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Purpose: Proliferative retinal angiogenesis may severely impair the retina. Previous studies have indicated that matrix metalloproteinase (MMP)-2 and MMP-9 play important roles in the process of retinal angiogenesis. In this study, we suppressed MMP-2 and MMP-9 expression with RNA interference (RNAi) and then observed the inhibitory effects on the invasion and migration of human retinal microvascular endothelial cells (HRMECs). Methods: Small interfering RNAs against MMP-2 mRNA and MMP-9 mRNA were synthesized. After transfection, the MMP-2 and MMP-9 expression in HRMECs was examined by real-time polymerase chain reaction and Western blot analysis. Cell migration and invasion were measured with a migration assay and a scratch wound assay, respectively. Results: RNAi against MMP-2 and MMP-9 successfully inhibited the mRNA and protein expression of MMP-2 and MMP-9 in HRMECs. MMP-2 and MMP-9 knockdown could inhibit the invasion and migration of HRMECs. Conclusions: These findings suggest that the RNAi approach towards MMP-2 and MMP-9 may be a potentially effective therapeutic method for the treatment of proliferative retinal angiogenesis.

Retinal organoids (ROs) derived from human inducible pluripotent stem cells (hiPSCs) exhibit considerable therapeutic potential. However, current quality control of ROs during in vitro differentiation is largely limited to the detection of molecular markers, often by immunostaining, polymerase chain reaction (PCR) assays and sequencing, often without proper functional assessments. As such, in the current study, we systemically characterized the physiological maturation of photoreceptor-like cells in hiPSC-derived ROs. By performing patch-clamp recordings from photoreceptor-like cells in ROs at distinct differentiation stages (ie, Differentiation Day [D]90, D150, and D200), we determined the electrophysiological properties of the plasma membrane and several characteristic ion channels closely associated with the physiological functions of the photoreceptors. Ionic hallmarks, such as hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and cyclic nucleotide-gated (CNG) channels, matured progressively during differentiation. After D200 in culture, these characteristic currents closely resembled those in macaque or human native photoreceptors. Furthermore, we demonstrated that the hyperpolarization-activated inward current/depolarization-activated outward current ratio (I−120/I+40), termed as the inward-outward current (IOC) ratio hereon, accurately represented the maturity of photoreceptors and could serve as a sensitive indicator of pathological state. Thus, this study provides a comprehensive dataset describing the electrophysiological maturation of photoreceptor-like cells in hiPSC-derived ROs for precise and sensitive quality control during RO differentiation.

Retinitis pigmentosa (RP) is a form of inherited retinal degenerative disease that ultimately involves the macula, which is present in primates but not in the rodents. Therefore, creating nonhuman primate (NHP) models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future. Here we applied adeno-associated virus (AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque (Macaca mulatta) to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP. Through a series of studies, we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression. More importantly, the mutant macaque retinae displayed clinical RP phenotypes, including photoreceptor degeneration, retinal thinning, abnormal rod subcellular structures, and reduced photoresponse. Therefore, we suggest somatic editing of the RHO gene is able to phenocopy RP, and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.