Restoration falls into two main categories.
Endogenous – Cell replacement is triggered from cells within the eye.
Exogenous – Cell replacement with cells from outside the eye.
Some current approaches:
Exogenous:
ACTC, a public company is currently in Phase I/II trials using hESC to generate mature RPE cells that are delivered in suspension (with no scaffold for cell organization) via subretinal injection.
Phase II trials are underway for dry AMD and Stargardt’s macular dystrophy. Safety phase has been successfully completed. Ability of the cells introduced in suspension to survive and integrate into the human retina and their efficacy are as yet unknown.
Dr. Seabra, UK and Portugal, is in the second year of his CRF grant project. He is experimenting with creating iPSC from fat cells taken from the CHM mouse. He is then converting those into
RPE cells and doing a gene augmentation therapy on those cells. If time and budget allows he plans to attempt transplanting those cells back into the CHM mouse. A challenge with this autograft approach is that cells will have to be developed for each patient individually and the
RPE transplantation alone does not address PR loss.
Michael Young, Harvard, is working with Renuron (public company) on creating retinal precursor cells from human fetal eye tissue and using those to generate a two layer, photoreceptor plus RPE retinal graft. This allograft approach would potentially produce a supply of cells that could be used for any RDD patient. Dr. Young has stated they are working toward human trials.
David Gamm at the University of Wisconsin is producing iPS cells from diseased carrying patient samples for study. For transplantation Dr. Gamm is creating a two layer approach and using a scaffolding matrix in order to help organize the cells until they integrate into the adjacent cell layers after transplant. His primary approach is to generate a two layer PR plus
RPE retinal patch for transplantation using mature RPE and PR precursor cells. Precursor PR cells integrate better adjacent cell layers. He has also been successful at generating a two layer transplant patch of RPE plus choroid cells and may attempt a three layer patch with choroid, RPE and PR. He expects to proceed to wildtype large animal studies in 12-24 months.
Dr. Gamm is producing cells from what he calls “superdonors” people whose cells and tissues provide an immune match for a significant percentage of the general public. When properly matched with the recipient, the cells are less likely to be rejected. The FFB recently announced
$900k in funding for his work. Dr. Jean Bennett has asked Dr. Gamm to consider generating
RPE from CHM iPS cells which is an opportunity to perhaps gain access to these cells for other researchers.
David O. Clegg with Regenerative Patch Technologies (private company) is working toward human trials. Their technology involves creating RPE from hESC and growing as a monolayer
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on a synthetic substrate scaffold (described as Bruch’s membrane like) to create a “RPE patch”. The surgical technique has been practiced on pigs and Dr. Clegg has announced that he has enough GMP product for phase I trials. He announced at ARVO 5/2013 that he intended to be in phase I trials by year end 2013. Transplanted RPE would need to be under viable PR cells in order to have an effect unless a two step approach of transplanting RPE followed by a transplant of PR cells or some type of endogenous inducement of non PR cells to make PR cells over the RPE patch. This study should be followed and there could be initial result reporting in 2014.
Dr. Derek Van Der Kooy, University of Alberta is working on endogenous repair of th retina.
Quiescnt retinal stem cells are present in the ciliary margin of the adult mouse and human eye, in a region similar to where proliferating adult retinal stem cells persist in fish and amphibian retinas. The adult mammalian retinal stem cells can be prospectively isolated, and thus do not arise from a transdifferentiation process common to all peripheral RPE cells.
Single adult retinal stem cells can give rise to all retinal cell types including RPE cells, but only a minority of these become photoreceptors when differentiated in vitro. Differentiation of retinal stem cell progeny in the presence of taurine and retinoic acid in vitro can increase greatly the proportions of photoreceptors and produce rod specific progenitor clones.
Furthermore, when these adul retinal stem cells derived photoreceptor progeny are transplanted into mouse eyes, they show integration and differentiation into the outer retinal layer. More recent work has been directed at delivering these retinal stem cell progeny into the subretinal space within a biodegradable gel that improves the spread of the donor cells across the retina, as well as their survival and integration. Finally, we have begun a search for the factors that limit the proliferation of the endogenous retinal stem cells in vivo in the adult eye, and have found that the adult lens and the cornea specifically secrete factors that inhibit adult retinal stem cell proliferation.
Stephen Tsang, Columbia University, believes that cell transplantation in the human retina has the potential to restore lost vision and provide treatment for advanced stages of retinal degeneration featuring significant photoreceptor neuronal loss, noting that a major obstacle for this approach is the ability to produce sufficient patient specific photoreceptor cells for transplantation. Adult retinal stem cells, which reside in the ciliary body of the adult human eye, are one potential source of photoreceptors. Fish regenerate retinal neurons from a population of stem cells that are intrinsic in the ciliary body, which surrounds the lens of the eye and maintains proper pressure in the eyeball; these cells reside within the differentiated retina throughout the lifetime of the animal. The progeny of fish stem cells can divide and migratory progenitors are the antecedents of photoreceptor precursors. It is these intrinsic adult retinal stem cells that allow the fish to regenerate photoreceptor neurons spontaneously when existing neurons are killed. Stem cell transplantation therefore has the potential to restore lost vision and provide treatment for advanced stages of retinal degeneration even in cases of significant photoreceptor loss in humans.
