Results By the direct differentiation,Nestin-positive NSCs were generated after 14～16days.The neural stem cells could be differentiated into neurons and glial cells.MAP2 andGFAP were detected after the differentiation of neural stem cells.Conclusion hESCs can successfully differentiate into neural stem cells without EBculture period in vitro.This method has the better effect than the traditional one and will beused for the cell transplantation in the research of nervous system diseases. PartⅡEffect of VEGF on Neural Differentiation ofHuman Embryonic Stem Cells in vitroObjective To observe the effect of VEGF on neural differentiation of human embryonicstem cells in vitroMethods After embryonic body formation,hESCs were differentiated into neuronalcells.10ng/mL VEGF or 10ng/mL VEGF + 10ng/mLVEGFR2/Fc were added at every stageof differentiation.The markers of every stage were detected through RT-PCR.Nestin andMAP2 expressing cells were detected via immunofluorescence method.The percentages ofthe imunostaining positive cells were determined by flow cytometer (FCM).Results The markers of different stages were detected by RT-PCR.The expression ofVEGFR2 was detected in human neural stem cells using RT-PCR.The percentage of Nestinand MAP2 positive cells in VEGF treated group were much higher than that in routineinduction group and VEGF+VEGFR2/Fc treated group.There was no difference betweenroutine induction group and VEGF+VEGFR2/Fc treated group.Conclusion hNSCs express VEGFR2.Low dose (10ng/mL) of VEGF,via VEGFR2,stimulates the proliferation of NSCs and the neuronal cells differentiation from humanembryonic stem cells in vitro.PartⅢThe Mechanism of VEGF on Neural Differentiation ofHuman Embryonic Stem in vitroObjective To study the mechanism of VEGF on the neural differentiation of hESCs invitroMethods hESCs were cultured and differentiated into neurons through the embryonic body.Before the VEGF (10ng/mL) was added,γ-secretase inhibitor and p38 MAPK inhibitorwere given at every stage of induction for 1h.The markers of every stage were detected andthe percentages of the imunostaining positive cells were counted throughimmunofluorescence.The expressions of HeslmRNA and p38αmRNA in each group weredetected by RT-PCR.Results The percentage of Nestin in theγ-secretase inhibitors pretreatment group wassimilar with the conventional group.The applications of p38 MAPK inhibitor produced thesimilar number of neural stem cells with the VEGF group;RT-PCR revealed that theexpression of HeslmRNA in the VEGF-treated group was higher thanγ-secretase inhibitorpreconditioning group and the conventional group.After the differentiation of NSCs intoneurons,the neurons generated fromγ-secretase inhibitors group and the VEGF-treated grouphad no significant difference,while the neurons of p38 MAPK inhibitor pretreatment groupand conventional induction group were similar.RT-PCR analysis showed that the expressionof p38αmRNA in the VEGF-treated group was higher than p38 MAPK inhibitorpreconditioning group and the conventional group.Conclusion Through the activation of Notch signal transduction pathway,VEGF play arole in promoting human embryonic stem cell differentiated into neural stem cells.Theinhibitor of Notch signaling can antagonize the effect of VEGF in this process.In thedifferentiation of human neural stem cells into neurons,VEGF can promote neural stem cellsto differentiate into more neurons via p38MAPK way.p38 MAPK inhibitors can block therole of VEGF in this process.