Michael Baron

Adult ciliary epithelial cells, previously identified as retinal stem cells with potential for retinal repair, fail to differentiate into new rod photoreceptors.

The ciliary margin in lower vertebrates is a site of continual retinal neurogenesis and a stem cell niche. By contrast, the human eye ceases retinal neuron production before birth and loss of photoreceptors during life is permanent and a major cause of blindness. The discovery of a proliferative cell population in the ciliary epithelium (CE) of the adult mammalian eye, designated retinal stem cells, raised the possibility that these cells could help to restore sight by replacing lost photoreceptors. We previously demonstrated the feasibility of photoreceptor transplantation using cells from the developing retina. CE cells could provide a renewable source of photoreceptors for transplantation. Several laboratories reported that these cells generate new photoreceptors, whereas a recent report questioned the existence of retinal stem cells. We used Nrl.gfp transgenic mice that express green fluorescent protein in rod photoreceptors to assess definitively the ability of CE cells to generate new photoreceptors. We report that CE cells expanded in monolayer cultures, lose pigmentation, and express a subset of eye field and retinal progenitor cell markers. Simultaneously, they continue to express some markers characteristic of differentiated CE and typically lack a neuronal morphology. Previously reported photoreceptor differentiation conditions used for CE cells, as well as conditions used to differentiate embryonic retinal progenitor cells (RPCs) and embryonic stem cell‐derived RPCs, do not effectively activate the Nrl‐regulated photoreceptor differentiation program. Therefore, we conclude that CE cells lack potential for photoreceptor differentiation and would require reprogramming to be useful as a source of new photoreceptors. STEM Cells 2010;28:1048–1059

Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells

Retinal degenerative disease causing loss of photoreceptor cells is the leading cause of untreatable blindness in the developed world, with inherited degeneration affecting 1 in 3000 people. Visual acuity deteriorates rapidly once the cone photoreceptors die, as these cells provide daylight and colour vision. Here, in proof-of-principle experiments, we demonstrate the feasibility of cone photoreceptor transplantation into the wild-type and degenerating retina of two genetic models of Leber congenital amaurosis, the Crb1rd8/rd8 and Gucy2e−/− mouse. Crx-expressing cells were flow-sorted from the developing retina of CrxGFP transgenic mice and transplanted into adult recipient retinae; CrxGFP is a marker of cone and rod photoreceptor commitment. Only the embryonic-stage Crx-positive donor cells integrated within the outer nuclear layer of the recipient and differentiated into new cones, whereas postnatal cells generated a 10-fold higher number of rods compared with embryonic-stage donors. New cone photoreceptors displayed unambiguous morphological cone features and expressed mature cone markers. Importantly, we found that the adult environment influences the number of integrating cones and favours rod integration. New cones and rods were observed in ratios similar to that of the host retina (1:35) even when the transplanted population consisted primarily of cone precursors. Cone integration efficiency was highest in the cone-deficient Gucy2e−/− retina suggesting that cone depletion creates a more optimal environment for cone transplantation. This is the first comprehensive study demonstrating the feasibility of cone transplantation into the adult retina. We conclude that flow-sorted embryonic-stage Crx-positive donor cells have the potential to replace lost cones, as well as rods, an important requirement for retinal disease therapy.

The biological impact of blood pressure associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle.

Abstract Elevated blood pressure (BP) is a major global risk factor for cardiovascular disease. Genome-wide association studies have identified several genetic variants at the NPR3 locus associated with BP, but the functional impact of these variants remains to be determined. Here we confirmed, by a genome-wide association study within UK Biobank, the existence of two independent BP-related signals within NPR3 locus. Using human primary vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) from different individuals, we found that the BP-elevating alleles within one linkage disequilibrium block identified by the sentinel variant rs1173771 was associated with lower endogenous NPR3 mRNA and protein levels in VSMCs, together with reduced levels in open chromatin and nuclear protein binding. The BP-elevating alleles also increased VSMC proliferation, angiotensin II-induced calcium flux and cell contraction. However, an analogous genotype-dependent association was not observed in vascular ECs. Our study identifies novel, putative mechanisms for BP-associated variants at the NPR3 locus to elevate BP, further strengthening the case for targeting NPR-C as a therapeutic approach for hypertension and cardiovascular disease prevention.

An Ensemble-Based Protocol for the Computational Prediction of Helix–Helix Interactions in G Protein-Coupled Receptors using Coarse-Grained Molecular Dynamics

The accurate identification of the specific points of interaction between G protein-coupled receptor (GPCR) oligomers is essential for the design of receptor ligands targeting oligomeric receptor targets. A coarse-grained molecular dynamics computer simulation approach would provide a compelling means of identifying these specific protein–protein interactions and could be applied both for known oligomers of interest and as a high-throughput screen to identify novel oligomeric targets. However, to be effective, this in silico modeling must provide accurate, precise, and reproducible information. This has been achieved recently in numerous biological systems using an ensemble-based all-atom molecular dynamics approach. In this study, we describe an equivalent methodology for ensemble-based coarse-grained simulations. We report the performance of this method when applied to four different GPCRs known to oligomerize using error analysis to determine the ensemble size and individual replica simulation time required. Our measurements of distance between residues shown to be involved in oligomerization of the fifth transmembrane domain from the adenosine A2A receptor are in very good agreement with the existing biophysical data and provide information about the nature of the contact interface that cannot be determined experimentally. Calculations of distance between rhodopsin, CXCR4, and β1AR transmembrane domains reported to form contact points in homodimers correlate well with the corresponding measurements obtained from experimental structural data, providing an ability to predict contact interfaces computationally. Interestingly, error analysis enables identification of noninteracting regions. Our results confirm that GPCR interactions can be reliably predicted using this novel methodology.

Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells

The protocols described in this unit provide detailed information on how to isolate and expand, in culture, ciliary epithelial cells (CECs), previously identified as retinal stem cells, from the adult mouse eye, and embryonic retinal progenitor cells (RPCs) from the developing retina. CECs are initially cultured in floating conditions as neurospheres and then expanded in monolayer cultures. RPCs are cultured in floating conditions. Detailed protocols for retinal differentiation, as well as exogenous gene expression using lentivirus are also described.