Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig

Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig. Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous photoreceptor rosettes within GFP-positive grafts, indicative of the development of cellular polarity and self-assembly into rudiments of outer retinal tissue. Conclusion. Together, these data support the tolerance of RPCs as allografts and demonstrate the high level of rod photoreceptor development that can be obtained from cultured RPCs following transplantation. Strategies for further progress in this area, together with possible functional implications, are discussed

Subretinal Implantation of Electrospun, Short Nanowire, and Smooth Poly(ε-caprolactone) Scaffolds to the Subretinal Space of Porcine Eyes

Biodegradable scaffolds play an important adjunct role in transplantation of retinal progenitor cells (RPCs) to the subretinal space. Poly(ε-Caprolactone) (PCL) scaffolds with different modifications were subretinally implanted in 28 porcine eyes and evaluated by multifocal electroretinography (mfERG) and histology after 6 weeks of observation. PCL Short Nanowire, PCL Electrospun, and PCL Smooth scaffolds were well tolerated in the subretinal space in pigs and caused no inflammation and limited tissue disruption. PCL Short Nanowire had an average rate of preserved overlying outer retina 17% higher than PCL Electrospun and 25% higher than PCL Smooth. Furthermore, PCL Short Nanowire was found to have the most suitable degree of stiffness for surgical delivery to the subretinal space. The membrane-induced photoreceptor damage could be shown on mfERG, but the reductions in P1 amplitude were only significant for the PCL Smooth. We conclude that of the tested scaffolds, PCL Short Nanowire is the best candidate for subretinal implantation

The Influence of Brightness on Functional Assessment by mfERG: A Study on Scaffolds Used in Retinal Cell Transplantation in Pigs

To determine the effect of membrane brightness on multifocal electroretinograms (mfERGs), we implanted poly lactic-co-glycolic acid (PLGA) membranes in the subretinal space of 11 porcine eyes. We compared membranes with their native shiny white color with membranes that were stained with a blue dye (Brilliant Blue). Histological and electrophysiological evaluation of the overlying retina was carried out 6 weeks after implantation. Histologically, both white and blue membranes degraded in a spongiform manner leaving a disrupted outer retina with no preserved photoreceptor segments. Multifocal ERG revealed the white membranes to have a significantly higher P1-amplitude ratio than the blue (P = 0.027), and a correlation between brightness ratio and P1-amplitude ratio was found (r = 0.762). Based on our findings, we conclude that bright subretinal objects can produce normal mfERG amplitude ratios even when the adjacent photoreceptors are missing. Functional assessment with mfERG in scaffold implant studies should therefore be evaluated with care

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE)

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).This project was funded by the National Health and Medical Research Council (NHMRC; 1093017), the Walking On Sunshine Foundation, Anne Stanton, Nicola Laws and Lloyd Owen in Memorial and Civic Solutions. This study was also funded by Fight for Sight, Denmark. A.L.P. is supported by Highland Island Enterprise (HMS9353763). This work was supported by an NHMRC Program Grant (G.V.L., G.J.M., R.A.S. and N.K.H.). G.V.L. is supported by an NHMRC Practitioner Fellowship and The University of Sydney, Medical Foundation. R.A.S. is supported by an NHMRC Practitioner Fellowship. Support from Melanoma Institute Australia and The Ainsworth Foundation is also gratefully acknowledged. J.S.W. is supported by a NHMRC early career fellowship (1111678). N.W. is supported by an NHMRC Senior Research Fellowship (1139071). N.K.H. is supported by an NHMRC Senior Principal Research Fellowship (1117663)

RPE and Stem Cell Therapy

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