S. Girman

Long‐Term Safety and Function of RPE from Human Embryonic Stem Cells in Preclinical Models of Macular Degeneration

Assessments of safety and efficacy are crucial before human ESC (hESC) therapies can move into the clinic. Two important early potential hESC applications are the use of retinal pigment epithelium (RPE) for the treatment of age‐related macular degeneration and Stargardt disease, an untreatable form of macular dystrophy that leads to early‐onset blindness. Here we show long‐term functional rescue using hESC‐derived RPE in both the RCS rat and Elov14 mouse, which are animal models of retinal degeneration and Stargardt, respectively. Good Manufacturing Practice‐compliant hESC‐RPE survived subretinal transplantation in RCS rats for prolonged periods (>220 days). The cells sustained visual function and photoreceptor integrity in a dose‐dependent fashion without teratoma formation or untoward pathological reactions. Near‐normal functional measurements were recorded at >60 days survival in RCS rats. To further address safety concerns, a Good Laboratory Practice‐compliant study was carried out in the NIH III immune‐deficient mouse model. Long‐term data (spanning the life of the animals) showed no gross or microscopic evidence of teratoma/tumor formation after subretinal hESC‐RPE transplantation. These results suggest that hESCs could serve as a potentially safe and inexhaustible source of RPE for the efficacious treatment of a range of retinal degenerative diseases. STEM CELLS 2009;27:2126–2135

Cells Isolated from Umbilical Cord Tissue Rescue Photoreceptors and Visual Functions in a Rodent Model of Retinal Disease

Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human‐derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue‐derived cells, placenta‐derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical‐derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue‐derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue‐derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.

Long-term Vision Rescue by Human Neural Progenitors in a Rat Model of Photoreceptor Degeneration

PURPOSEnAs a follow-up to previous studies showing that human cortical neural progenitor cells (hNPC(ctx)) can sustain vision for at least 70 days after injection into the subretinal space of Royal College of Surgeons (RCS) rats, the authors examined how functional rescue is preserved over long periods and how this relates to retinal integrity and donor cell survival.nnnMETHODSnPigmented dystrophic RCS rats (n = 15) received unilateral subretinal injections of hNPC(ctx) at postnatal day (P) 21; control rats (n = 10) received medium alone and were untreated. All animals were maintained on oral cyclosporine A. Function was monitored serially by measuring acuity (using an optomotor test) and luminance thresholds (recording from the superior colliculus) at approximately P90, P150, and P280. Eyes were processed for histologic study after functional tests.nnnRESULTSnAcuity and luminance thresholds were significantly better in hNPC(ctx)-treated animals than in controls (P < 0.001) at all time points studied. Acuity was greater than 90%, 82%, and 37% of normal at P90, P150, and P270, whereas luminance thresholds in the area of best rescue remained similar the whole time. Histologic studies revealed substantial photoreceptor rescue, even up to P280, despite progressive deterioration in rod and cone morphology. Donor cells were still present at P280, and no sign of donor cell overgrowth was seen.nnnCONCLUSIONSnLong-term rescue of function and associated morphologic substrates was seen, together with donor cell survival even in the xenograft paradigm. This is encouraging when exploring further the potential for the application of hNPC(ctx) in treating retinal disease.

Transplantation of human central nervous system stem cells – neuroprotection in retinal degeneration

Stem cells derived from the human brain and grown as neurospheres (HuCNS‐SC) have been shown to be effective in treating central neurodegenerative conditions in a variety of animal models. Human safety data in neurodegenerative disorders are currently being accrued. In the present study, we explored the efficacy of HuCNS‐SC in a rodent model of retinal degeneration, the Royal College of Surgeons (RCS) rat, and extended our previous cell transplantation studies to include an in‐depth examination of donor cell behavior and phenotype post‐transplantation. As a first step, we have shown that HuCNS‐SC protect host photoreceptors and preserve visual function after transplantation into the subretinal space of postnatal day 21 RCS rats. Moreover, cone photoreceptor density remained relatively constant over several months, consistent with the sustained visual acuity and luminance sensitivity functional outcomes. The novel findings of this study include the characterization and quantification of donor cell radial migration from the injection site and within the subretinal space as well as the demonstration that donor cells maintain an immature phenotype throughout the 7u2003months of the experiment and undergo very limited proliferation with no evidence of uncontrolled growth or tumor‐like formation. Given the efficacy findings and lack of adverse events in the RCS rat in combination with the results from ongoing clinical investigations, HuCNS‐SC appear to be a well‐suited candidate for cell therapy in retinal degenerative conditions.

