Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe retinal pigment epithelium (RPE) is a cellular monolayer between the retina and the underlying choroidal vasculature. The RPE participates actively in the visual process, notably by supporting the diurnal replenishment of the photoreceptors (). RPE dysfunction significantly contributes to the pathophysiology of age-related macular degeneration (AMD), a leading cause of blindness (). There are currently no disease-altering therapies available for the vast majority (over 85%) of AMD patients that suffer from the dry form of the disease, which is characterized by extracellular deposits termed drusen beneath the RPE and subsequent RPE atrophy in the macula. The remaining approximately 15% of patients have wet AMD, in which neovascularization invades from the choroid; for these patients, repeated intravitreal injections with antiangiogenic drugs offer a highly effective, albeit palliative, treatment (). Replacement of dysfunctional submacular RPE with a cell-based therapeutic agent represents a potentially curative treatment strategy (). Some previous attempts in patients have been shown to improve vision, but most were limited by immune reactions, surgical complications, late-stage disease, or lack of an adequate RPE cell source (). Translocation of an autologous patch of RPE/choroid remains clinically the most popular approach, because some patients benefit from the procedure, despite its high complication rates ().With the development of RPE differentiation protocols from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (), RPE transplantation has experienced a powerful renaissance, as scientists and clinicians envision an unlimited supply of RPE for transplantation. However, much is still not understood with regard to the physiology of stem-cell-derived RPE () and transplantation into patients is in the early stages. Pilot data from a phase I/II trial ( and ) with a suspension of hESC-derived RPE injected in patients with dry AMD or Stargardt’s disease suggest a favorable safety profile and some limited improvement in vision (); further dose-escalation in this multicenter study is on-going. This is encouraging, given that prior studies using RPE cell suspensions showed they failed to survive or function on aged submacular Bruch’s membrane () and are more likely to be rejected than are RPE monolayers ().A cultured human RPE monolayer that exhibits the physiology of its native counterpart could be a valuable alternative to an RPE-cell suspension. This type of culture has been readily attained using fetal- or pluripotent-stem-cell-derived RPE. However, establishing such cultures from adult RPE has proven difficult and inconsistent, due to its propensity to undergo epithelial-mesenchymal transition (reviewed in ). We have optimized culture conditions that robustly activate a subpopulation of adult human RPE stem cells (RPESC), expand, and then differentiate them into highly pure RPE monolayers that exhibit physiological features of native RPE (). This protocol allows us to explore the potential of adult RPESC-derived RPE for cell-replacement therapy. To date, we do not know which cell source will turn out to be therapeutically successful, and therefore, testing all potential candidates is important. Using a cell source derived from the adult human RPE may possess several potential advantages, such as fewer ethical concerns compared to hESC and fetal human RPE (hRPE), the possibility of routine histocompatibility leukocyte antigen matching or even autologous transplantation (using a patient’s own remaining healthy RPESCs) to minimize immunosuppression, reduced proliferative potential than hESCs or human iPSCs and therefore reduced tumorigenesis risk, and reduced threat of generating abnormal cell types.RPE monolayers grown on cell carriers would facilitate surgical handling and long-term functionality by substituting some or all of the functions of the aged Bruch’s membrane (href="#bib3" rid="bib3" class=" bibr popnode">Binder et al., 2007). Coimplantation of differentiated RPE monolayers on a substrate has been attempted in animal models only in a few instances and with limited success (href="#bib2" rid="bib2 bib11 bib31" class=" bibr popnode">Bhatt et al., 1994; Diniz et al., 2013; Nicolini et al., 2000). Improvements would involve employing a biocompatible matrix that exhibits minimal deformation after transplantation, longer-term assessment postsurgery, and use of a large-eyed animal model for better assessment of surgical technique.We have previously reported on a method and instrumentation to deliver ultrathin rigid-elastic cell carriers (polyester [PET]) into the subretinal space (SRS) of rabbits (href="#bib42" rid="bib42" class=" bibr popnode">Stanzel et al., 2012). Here, we demonstrate this technology can be used to deliver monolayers of human RPE on permeable polyester carriers into the SRS of the rabbit. Notably, we find RPE isolated from adult cadaver donors can expand 20-fold and survive as a polarized RPE monolayer for 1 month after transplantation, therefore representing a clinically relevant RPE cell source. Transplants were followed with state-of-the-art ophthalmic imaging technology, including spectral domain optical coherence tomography (SD-OCT), confocal scanning-laser ophthalmoscopy (cSLO), color funduscopic photography, and histology. In addition, the influence of local and systemic immunosuppression on retinal tissue alterations following xenografting was evaluated.