Diabetes is characterized by ‘glucotoxic’ loss of pancreatic β-cell function and insulin content, but underlying mechanisms remain unclear. A mouse model of insulin-secretory deficiency induced by β-cell inexcitability (KATP gain-of-function) demonstrates development of diabetes and reiterates the features of human neonatal diabetes. In the diabetic state, β-cells lose their mature identity and dedifferentiate to neurogenin3-positive and insulin-negative cells. Lineage-tracing experiments show that dedifferentiated cells can subsequently re-differentiate to mature neurogenin3-negative, insulin-positive, β-cells after lowering of blood glucose by insulin therapy. We demonstrate here that β-cell dedifferentiation, rather than apoptosis, is the main mechanism of loss of insulin-positive cells, and re-differentiation accounts for restoration of insulin content and antidiabetic-drug responsivity in these animals. These results may help explain gradual decrease in β-cell mass in long-standing diabetes, and recovery of β-cell function and drug responsivity in type-2 diabetic patients following insulin therapy, and suggest an approach to rescuing ‘exhausted’ β-cells in diabetes.

Cell type-specific investigations commonly use gene reporters or single-cell analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while single-cell RNA (scRNA)-sequencing (seq) frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from human pluripotent stem cells (hPSCs), we performed scRNA-seq on optic vesicle (OV)-like structures cultured under cGMP-compatible conditions. However, efforts to apply traditional scRNA-seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell-to-cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC-derived retinal cell types. Stem Cells 2018;36:313-324.

Mutations in the KCNJ13 gene that encodes the inwardly rectifying potassium channel Kir7.1 cause snowflake vitreoretinal degeneration (SVD) and leber congenital amaurosis (LCA). Kir7.1 controls the microenvironment between the photoreceptors and the retinal pigment epithelium (RPE) and also contributes to the function of other organs such as uterus and brain. Heterologous expressions of the mutant channel have suggested a dominant-negative loss of Kir7.1 function in SVD, but parallel studies in LCA16 have been lacking. Herein, we report the identification of a novel nonsense mutation in the second exon of the KCNJ13 gene that leads to a premature stop codon in association with LCA16. We have determined that the mutation results in a severe truncation of the Kir7.1 C-terminus, alters protein localization, and disrupts potassium currents. Coexpression of the mutant and wild-type channel has no negative influence on the wild-type channel function, consistent with the normal clinical phenotype of carrier individuals. By suppressing Kir7.1 function in mice, we were able to reproduce the severe LCA electroretinogram phenotype. Thus, we have extended the observation that Kir7.1 mutations are associated with vision disorders to include novel insights into the molecular mechanism of disease pathobiology in LCA16.

Oxytocin and OXTR are present in the posterior retina, and OXT induces an increase in hfRPE [Ca(2+)]i. These results suggest that the OXT-OXTR signaling pathway is active in the retina. We propose that OXT activation of the OXTR occurs in the posterior retina and that this may serve as a paracrine signaling pathway that contributes to communication between the cone photoreceptor and the RPE.

Oxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G-protein coupled receptor. Our localization of OXTR to the retinal pigment epithelium (RPE), in close proximity to OXT in the adjacent photoreceptor neurons, leads us to propose that OXT plays an important role in RPE-retinal communication. An increase of RPE [Ca2+]i in response to OXT stimulation implies that the RPE may utilize oxytocinergic signaling as a mechanism by which it accomplishes some of its many roles. In this study, we used an established human RPE cell line, a HEK293 heterologous OXTR expression system, and pharmacological inhibitors of Ca2+ signaling to demonstrate that OXTR utilizes capacitative Ca2+ entry (CCE) mechanisms to sustain an increase in cytoplasmic Ca2+. These findings demonstrate how multiple functional outcomes of OXT-OXTR signaling could be integrated via a single pathway. In addition, the activated OXTR was able to inhibit the Kir7.1 channel, an important mediator of sub retinal waste transport and K+ homeostasis.