Optogenetic Stimulation Inhibits Self-renewal Of Mouse Neural Stem Cells And Embryonic Stem Cells

Background:The death of photoreceptor cells caused by retinal degenerative diseases often resultsin a complete loss of retinal responses to light. Despite advancements made in ourunderstanding of ocular biology, therapeutic options for many debilitating retinal diseasesremain limited. Stem cell-based therapies are a potential avenue for treatment of retinaldisease. Over the past decade, significant advancements have been made using differenttypes of stem cells with varying capacities to differentiate into these target retinal cell types.However, current methods to examine and regulate the functional integration and plasticityof stem cell-derived neurons in retina are cumbersome and technically challenging. Here,we engineered stem cells and their derivatives to express the light-gated channelrhodopsin-2(ChR2) protein to overcome these deficiencies. Optogenetic targeting of stem cell-derivedneurons with ChR2linked to the GFP fluorophore allowed reliable cell tracking as well aslight-induced spiking at physiological frequencies. We want to demonstrate stem cell-derived neurons become functionally integrated in retina. Optically induced excitatorypostsynaptic currents could be elicited in ChR2+neurons, revealed complex, bi-directionalsynaptic interactions between grafted cells and host neurons and extensive synapticconnectivity within the graft. However, it is essential to clarify the effect of opticalstimulation on proliferation and self-renewal of neural stem cells and embryonic stem cellsbefore we clarifying the functional integration.Understanding the detailed mechanisms that govern stem cells fate between self-renewal and differentiation has been an urgent task for both basic research and clinicalapplications. Based on our current understanding, a set of core transcription factors (TFs),especially Oct4, Sox2and Nanog, form an auto-regulatory network to maintain thepluripotent state of mESCs and p21and Sox2regulate the stem state of NSCs. Besides theinner regulators in governing stem cell state, many extra-cellular factors have been identified that trigger the transition of stem cells from self-renewal to differentiation via themodulation of the core TFs. However, the signaling pathways involved in this processrequire further investigation. A previous study demonstrated that ESCs possess outward Kvcurrents, and when the K+channels were blocked, proliferation of mESCs was significantlyinhibited. Furthermore, membrane potential oscillation is tightly correlated with cellproliferation-related events, such as mitosis, DNA synthesis and overall cell cycleprogression. In particular, depolarization could modulate the proliferation process individing cells. The development and maturation of neural cells are regulated by membraneion channel activity. Changes of ion channel activity have been detected in proliferatingglial progenitors in vivo and in vitro.ChR2has been found to enhance differentiation of mESCs upon the treatment withretinoic acid (RA), but the underlying mechanism is not clear. Stem cell-based optogeneticstechnology represents a significant advancement in regulating the physiological state ofstem cells. Thus, we want to examine whether the change in ion flux induced by blue lightcould trigger the transition of mESC fate from self-renewal to differentiation and inhibit theproliferation and cell cycle progress of NSCs.Methods:1. ChR2-GFP-V6.5mESCs and ChR2-GFP-C17.2NSCs established by transducingwith a lentiviral ChR2-GFP construct under the control of the UbC promoter and ChR2-GFP positive cells were selected by FACS.2. By employing Real-time PCR, imunostaining and AP staining, the expression ofstem cell marker were detected.3. The cell cycle and apoptosis rate of cells after blue light stimulation was verified byflow cytometry, and the cell number and cell viability was evaluated simultaneously withtypan blue exclusion assay.4. Blue light induced inwards current was assayed with patch-clamp.5. By employing Western-blot, the ERK and p21, p27expression level was analyzed.Results:1. After transducing with lentivirous and sorted by FACS, single cell derived ChR2-GFP-V6.5mESCs and ChR2-GFP-C17.2NSCs expressed the ChR2-GFP protein inmembrane and the470nm blue light could generate inward current on both cell lines. 2. The ChR2-GFP-V6.5mESCs and ChR2-GFP-C17.2NSCs expressed stem cellmarkers and the cell proliferate was not affected.3. Optical stimulation inhibited proliferation rate and disrupted cell cycle progressionof ChR2-GFP-V6.5mESCs and ChR2-GFP-C17.2NSCs. Light stimulation led todecreased proportion of cells in the S phase. Optical stimulation inhibited the self-renewal,AP positive clone forming and expression of core TFs Oct4/Sox2/Nanog/Klf4/Esrrb ofChR2-GFP-V6.5mESCs. Optical stimulation also reduced the proportion of Ki67positivecells in ChR2-GFP-C17.2NSCs.4. Optical stimulation does not affect the survival of ChR2-GFP-V6.5mESCs andChR2-GFP-C17.2NSCs and not induce apoptosis in both cell lines.5. Blue light stimulation resulted in an increased transcription level of variousdifferentiation markers (including Fox2, Gata6, Branchyury, Gata2, Hand1, Fgf5, Cdx2andNestin), especially markers of the trophectoderm and ectoderm fates (Cdx2, Fgf5andNestin) and the ERK activity was significantly up-regulated after blue light stimulation ofChR2-GFP-V6.5mESCs.6. Blue light evoked membrane depolarization of ChR2-GFP-C17.2NSCs and bluelight significantly increased both p21and p27expression level.Conclusion:In conclusion, by utilizing optogenetic techniques, we have demonstrated that cellmembrane depolarization plays an important role in the regulation of mESC and NSCsproliferation, self-renewal and initiation of differentiation. Our results demonstrate thatintracellular signal transduction pathways associated with the activation of ERK areinvolved in the transition of mESCs from self-renewal to differentiation that was induced bymembrane depolarization and p21/p27upregulation involved in the inhibition of NSCs byblue light stimulation.