Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Abstract

Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.

Migration, Integration and Maturation of Photoreceptor Precursors Following Transplantation in the Mouse Retina

“…Rod a-transducin is one of the last components of the phototransduction pathway to be expressed in development and is later than a number of proteins involved in outer segment formation, such as peripherin-2 [59]. Further studies at the ultrastructural level, similar to those performed by Ader and colleagues at 3 weeks post-transplantation [5], would be required to confirm the time-course of outer segment elaboration. Nonetheless, the onset of visual function, as assessed by optokinetic head tracking, correlates very well with the expression of rod atransducin in the integrated donor cells.…”

“…By transplanting cells isolated from the developing retina at a stage when rod photoreceptors are immature, we have shown that the non-neurogenic, adult retinal environment is able to incorporate and maintain new photoreceptors for many months [1,2]. These new photoreceptors form synaptic connections with inner retinal neurons, generate outer segments, and are capable of restoring rod-mediated vision in models of retinal degeneration [3][4][5][6]. Importantly, we were able to show that these results are only achieved using postmitotic photoreceptor precursor cells but not progenitor cells or photoreceptors at other stages of development [1,3,7,8], a finding supported by others [9,10].…”

Cell therapy using retinal progenitor cells shows therapeutic effect in a chemically-induced rotenone mouse model of Leber hereditary optic neuropathy

“…[12][13][14]18,19 We investigated whether newly-specified RGCs could be used intravitreally to treat optic neuropathies, using a rotenone-induced model of LHON. Retinas were dissociated from embryonic day 16 and 17 (E16 and E17) and postnatal Day2 (pn2) eGFP mice; within the timeframe for RGC specification.…”

hESC-derived photoreceptors survive and integrate better in immunodeficient retina

“…Fetal retinal sheet transplants have resulted in long-term visual improvements in different animal models of retinal degeneration (4,(6)(7)(8) and in patients (9), and have shown to integrate and synaptically connect with a degenerated retina (6,8). (II) The second approach is injection of dissociated photoreceptor precursors (10)(11)(12)(13)(14)(15). Studies have shown that a small percentage of subretinally injected photoreceptor precursor cells can integrate in the photoreceptors layer, form synaptic terminals (10,15) and outer segments (14,15).…”

“…(II) The second approach is injection of dissociated photoreceptor precursors (10)(11)(12)(13)(14)(15). Studies have shown that a small percentage of subretinally injected photoreceptor precursor cells can integrate in the photoreceptors layer, form synaptic terminals (10,15) and outer segments (14,15). However, this requires the presence of an outer nuclear layer in the host retina; transplanted photoreceptor progenitors do not develop proper morphology in recipients with severe loss of photoreceptors (10,11,16).…”