Our research paves the way toward
"retinoplasty," reconstruction of interfaces between photoreceptors and their environment
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after the onset of retinal degeneration. Our approach involves the culture of human retinal stem cells from the ciliary body in eye-bank globes, and using those cultured cells to determine the combination of transcription factors involved in regulating their proliferation and differentiation into light-sensing photoreceptor neurons. These experiments will identify the effectors regulating human retinal stem cell differentiation and proliferation, as well as testing the ability of in vitro generated stem cells to repopulate the diseased retina. Future applications may include patient-specific stem cells obtained from fine-needle aspiration of their ciliary bodies in the operating room. Based on our findings, we foresee the ability to manipulate the patients' own stem cells to cure their specific disease. This approach will solve the problem of limited supply of allograft rejection by using a patient's own cells.
Sally Temple, Neuroscience, Neural Stem Cell Institute, Rensselaer, NY, and Athghin Biotech is working on producing RPE cells from tissue specific stem cells isolated from adult cadaver donor eyes that contain a subpopulation (around 3%) of cells that are highly proliferative, selfrenewing and multipotent, defining the RPE stem cell (RPESC). RPESCs can proliferate and differentiate to produce a cobblestone RPE-like epithelium in vitro. In this study, we use adult
RPESCs as a unique cell source for iPSC production. The resulting RPESC-derived iPSCs can be differentiated back into RPE cells, producing an essentially unlimited cell supply. Because the
RPE is the disease-affected tissue in Age-Related Macular Degeneration (AMD), the leading cause of blindness in people over 50, these RPESC-derived cells may produce a superior iPSCbased model of AMD compared to iPSCs derived from other tissues.
Methods: We routinely isolate adult RPE from cadaver donor eyes of different ages and gender. iPSC were derived from human adult RPESCs using a Sendai virus-based reprogramming strategy. The resulting cells were characterized by standard protocols, including immunostaining, FACS, qPCR, in vitro differentiation into the three germ layers and teratoma formation in vivo.
Results: Adult human RPESC-derived RPE cells were successfully used as donor tissue to derive iPSCs expressing pluripotency markers, including Tra-1-60, Nanog and SSEA4, and able to give rise to the three germ layers in vitro and in vivo. No residual donor RPE cells were detected in the iPSC cultures. Moreover, we obtained iPSCs from RPESCs isolated from AMD cadaver donor eyes.
Conclusions: We were able to obtain iPSC from elderly donor using a mix of 4 factors, demonstrating that we can successfully reprogram RPE from donors with ages ranging from 56 to 91 years old. To our knowledge, these are the first adult human CNS-derived iPSC, and the collection is particularly valuable as it includes elderly and AMD donors. In contrast to other sources of CNS stem cells in the hippocampal dentate gyrus and the subventricular zone, the
RPESCs can be obtained relatively easily from live patients with minimal surgery, or from donor cadaver eyes. RPESC-derived iPSC could be used to reveal the molecular pathways
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involved with aging and that underlie AMD. The essentially unlimited RPESC-iPSC supply makes large-scale studies and drugs screens possible.
Exogenous
The adult retina contains a small resident population of stem cells. Early work is underway on methods to cause these cells, as well as other retinal cell types to differentiate and proliferate in the retina.
Three examples:
David Hyde, Notre Dame is working on in vivo retinal regeneration. Damaging the adult zebrafish retina induces the resident Muller glia to dedifferentiate and reenter the cell cycle, which produces neuronal progenitor cells that continue to proliferate and migrate to the proper retinal layer, where they differentiate into the missing neuronal cell types. This regeneration only occurs in the retinal region that was damaged and the number of regenerated neurons are restored to their approximate number prior to the damage. While several different proteins are known to function in retinal regeneration, the signals that initiate Muller glia proliferation and regeneration remain unknown. Using both gene microarray and proteomic analyses, we identified both an activator and a repressor of the initial Muller glia proliferation. Using electroporated morpholinos, we identified and characterized he signal transduction pathways that are downstream of the activator and repressor. Elucidating these processes may ultimately provide critical information to regulate neuronal regeneration from either endogenous or exogenous multipotent cells in the mammalian retina.
Dr. Kang Zhang, U Cal, San Diego, is working on stem cell and working on creating 600+ iPS cell lines that will be genotyped and made available. He is also working on in vivo cell reprogramming of glial cells.
Dr. Thomas Reh, University of Washington. Over the past 20 years many of the key transcription factors that control the development of the retina have been discovered. In addition, there have been major advances in reprogramming cells to more stem-like states using these developmentally important transcription factors. Dr. Reh is progressing in his lab in reprogramming Muller glia to retinal progenitors using proneural transcription factors as a strategy to repair damaged retina.
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