Coordination Of RA And Wnt Signaling In The Proliferation And Differentiation Of Neural Stem Cells In Vitro

Neural stem cells are undifferentiated cells that have the capacity for self-renewal, self-proliferation and the potential to give rise to all the main cell types of the central nervous system(CNS) including neural progenitor cells(NPCs) and glial progenitor cells. In vitro culturing of neural stem/progenitor cells, epidermal growth factor(EGF) and basic fibroblastic growth factor(bFGF) have proliferative effects on NPCs, and promote cultured neural progenitors to give rise to new clones of immature cells that proliferate in cell aggregates called neurospheres. The cells within these neurospheres will differentiate to neurons, astrocytes, and oligodendrocytes upon the growth factors removal. Therefore, current stem cells research focus on how to induce the NPCs to differentiate to proper neurocytes with the goal of treating neurodegenerative disease in the future. In neurosphere cultures, multiple cell-intrinsic and cell-extrinsic factors regulate NPCs proliferation, migration, and neuronal differentiation. NPCs cultures thus provide an excellent model to study the production of diverse CNS cells during distinct phases in neurodevelopmental diseases. The transplantation of NSCs collected from the embryo, fetus and adult body will establish a potential feasible prospect for the traumatic brain injury, and spinal cord injury and neurodegenerative diseases.Retinoic acid(RA) is a vitamin A-derived, small lipophilic molecule that plays a vital role on embryonic development. RA promotes proliferation of embryonic stem cells in vitro in mice, and also induces stem cells differentiate to the glial cell precursor cells and oligodendrocyto-GABAergic neurons in the development of the central nervous system. Wnt signaling pathway is a well-conserved signaling pathway in organogenesis. Wnt signal promotes the proliferation and differentiation processes both during embryonic neurogenesis and in adult nervous system, and has a crucial effect on neurodevelopmental and neurodegenerative diseases. In culture of embryonic stem cells in vitro, Wnt ligand accelerates stem cells differentiate to oligodendrocyto-GABAergic neurons besides the effects of the self-renewal, self-proliferation and cell aggregated neurospheres. However, there are still no literatures about the combined effect of RA and Wnt ligands on neurogenesis and differentiation in NSCs, therefore we explored the joint role of RA and Wnt3 a on the cultured embryonic NSCs in vitro in our experiment.PartⅠ the effects of RA signaling and Wnt signaling on theproliferation and differentiation of NSCsMethods:Neural stem cells(NSCs) were primarily cultured from embryonic midbrain in vitro. Under the conditions of RA(5μM) and Wnt3a(20ng/ml) stimulation, the cultured neural stem cells were induced to differentiate in vitro. BrdU incorporation assay was processed to test the proliferative capacity of neural stem cells. Immunocytochemistry and Western Blot of Dlx2(a marker for the common progenitor of oligodendrocyte and GABAergic neurons), NG2(a marker of oligodendrocyte precusors), GAD67(a marker of GABAergic neurons), TUJ1(a neuronal marker), and GFAP(a marker of astrocyte) were performed to examine the differentiation of oligodendrocytes, GABAergic neurons, neurons and astrocytes.Result:Both RA and Wnt3 a stimulation significantly increased the percentages of BrdUlabeling neural stem cells(P<0.05), GAD67- and TUJ1-positive neurons(P<0.05). However, RA stimulation inhibited the differentiation of Dlx2 and NG2-positive cells from NSCs while Wnt3 a stimulation promoted the differentiation. The combinative stimulation of RA and Wnt3 a showed no difference with the control group in proliferation and differentiation. And the percentage of GFAP-positive cells showed the opposite results in comparison with the differentiatiation of oligodentroocyto-GABAnegic neurons. Conclusion:Both RA and Wnt3 a individual stimulation promoted the proliferation and differentiation of oliogodendrocyto-GABAnergic neurons in NSCs, and showed suppressive effect in astrocytes from neural stem cells to some extent. However, the combination of RA and Wnt3 a showed the opposite outcomes in proliferation and differentiation of neural stem cells in comparision with individual stimulation of RA or Wnt3 a. The results indicated a potential mutual antagonism between intracellular RA and Wnt signaling.PartⅡ The mechanisms for RA /Wnt interaction, and the rescueeffect of β-catenin over-expression on RA/Wnt coadministrationMethods:As described previously, NSCs were primarily cultured from embryonic midbrain in vitro. Western Blot of β-catenin, Cyclin D1 and Axin2, three vital Wnt/β-catenin signaling proteins, and Raldh3, cyp 26B1, two RA signaling key enzymes was performed.Under the conditions of β-catenin over-expression, the proliferation and differentiation of cultured NSCs were detected by BrdU labeling and immunocytochemistry of Dlx2(a marker for the common progenitor of oligodendrocyte and GABAergic neurons), NG2(a marker of oligodendrocyte precusors), GAD67(a marker of GABAergic neurons), TUJ1(a neuronal marker), and GFAP(a marker of astrocyte)Result:Western Blot showed that RA significantly increased the expression of β-catenin, Cyclin D1, and Axin2, while Wnt3 a exhibited inhibitory effects on RA signaling by up regulation of cyp 26B1 and down regulation of Raldh3. RA/Wnt3 a coadministration inhibited the espreesion ofβ-catenin. β-catenin over-expreesion showed more BrdU-, Dlx2-, NG2-, GAD67-, and TUJ1-positive cells(P<0.05) and little GFAP-positive cells(P<0.05) than the combination of RA and Wnt3 a. Conclusion:The mutual antagonism between RA signaling and Wnt signaling was possibly mediated by β-catenin as it can rescue the effects if RA/Wnt3 a coadministration on the proliferation and differentiation of NSCs. Considering that Wnt3 a down-regulates the expression of Raldh3 gene and up-regulates the expression of cyp 26B1 gene, the effects of Raldh3 and cyp 26B1 on the mutual antagonism between RA and Wnt signaling could not be excluded.