Optimisation of Substrate and Growth Conditions of Retinal Pigment Epithelial Cells Destined for Transplantation

Retinal Pigment Epithelium (RPE) transplantation efforts have been ongoing for three decades . To this day no feasible cure exists for diseases such as age-related macular degeneration (AMD). The key to treatment of AMD is to replenish the RPE. Establishing cells that accurately represent their native tissue is a considerable challenge. RPE cells undergo de-differentiation in culture losing important characteristics after repeated passage. Furthermore, RPE cells are anchorage-dependant and require a substrate for survival. Attempts at replenishing the RPE using suspensions have been met with scepticism due to the high degree of apoptosis. ARPE-19, a human RPE line, retains several phenotypic characteristics of primary RPE although current passages have lost some established features. A variety of polymers with diverse chemistries, specialised coatings, and optimised media were tested to optimally grow ARPE-19 cells. Using information gathered from these experiments, optimal conditions were selected for Human Embryonic Stem Cell (HESC)-derived RPE cells. Cell/substrate composites were transplanted in pigs to validate their efficacy. Differentiation of ARPE-19 cells was enhanced by utilising a superior substrate, polyester filter, together with an optimal growth medium containing pyruvate. HESCderived RPE grown in optimal conditions developed differentiation characteristics identical to native RPE. This was assessed by morphology, immunohistochemical profile, trans-epithelial resistance, electron-microscopy, and growth factor secretion. A higher porosity version of this filter supported growth and differentiation of HESC-RPE and this was chosen as the basis for a transplantation project due to its good permeability. Transplanted HESC-RPE/polyester composites survived surgical delivery in a pig model and were eventually chosen for a phase I clinical trial. Conclusion: This thesis investigated optimal conditions for the human RPE line ARPE- 19. Optimal growth conditions were then applied to HESC-RPE cells which achieved a high level of differentiation. Composites were transplanted in pigs and led to their selection for use in clinical trials

A role for the ciliary marginal zone in the melanopsin-dependent intrinsic pupillary light reflex.

Maintenance of pupillary constriction in light-adapted rodents has traditionally been thought to involve a reflex between retina, brain and iris, with recent work identifying the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the major conduits for retinal input to the brain. There is also a less well-understood phenomenon whereby the iris of some mammals, including mice, will constrict to light when either the eye, or the iris itself is physically isolated from the brain. The intrinsic pupillary light reflex (iPLR) is the term given to pupil constriction in the absence of retinal input to the brain. Here, using an intraocular axotomy approach, we show that the iPLR in conscious mice spans a dynamic range over 3 log units of irradiance. This iPLR response is absent in melanopsin knockout (MKO) mice and can be significantly inhibited by atropine. Immunohistochemistry for cfos and melanopsin, in combination with light exposure revealed a population of small ipRGCs in the retinal ciliary marginal zone (CMZ), which remain responsive to light in axotomised mice. We report that damage to the CMZ in a novel in vitro preparation removes a significant component of the iPLR response, while a detailed immunohistochemical analysis of the CMZ in wildtype mice revealed a melanopsin-rich plexus, which was consistently most intense in nasal retina. There were clear examples of melanopsin-positive, direct retino-ciliary projections, which appear to emanate from Brn3b negative, M1 type ipRGCs. These cells are clustered along the melanopsin-rich plexus nasally and may channel ipRGC signals from retina into the iris via ciliary body. Comparison between wildtype and MKO mice reveals that the ciliary body is also weakly stained for melanopsin. Our results show that the full extent of iPLR in mice requires cholinergic neurotransmission and intact signalling at the CMZ / ciliary body. This response may be mediated to some extent by ipRGCs, which send direct projections from the retina into ciliary body. In addition to the melanopsin-mediated iris sphincter constriction suggested by others, we propose a new mechanism, which may involve constriction of the ciliary body and ipRGC-mediated relaxation of the iris dilator muscle

Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients

Mislocalisation of BEST1 in iPSC-derived retinal pigment epithelial cells from a family with autosomal dominant vitreoretinochoroidopathy (ADVIRC)

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene, which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here, we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T > C, p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE, however in patient-derived iPSC-RPE, BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery, from an early developmental stage, could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients

Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD

Calciotropic hormones raise the chemically detectable [Pi] in UMR 106-06 osteoblast-like cells.

Uptake of orthophosphate (Pi) by osteoblast-like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast-like cells (UMR 106-06) that PTH (10(-11) to 10(-7) M) increases both 32Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D3 (1,25D) (10(-12) to 10(-6) M) and salmon calcitonin (CT) (10(-12) to 10(-6) g ml-1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular 32Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy- and 25 hydroxyvitamin D3. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth

Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast-like cells: effect of Pi, Na+ and K+.

Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106-01 and UMR 106-06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106-01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [32Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular 32P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106-01 and DET, but the decrease was smaller than that in [32Pi]-uptake. Ouabain decreased [32Pi]-uptake and cellular [Pi] in DET, but not in UMR 106-01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts

Dissecting a role for melanopsin in behavioural light aversion reveals a response independent of conventional photoreception.

Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA) is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT), melanopsin knockout (Opn4(-/-)) and rd/rd cl (melanopsin only (MO)) mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO) mice, lacking three elements deemed essential for conventional photoreception (Opn4(-/-) Gnat1(-/-) Cnga3(-/-)). Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice