Optogenetic control of neural differentiation in Opto‐mGluR6 engineered retinal pigment epithelial cell line and mesenchymal stem cells

Abstract

In retinal degenerative disorders, when neural retinal cells are damaged, cell transplantation is one of the most promising therapeutic approaches. Optogenetic technology plays an essential role in the neural differentiation of stem cells via membrane depolarization. This study explored the efficacy of blue light stimulation in neuroretinal differentiation of Opto‐mGluR6‐engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs were selected for optogenetic study due to their capability to differentiate into retinal‐specific neurons. BMSCs were isolated and phenotypically characterized by the expression of mesenchymal stem cell‐specific markers, CD44 (99%) and CD105 (98.8%). mRPE culture identity was confirmed by expression of RPE‐specific marker, RPE65, and epithelial cell marker, ZO‐1. mRPE cells and BMSCs were transduced with AAV‐MCS‐IRES‐EGFP‐Opto‐mGluR6 viral vector and stimulated for 5 days with blue light (470 nm). RNA and protein expression of Opto‐mGluR6 were verified. Optogenetic stimulation‐induced elevated intracellular Ca2+ levels in mRPE‐ and BMS‐treated cells. Significant increase in cell growth rate and G1/S phase transition were detected in mRPE‐ and BMSCs‐treated cultures. Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rxrg, Nr2f2, Ascl1, Hes5, and Sox8 were overexpressed in treated BMSCs and Barlh2, Rorb, and Sox8 were overexpressed in treated mRPE cells. Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal‐specific neuron markers, in mRPE and BMS cell cultures were demonstrated. Differentiation of ganglion, amacrine, photoreceptor cells, and bipolar and Muller precursors were determined in BMSCs‐treated culture and were compared with mRPE. mRPE cells represented more abundant terminal Muller glial differentiation compared with BMSCs. Our results also demonstrated that optical stimulation increased the intracellular Ca2+ level and proliferation and differentiation of Opto‐mGluR6‐engineered BMSCs. It seems that optogenetic stimulation of mRPE‐ and BMSCs‐engineered cells would be a potential therapeutic approach for retinal degenerative disorders.

microRNA-96 targets the INS/AKT/GLUT4 signaling axis: Association with and effect on diabetic retinopathy

“…mRPE cells were transfected with the pAAV-2-MSC-eGFP-mmu-miR-96 or pAAV-2-MSC-eGFP vector. At 24, 48, and 72 h post transfection, the cells were trypsinized and subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to a previously reported protocol [ 46 ]. The results were acquired from at least three independent experiments with at least three technical replicates per experiment.…”

Emerging optogenetics technologies in biomedical applications

“…56,57 These vectors include adenoviral vectors (AAV), retroviral vectors, lentiviral vectors, and others. [58][59][60][61][62] Of these, AAV has proven to be a safe and effective vector for transduction. [63][64][65] When used as a vector, recombinant AAV (rAAV) deletes all AAV protein sequences and adds the target protein.…”

“…Currently, viral vectors are the most widely used method for protein delivery 56,57 . These vectors include adenoviral vectors (AAV), retroviral vectors, lentiviral vectors, and others 58–62 . Of these, AAV has proven to be a safe and effective vector for transduction 63–65 .…”

Editorial – Optogenetic Therapy for Retinal Degenerative Diseases

“…Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal-specific neuron markers in mRPE and BMS cell cultures were also demonstrated. [ 11 ]…”

“…This study revealed that optical stimulation increased the intracellular Ca level and proliferation and differentiation of Opto-mGluR6-engineered BMSCs. [ 11 ]…”