New Approaches to the Transplantation of Stem Cells and their Progeny for the Treatment of Retinal Degeneration

Cellular transplantation for photoreceptor replacement in retinal disease is limited by poor distribution, survival and integration of cells in vivo after standard delivery in saline vehicle. We were interested in addressing each of these barriers in order to improve transplant efficacy. To this end, we designed the first injectable biomaterial-based cell delivery vehicle to transplant adult stem cell progeny into the subretinal space of adult retina. A minimally-invasive and bio-resorbable blend of hyaluronan and methylcellulose (HAMC) was found to overcome cellular aggregation and non-contiguous distribution. The ability to direct stem cell differentiation toward a particular retinal lineage is another challenge facing clinical application. We showed that prospectively and clonally isolated multipotent mouse and human retinal stem cells (RSCs) could be directed toward a mature rod photoreceptor fate with the highest efficiency reported to date (>90%). Combinations of taurine and retinoic acid directed rod differentiation similar to rod development in vivo. RSC-derived rods exhibited morphology, protein and gene expression consistent with primary cultures of rods in vitro. When combined with the HAMC delivery vehicle, greater cell survival and improved integration of post-mitotic RSC-derived rods was observed in vivo compared to saline delivery. Improved donor rod survival was ascribed to the CD44 receptor – HAMC interaction in vitro and in vivo. Combined with HAMC delivery, disruption of the glial limiting membrane improved cell integration and resulted in the highest levels of integration of adult stem cell-derived rod photoreceptors relative to previous reports in the literature. Mature rod-lineage committed cells demonstrated higher integration potential compared to immature rods in this context. The integrated cells expressed Rhodopsin and elaborated outer segments. In the absence of the glial limiting membrane, rod integration depended on pro-survival signals from the environment. This work demonstrates that adult RSCs show promise for regenerative medicine strategies in the adult retina.Ph

Beyond Sector Retinitis Pigmentosa: Expanding the Phenotype and Natural History of the Rhodopsin Gene Codon 106 Mutation (Gly-to-Arg) in Autosomal Dominant Retinitis Pigmentosa

Sector and pericentral are two rare, regional forms of retinitis pigmentosa (RP). While usually defined as stable or only very slowly progressing, the available literature to support this claim is limited. Additionally, few studies have analyzed the spectrum of disease within a particular genotype. We identified all cases (9 patients) with an autosomal dominant Rhodopsin variant previously associated with sector RP (RHO c.316G > A, p.Gly106Arg) at our institution. Clinical histories were reviewed, and testing included visual fields, multimodal imaging, and electroretinography. Patients demonstrated a broad phenotypic spectrum that spanned regional phenotypes from sector-like to pericentral RP, as well as generalized disease. We also present evidence of significant intrafamilial variability in regional phenotypes. Finally, we present the longest-reported follow-up for a patient with RHO-associated sector-like RP, showing progression from sectoral to pericentral disease over three decades. In the absence of comorbid macular disease, the long-term prognosis for central visual acuity is good. However, we found that significant progression of RHO p.Gly106Arg disease can occur over protracted periods, with impact on peripheral vision. Longitudinal widefield imaging and periodic ERG reassessment are likely to aid in monitoring disease progression

Challenges Facing Physician Scientist Trainees: a Survey of Trainees in Canada’s Largest Undergraduate and Postgraduate Programs in a Single Centre

Purpose: A number of indicators suggest that the physician scientist career track is threatened. As such, it is an opportune time to evaluate current training models. Perspectives on physician scientist education and career path were surveyed in trainees at the University of Toronto, home to Canada’s longest standing physician scientist training programs. Methods: Trainees from the Clinician Investigator Program (CIP) and MD/PhD Program at the University of Toronto were surveyed. Liekert-style closed-ended questions were used to assess future career goals, present and future perspectives and concerns about and beliefs on training. Demographic information was collected regarding year of study, graduate degree program and focus of clinical and health research. Statistical analysis included non-parametric tests for sub-group comparisons. Results: Both groups of trainees were motivated to pursue a career as a physician scientist. While confident in their decision to begin and complete physician scientist training, they expressed concerns about the level of integration between clinical and research training in the current programs. They also expressed concerns about career outlook, including the ability to find stable and sustainable careers in academic medicine. Trainees highlighted a number of factors, including career mentorship, as essential for career success. Conclusion: These findings indicate that while trainees at different stages consistently express career motivation, they identified concerns that are program- and training stage-specific. These concerns mirror those highlighted in the medical education literature regarding threats to the physician scientist career path. Understanding these different and changing perspectives and exploring those differences could form an important basis for trainee program improvements both nationally and internationally

