Imaging Transplanted Photoreceptors in Living Nonhuman Primates with Single-Cell Resolution

Abstract

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.

cGMP-grade human iPSC-derived retinal photoreceptor precursor cells rescue cone photoreceptor damage in non-human primates

“…This is in contrast to previous work where cGMP-iPSC-derived photoreceptor precursors transplanted into immune-deficient murine models predominantly differentiated into rod photoreceptors [ 15 ]. In other NHP studies whereby retinal cell sheets and photoreceptor precursors were transplanted, the transplanted cells differentiated into cone photoreceptors first, with only one study reporting differentiation into rod photoreceptors only after an additional 28 days after the detection of cone photoreceptors [ 21 , 50 ]. Thus, it is possible that with longer follow-up times post-transplantation, we might be able to observe transplanted cells differentiating into rod photoreceptors.…”

“…Limitations of the cat model include the presence of the reflective tapetum lucidum, [ 48 ], while porcine eyes lack a fovea and are not optimal for pre-clinical studies due to inconsistencies in disease development patterns [ 48 , 49 ]. In contrast, the ocular anatomy of NHPs resemble humans the most, making them most suitable for the evaluation of retinal cell-based therapies [ 44 , 50 – 53 ]. To date there has been only one other study of photoreceptor precursor transplants in NHPs.…”

Stem Cell Therapy for Retinal Degeneration: The Evidence to Date

“…11 In a study involving non-human primates, subretinal transplantation of hESC-derived retinal organoids was well tolerated and the transplanted cells integrated into the retinal layer in the injury site created by laser ablation. 12 iPSCs iPSCs, similar to ESCs, have pluripotent differentiation ability but without ethical concerns. The human iPSC-derived retina was transplanted into the subretinal space of monkeys with laser-induced retinal injury and immune-deficient rats with RP.…”

“… 11 In a study involving non-human primates, subretinal transplantation of hESC-derived retinal organoids was well tolerated and the transplanted cells integrated into the retinal layer in the injury site created by laser ablation. 12 …”

Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy

“…Yet, significant untapped potential remains for the future use of gene delivery to retinal organoids in disease modeling and therapy (Dalkara et al, 2016 ). Over the last 5 years, efforts have been directed to the transplantation of photoreceptors derived from 3D retinal organoids (Gonzalez-Cordero et al, 2017 ; Gagliardi et al, 2018 ; Lakowski et al, 2018 ; Collin et al, 2019 ; Aboualizadeh et al, 2020 ) resulting in different levels of success but the formation of light-sensitive outer segments (Mandai et al, 2017 ; Iraha et al, 2018 ) and interaction with retinal pigment epithelium (RPE), which is necessary for the appropriate functioning of photoreceptor cells (Strauss, 2005 ) remain challenges for cell replacement with therapeutic outcomes. Microbial opsins can circumvent these issues as we have recently proposed (Garita-Hernandez et al, 2019 ).…”