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Abstract Age-related macular degeneration (AMD) is associated with the dysfunction and degeneration of retinal pigment epithelium (RPE) cells. Here, we examined how the formation and expansions of cell clusters are regulated by the differentiation of the RPE cells. In this study, ARPE-19 cells were cultivated in standard or differentiation media, i.e., without or with nicotinamide, to evaluate the spreading of cell clusters specified with differentiated cell phenotypes. Mitochondria membrane potential (MMP) and the distribution of the RPE cell clusters was also monitored with or without rotenone, a mitochondrial electron transport chain (ETC) complex I inhibitor. Cultured ARPE-19 cells generated scattered cell clusters composed mostly of smaller size cells expressing the differentiation markers mouse anti-cellular retinaldehyde–binding protein (CRALBP) and Bestrophin only in differentiation medium. After the increase of the number of clusters, the clusters appeared to paracellularly merge, resulting in expansion of the area occupied by the clusters. Of note, the cells within the clusters selectively had high MMP and were in accordance with the expression of RPE differentiation markers. Rotenone repressed the formation of the clusters and decreased intracellular MMP. The above results suggest that clustering of RPE cells with functional mitochondria plays a pivotal role in RPE cell differentiation process and the ETC complex I inhibition greatly influences the composition of RPE cells that are degenerated or differentiation disposed.

Abstract This study aims to clarify the immunogenicity in acquired and innate immune responses of cultured human corneal endothelial cells (hCECs) applied for cell injection therapy, a newly established modality for corneal endothelium failures. Thirty-four patients with corneal endothelial failure received injection of allogeneic hCEC suspension into anterior chamber. No sign of immunological rejection was observed in all 34 patients during the 5–8 years postoperative follow-up period. Cell injection therapy was successful in 2 patients treated for endothelial failure after penetrating keratoplasty and one patient with Descemet membrane stripping automated endothelial keratoplasty failure. ELISPOT assays performed in allo-mixed lymphocyte reaction to the alloantigen identical to that on the injected hCECs, elicited sparse IFN-γ-specific spots in the peripheral blood mononuclear cells of patients who received hCEC injection. The therapy generated simple and smooth graft-host junctions without wound stress. The injection of C57BL/6 CECs into the anterior chamber of BALB/c mice, which rejected C57BL/6 corneas 6 weeks ago, induced no sign of inflammatory reactions after the second challenge of alloantigen. Collectively, injection of the hCEC cell suspension in the aqueous humor induces immune tolerance that contributes to the survival of the reconstituted endothelium.

Abstract This study aimed to uncover the mechanism responsible for the clinical efficacy of cell injection therapy with fully differentiated cultured cells. Analysis of polarized expression of ion transporters on cultured human corneal endothelial cells (CECs) subpopulations (SPs) was performed. The intracellular pH (pHi) between two CEC SPs, distinct in the proportion of differentiated cells, was measured, and the association with mitochondrial respiration homeostasis was investigated. The effects of the ion transporter inhibition by their selective inhibitors or siRNA transfection were also explored. Na+/K+-ATPase, Aquaporin 1, SLC4A11, NBCe1, NHE1 as transporters, and ZO-1, were all selectively expressed in differentiated SPs, but were almost null in the cell-state-transitioned SPs. We also confirmed that the pHi of CEC SPs affected their mitochondrial respiration by modulating the expression of these ion transporters via inhibitors or siRNA transfection. Ion and water transporters might participate in the maintenance of pHi and mitochondria homeostasis in differentiated SPs, which may contribute, combined with integral barrier functions, to efficient water efflux. The differences in intracellular pH between the two SPs is attributed to variations in the expression profile of specific ion transporters and mitochondrial functions, which may associate with the efficacy of the SPs in cell injection therapy.

Abstract This study investigated the pathophysiological features of pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD) by analysing and comparing cytokine profiles in aqueous humour (AH) collected from 18 PNV, 18 nAMD and 11 control patients. Responses to intravitreal injection of aflibercept were also analysed in the PNV and nAMD groups. In the PNV group, vascular endothelial growth factor (VEGF)-A was significantly lower than in the nAMD group (p = 0.03) but was almost identical to that in the control group (p = 0.86). The nAMD group showed positive correlations between interleukin (IL)-6 and IL-8 (r = 0.78, p