Improvement of Retinal Injury After Subretinal Transplantation of Neurosphere-Derived Retinal Pigment Epithelium in the Age-RelatedMacular Degeneration Model

Background and Aim: Age-related macular degeneration (AMD) is themain cause of irreversible blindness. Stem cells therapy is a promisingtool in such diseases. The present study aims to improve the retinal injuryfollowing subretinal transplantation of Neurosphere-derived Retinalpigment epithelium cells (NRPELC) for replacement therapies in cases ofretinal degenerative diseases.Methods: Bone marrow stromal cells (BMSCs) were isolated frompigmented hooded male rats. These cells were transformed intoneurospheres and differentiated into pigmented spheres (PS) withsmall molecule induction. Finally, NRPELC were immunostained withRPE65, CRALBP, and OTX2 markers. Consequently, their expressionwas supported by RT-PCR and RT-qPCR. Moreover, the functionalityof NRPELC was evaluated by phagocytosis of fluorescein-labeledphotoreceptor outer segment (POS). Accordingly, 5 μL of BrdU-labeledNRPELC cells suspension transplanted into subretinal space throughscleral approach. The morphological and electroretinography (ERG)changes in RPE layer and neurosensory retina investigated in time cursefrom seven days to 90 days post-injection.Results: In vitro results showed that the cultured BMSCs differentiatedinto neurospheres. The neurosphere at the seven days was to express theneural markers (Sox2, Oct4, Nanog, and Nestin, P < 0.05). The pigmentedspheres obtained from neurosphere expressed the RPE markers (RPE65(100%±1), CRALBP (100%±2), and OTX2 (92%±2), P < 0.05) quite well.In addition, RT-qPCR supported the gene expression after 14 days invitro. Cultivation of PS showed expression of markers from 7 days to 90days after induction. Furthermore, NRPELC internalized the POS afterseven days in culture. In vivo results showed that BrdU-labeled NRPELCsurvived, migrated, and integrated 90 days post-transplantation into RPElayer. Consequently, Changes in a-wave and b-wave of ERG confirmedthe improvement of neurosensory retina function and morphology aftercell therapy.Conclusion: Mesenchymal stem cells therapies are rising trends inthe treatment of AMD, and future pre-clinical and clinical studies arerequired for standardization of the therapy and obtaining the long-timedata about the efficacy and safety.

Keywords:

Bone marrow stem cells , Neurosphere , Retinal pigmented epithelium , Age-related macular degeneration , Sodium iodate