Summary
Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX
+/tdTomato
human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time
in vivo
. Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.
Blinding disorders of the outer retina involve dysfunction and degeneration of photoreceptors. One potential approach to treat these forms of blindness is to repopulate the outer retina via a simple bolus injection of donor photoreceptors. However, this may not be ideal due to the highly polarized organization of photoreceptors that include apical light sensing photopigments and basal axon terminals. Furthermore, bolus injections create uncertainty with regard to the area, density, and retention of donor cells. Here, a novel and robust microfabrication process is developed to create 3D, micrometer-sized complex structures in ultrathin and biocompatible elastomer films (nonbiodegradable polydimethylsiloxane and biodegradable poly(glycerol-sebacate)) that can serve as polarizable photoreceptor delivery scaffolds, consisting of an array of cup-shaped photoreceptor capture wells that funnel into a microchannel. This "wine glass" scaffold design promotes efficient capture of human pluripotent stem-cell-derived photoreceptor cell bodies and guidance of basal axon extensions, ultimately achieving a uniform level of organization and polarization that is not possible with bolus injections or previously described scaffolds. In addition to future therapeutic applications, our scaffold design and materials provide a platform to generate reproducible and scalable in vitro models of photoreceptor-based diseases.
The clinical importance of the ocular hypotensive effects of 3-times-daily topical ophthalmic application of 0.005% latanoprost solution in dogs with glaucoma warrants investigation.
Polymeric scaffolds are revolutionizing therapeutics for blinding disorders affecting the outer retina, a region anatomically and functionally defined by light-sensitive photoreceptors. Recent engineering advances have produced planar scaffolds optimized for retinal pigment epithelium monolayer delivery, which are being tested in early-stage clinical trials. We previously described a three-dimensional scaffold supporting a polarized photoreceptor monolayer, but photoreceptor somata typically occupy multiple densely packed strata to maximize light detection. Thus, patients with severe photoreceptor degeneration are expected to extract greater benefits from higher-density photoreceptor delivery. Here, we describe the microfabrication of a biodegradable scaffold patterned for high-density photoreceptor replacement. The “ice cube tray” structure optimizes mechanical properties and cell-to-biomaterial load, enabling production of a multicellular photoreceptor layer designed for outer retinal reconstruction. Our approach may also be useful in the production of a multitude of micro- and nanoscale structures for multilayered cell delivery in other tissues.
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