Optogenetic Stimulation Promotes Proliferation and Neuroretinal differentiation of Retinal Pigment Epithelial Cells and Mesenchymal Stem Cells
Hoda Shams Najafabadi1 , Zahra-Soheila Soheili1 *, Mohammad Ismail Zibaii2 , Shahram Samiee3 , Hamid Ahmadieh4 , Mehdi Sadeghi1 , Ehsan Ranaei Pirmardan5 , Sepideh Taghizadeh1
- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
- Laser & Plasma Research Institute, Shahid Beheshti University, Tehran, Iran.
- Blood Transfusion Research Center High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
- Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Molecular Biomarkers Nano-imaging Laboratory, Brigham & Women's Hospital, Department of Radiology, Harvard Medical School, Boston, MA, USA.
Abstract: Neural retinal cell transplantation is one of the most promising therapeutic approaches in treatment of retinal degenerative disorders. In this optogenetic study, retinal pigment epithelial (RPE) cells and bone marrow mesenchymal stem cells (BMSCs) were recruited due to their differentiation ability into retinal specific neurons. This research explored the potential role of blue light stimulation on retinal neural differentiation of Opto-mGluR6 engineered human RPE and BMSCs.
Methods: hRPE and BMSCs were transduced by AAV-MCS-IRES-EGFP-OptomGluR6 viral vector. Forty-eight hours after transduction, sample cultures were immunostained by melanopsin marker to provide the melanopsin expression. Transduced cultures were also treated with 2 μM all trans retinal and were stimulated for 5 days with blue light (470 nm) in a humidified CO2 incubator. Whole cell patch clamp, calcium imaging and cell cycle analysis were performed. Total RNA was isolated and RT-qPCR for retinal specific neuron genes were performed. Imunnocytochemistry (ICC) assay for Rhodopsin, PKCa, Thy1, CRX, CD73, OPN1, recoverin and CRABP as retinal specific neuron markers were performed.
Results: hRPE and BMS cells were successfully transduced. Treated cells expressed Opto-mGluR6 protein in cells’ membrane. Effective depolarization was observed in Opto-mGluR6 transduced mRPE cells (68 pA) and BMSCs (42 pA) culture. Optical stimulation induced influx of Ca2+ into the cytoplasm of the treated cells. Optical stimulation could promote opto-mGluR6 transduced BMSCs transition from G1 phase of the cell cycle to S phase; however it did not have a significant effect on Opto-mGluR6 transduced mRPE cells. RT-qPCR revealed that Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rora, Rxrg, Nr2f2, Ascl1, Hes5 and Sox8 expressed 4.7, 8.5, 10.9, 23.8, 1.6, 3.1, 5.2, 0.5, 6, 2.2, 3.4, 5.2 and 5.2 fold in BMS cells culture and 0.1, 0.6, 0.4, 2.2, 0.4, 0.5, 2.3, 1.5, 0.7, 0.6, 1.5, 1.2 and 45.3 fold in hRPE cells culture. ICC assay confirmed expression of retinal specific neuron markers in BMSCs and hRPE cells cultures.
Conclusion: Optogenetic stimulation induced marked differentiation of ganglion, amacrine, photoreceptor, bipolar precursors and Muller precursor cells in BMSCs treated culture. mRPE cells represented dominant terminal Müller glial differentiation.