Heterochromia and ocular ischemia resulting from Neurofibromatosis I-related optic pathway glioma

NF1-OPG are typically benign optic nerve tumors arising in childhood which may be associated with visual loss from optic neuropathy, impaired extraocular motility, and proptosis. While optic disc edema, pallor, or choroidal folds are commonly observed, iris and retinal vascular sequelae arising from NF1-OPG are exceedingly rare (1-4)

The Next Generation of Molecular and Cellular Therapeutics for Inherited Retinal Disease

Inherited retinal degenerations (IRDs) are a diverse group of conditions that are often characterized by the loss of photoreceptors and blindness. Recent innovations in molecular biology and genomics have allowed us to identify the causative defects behind these dystrophies and to design therapeutics that target specific mechanisms of retinal disease. Recently, the FDA approved the first in vivo gene therapy for one of these hereditary blinding conditions. Current clinical trials are exploring new therapies that could provide treatment for a growing number of retinal dystrophies. While the field has had early success with gene augmentation strategies for treating retinal disease based on loss-of-function mutations, many novel approaches hold the promise of offering therapies that span the full spectrum of causative mutations and mechanisms. Here, we provide a comprehensive review of the approaches currently in development including a discussion of retinal neuroprotection, gene therapies (gene augmentation, gene editing, RNA modification, optogenetics), and regenerative stem or precursor cell-based therapies. Our review focuses on technologies that are being developed for clinical translation or are in active clinical trials and discusses the advantages and limitations for each approach

Directed differentiation of Photoreceptors and Retinal Pigment Epithelium from adult Mouse Retinal Stem Cells

The use of stem cell progeny for transplantation for retinal regenerative therapy will require techniques to direct the differentiation of these cells toward specific retinal cell types. Adult retinal stem cells (RSCs) represent a promising source of cells for retinal cell therapy to treat blinding eye diseases. Here, we describe the use of combinations of exogenous culture additives to direct RSCs along photoreceptor and retinal pigment epithelial (RPE) lineages with high efficiency

The adult retinal stem cell is a rare cell in the ciliary epithelium whose progeny can differentiate into photoreceptors

Self-renewing, multipotential retinal stem cells (RSCs) reside in the pigmented ciliary epithelium of the peripheral retina in adult mammals. RSCs can give rise to rhodopsin positive-cells, which can integrate into early postnatal retina, and represent a potentially useful option for cellular therapy. The ability to purify a stem cell population and direct the differentiation toward a particular cell lineage is a challenge facing the application of stem cells in regenerative medicine. Here we use cell sorting to prospectively enrich mouse RSCs based on size, granularity and low expression of P-cadherin and demonstrate that only rare cells with defined properties proliferate to form colonies. We show that clonally-derived mouse and human RSC progeny are multipotent and can differentiate into mature rhodopsin-positive cells with high efficiency using combinations of exogenous culture additives known to influence neural retinal development, including taurine and retinoic acid. This directed RSC differentiation follows the temporal sequence of photoreceptor differentiation in vivo, and the cells exhibit morphology, protein and gene expression consistent with primary cultures of rods in vitro. These results demonstrate that the RSC, an adult stem cell, can be enriched and directed to produce photoreceptors as a first step toward a targeted cell replacement strategy to treat retinal degenerative disease

Beyond Sector Retinitis Pigmentosa: Expanding the Phenotype and Natural History of the Rhodopsin Gene Codon 106 Mutation (Gly-to-Arg) in Autosomal Dominant Retinitis Pigmentosa

Sector and pericentral are two rare, regional forms of retinitis pigmentosa (RP). While usually defined as stable or only very slowly progressing, the available literature to support this claim is limited. Additionally, few studies have analyzed the spectrum of disease within a particular genotype. We identified all cases (9 patients) with an autosomal dominant Rhodopsin variant previously associated with sector RP (RHO c.316G > A, p.Gly106Arg) at our institution. Clinical histories were reviewed, and testing included visual fields, multimodal imaging, and electroretinography. Patients demonstrated a broad phenotypic spectrum that spanned regional phenotypes from sector-like to pericentral RP, as well as generalized disease. We also present evidence of significant intrafamilial variability in regional phenotypes. Finally, we present the longest-reported follow-up for a patient with RHO-associated sector-like RP, showing progression from sectoral to pericentral disease over three decades. In the absence of comorbid macular disease, the long-term prognosis for central visual acuity is good. However, we found that significant progression of RHO p.Gly106Arg disease can occur over protracted periods, with impact on peripheral vision. Longitudinal widefield imaging and periodic ERG reassessment are likely to aid in monitoring disease progression