Role Of Wnt/β-catenin Pathway In The Differentiation Of Rat Embryonic Stem Cells Into Neurons

Backgraund & Objective The discovery and successful culture of neuralstem cells(NSCs) in vitro provided a newly idea and method for reseachingthe development of nervous system and treatment of central nervous systemdisease and injury. NSCs were defined as undifferentiated neural cells thatwere endowed with a high potential for proliferation and the capacity forself-renewal, they also could differentiate into the main phenotypes of thenervous system, neuronal, astrocytic and oligodendrocytic, and retained theirmultipotency. At present, although many studies about NSCs have been done,but there still have a great deal of works need to be done for its application inclinic. Recent studies had shown that transplantation of NSCs into the injuredspinal cord could promote functional recovery in adult animal models.However, the majority of transplanted NSCs differentiated into astroglialphenotypes, which resulted in the formation of glial scar in the injuried spinalcord and prevented the recovery of neurofunction. Therefore, it became ahotspot to explore an effective method to induce NSCs directeddifferentiation into neurons in SCI reseaching. It had been confirmed thatautogene and foreign signal play synergistic effect in the proliferation and differentiation of NSCs. Wnt/β-catenin signaling pathway played importantroles in the neural development, and played synergistic effect with autogene(such as bHLH family and Notch signal molecule) and foreign signal (such asTGF-βsuperfamily member—bone morphogenetic protein), neurotrophicfactor, cell factor and chemical materials for instance all-trans-retinoicacid(ATRA). Recent reports had shown that ATRA could promote induceNSCs directed differentiation into neurons, and the crosstalk ofWnt/β-catenin signaling pathway and other signal molecules, but therelationships between these factors had not been fully elucidated. The aims ofthis study were: to investigate the roles of Wnt/β-catenin'signaling pathwayand other correlated signal molecules in the differentiation of ATRA-treatedrat embryonic stem cells into neurons.Methods 1. Rat embryonic stem cells was cultured with 1×10~4/ml, 1×10~5/ml,1×10~6/ml and 1×10~7/ml, respectively. The growth character of cells atdifferent seeding density was observed at different time point by MTT assay.The NSCs and its differentiated cells were detected by Immunocytochemistryto determine the optimal seeding density and passaging way.2. Rat embryonic stem cells were cultured with different ATRAconcentration, the reproductive activity of each groups was analyzed by BrdUstaining, the capacity of NSCs directed differentiation into neurons wasexplored by immunofluorescence double-labelling and flow cytometry todetermine the opimal concentration of ATRA.3. The expression of 13-catenin, CyclinD1, Axin, Wnt-1, Notchl, BMP2mRNA and protein were investigated by Real-time Flurescent QuantitivePolymerase Chain Reaction and Western-blot, respectively. The expressionofβ-catenin, CyclinD1, Wnt-1, Notchl, BMP2 at different time point were investigated by immunocytochemistry to explore the role of Wnt/β-cateninsignaling pathway and the relationships of Wnt/β-catenin signaling pathwayand other correlated signal molecules at the NSCs differentiation processingin ATRA treatment.Results 1. NSCs were isolated from cortex of rat embryos and were purifiedand characterized after bening cultured in vitro. After continuouslypassaging, NSCs retained strongly reproductive activity and multipotency.The optimal seeding density was 1×10~6/ml.2. NSCs could generate in DMED/F12 complete medium containing ATRA,but the capacity of proliferation was inferiorer than others cultured inDMED/F12 complete medium without ATRA. The differentiation ratio ofneurons was optimal in 1.01μmol/L ATRA treatment, but the majority of NSCscultured in DMED/F12 complete medium withoutATRA differentiated intoastroglial phenotypes.3. Wnt-1, a Writ ligend, obviously increased at earlier stage in ATRAtreatment Group, and Wnt-1 acted on the cell surface receptor Frizzled,activated Dishevelled(Dvl) binds, inhibiting the phosphorylation ofβ-cateninby repressing the formation of "destruction complex" with Axin, APC andGSK3, blockingβ-catenin degradation. All these causedβ-catenin toaccumulate and translocate to the nucleus, interacting with T cell specificfactor(Tcf)/lymphoid enhancer binding factor l(Lef-1) transcription factorand inducing the transcription of target genes such as cyclinD 1, inhibiting theexpression of Notchl and BMP2 at the same time. The above resultsilluminated that the promoting effect of ATRA on the differentiation of NSCsinto neurons may be related to the activation of Wnt/β-catenin signalingpathway and the suppression of Notchl and BMP2 signal molecules. Conclusions 1. Rat embryonic NSCs could purified and characterized afterbening cultured in vitro, and the optimal seeding density was 1×10~6/ml.2. ATRA treatment could promote the differentiaon and proliferation of NSCs,but the capacity of proliferation were inferiorer than others cultured inDMED/F12 complete medium without ATRA. ATRA treatment remarkablypromoted the differentiation of NSCs into neurons and the optimalconcentration was 1.0μmol/L.3. The promoting effect of ATRA on the differentiation of NSCs into neuronsmay be related to the activation of Wnt/β-catenin signaling pathway and thesuppression of Notchl and BMP2 signal molecules.