Preservation of visual responsiveness in the superior colliculus of RCS rats after retinal pigment epithelium cell transplantation

The dystrophic RCS rat undergoes progressive photoreceptor degeneration due to a primary defect in retinal pigment epithelial (RPE) cells. This has a major impact on central visual responsiveness. Here we have examined how functional deterioration is contained by subretinal transplantation of immortalized human RPE cells. Transplantation was done at three to four weeks of age prior to significant photoreceptor loss and recipients were kept on cyclosporin. At six months of age, sensitivity maps and multi-unit response properties were obtained across the visual field by recording at 76 equidistant sites encompassing the whole superior colliculus.A significant degree of functional protection, both in terms of area of responsive retina and response characteristics was observed following RPE transplantation. At best, the sensitivity, latency of onset, and response rise time were all maintained within normal ranges and this was achieved with no more than half of the normal complement of photoreceptors. Although partial, the degree of anatomical preservation (both in terms of outer nuclear layer thickness and area of rescue) correlated well with the level of preserved visual sensitivities. Sham injections also resulted in rescue, though the area of preservation was strictly confined to the needle injury site and the response properties were significantly worse than with RPE injections. This study shows that central physiological responsiveness and correlated retinal morphology can be preserved in an animal model of retinal disease by implantation of an immortalized cell line. The use of retinal sensitivity measurements provides a background for assessing higher visual functions in these animals and a direct comparison for human perimetry measures.

Human adult bone marrow-derived somatic cells rescue vision in a rodent model of retinal degeneration

Visual impairment associated with photoreceptor degeneration is a largely untreatable condition affecting millions of people worldwide. Cellular therapies offer an attractive alternative for the treatment of retinal degeneration. Human adult bone marrow-derived somatic cells (hABM-SCs) present particular advantages for interventional therapy to the eye because they are non-immunogenic, effective at low dose, maintain a stable phenotype and secrete factors known to promote photoreceptor cell survival. Here we assess the potential of hABM-SCs (developed by Garnet BioTherapeutics) to sustain vision in a rodent model of human retinal disease-the Royal College of Surgeons (RCS) rat. A cell suspension of 5 x 10(4) cells in 2 microl carrying medium was injected into the subretinal space of RCS rats at P21, control animals received medium alone and untreated. Animals were maintained with/without oral cyclosporine A through the experimental period. Visual functions including optokinetic response (OKR) and luminance threshold recording were conducted in both cell-treated and control animals. Histological study was performed afterward to examine donor cell survival and photoreceptor rescue. OKR measured at P60 and P90 in cell-injected groups with/without immunosuppression was significantly better than controls (P<0.01). Luminance threshold recordings around P90-100 also showed much lower threshold in cell-injected eyes with best result of 0.8 log units above background (compared with 0.4 log units in wild type retina), while sham-injected and untreated eyes gave values of 2.7 and 3.5 log units respectively. Histological examination revealed that there were 3-6 layers of photoreceptors in cell-injected eyes at P100; but only one layer in sham-injected and untreated controls. There was no evidence of unwanted pathology. This study has demonstrated that a human cell product that can be manufactured under conditions acceptable for clinical use and in quantities sufficient for treating large numbers of patients can rescue photoreceptors and sustain substantial visual function when injected into the subretinal space of a rodent model for retinal degeneration. Furthermore, we have shown that achieving a therapeutic benefit does not require the cells to persist and does not require immunosuppression.

Cortical visual functions can be preserved by subretinal RPE cell grafting in RCS rats.

Photoreceptor loss in the Royal College of Surgeons (RCS) rat is limited by transplantation of retinal pigment epithelial cell lines into the subretinal space prior to the onset of major photoreceptor loss. The purpose of this study was to examine to what extent visual cortical function was rescued by such transplantation and how the degree of rescue correlated with threshold responses recorded in the superior colliculus. To achieve this, single unit responses were recorded from the supragranular layers of cortical area, V1, at 7 months of age at a time when the cortex in these animals is normally non-responsive to specific visual stimulation. The best animals gave cortical responses that were very little different from normal. For the whole group studied, of the eight parameters measured for each cell, only three were significantly less well tuned than in normal non-dystrophic rats. In general, better single unit responses in the cortex were obtained with more photoreceptor rescue and this correlated with better threshold responses. These results indicate that discrete central visual responses can be preserved by subretinal transplantation of a cell line which limits chronic loss of input signal associated with progressive photoreceptor loss.

Time course of deterioration of rod and cone function in RCS rat and the effects of subretinal cell grafting: a light- and dark-adaptation study

To examine how rod and cone function are differentially affected during retinal degeneration, and after subretinal cell grafting, we obtained light- and dark-adaptation curves by recording threshold multiunit responses from the superior colliculus of anesthetized rats. Unoperated RCS dystrophic and non-dystrophic rats were used and the effects of subretinal grafting in dystrophic rats of cells known to limit photoreceptor degeneration were examined. The adaptation curves showed that rod function was severely compromised in unoperated dystrophic RCS rats at low luminance levels, even as early as 21 days of age and that cone thresholds became gradually elevated over time. While cell transplantation preserved both rod and cone photoreceptors, rod function did not recover, although further deterioration of cone threshold responses was prevented. This raises concern that measures of outer nuclear layer thickness may not in themselves be an accurate measure of visual capabilities and efficacy of a restoration strategy.

Morphological and Functional Rescue in RCS Rats after RPE Cell Line Transplantation at a Later Stage of Degeneration

PURPOSEnIt is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19.nnnMETHODSnDystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression.nnnRESULTSnLater grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors.nnnCONCLUSIONSnRPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Small Molecule Mediated Proliferation of Primary Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.