Systemic immunosuppression promotes survival and integration of subretinally implanted human ESC-derived photoreceptor precursors in dogs.

Regenerative therapies aimed at replacing photoreceptors are a promising approach for the treatment of otherwise incurable causes of blindness. However, such therapies still face significant hurdles, including the need to improve subretinal delivery and long-term survival rate of transplanted cells, and promote sufficient integration into the host retina. Here, we successfully delivered in vitro-derived human photoreceptor precursor cells (PRPCs; also known as immature photoreceptors) to the subretinal space of seven normal and three rcd1/PDE6B mutant dogs with advanced inherited retinal degeneration. Notably, while these xenografts were rejected in dogs that were not immunosuppressed, transplants in most dogs receiving systemic immunosuppression survived up to 3-5 months postinjection. Moreover, differentiation of donor PRPCs into photoreceptors with synaptic pedicle-like structures that established contact with second-order neurons was enhanced in rcd1/PDE6B mutant dogs. Together, our findings set the stage for evaluating functional vision restoration following photoreceptor replacement in canine models of inherited retinal degeneration.

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

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.

[Photoreceptor cell transplantation for future treatment of retinitis pigmentosa]. / Nouvelle approche thérapeutique pour les rétinites pigmentaires - La transplantation de photorécepteurs dérivés de cellules souches.

In inherited retinal diseases such retinitis pigmentosa, characterized by progressive loss of light sensitive neurons (photoreceptors), cell therapy is now considered as an attractive strategy. Photoreceptor cell replacement would be valuable for restoring function to retinas in a way that is independent from the cause of the disease. With advances in stem cell biology, considerable strides have been made towards the generation of retinal cells, in particular with the development of 3D culture systems allowing the generation of retinal organoids from pluripotent stem cells. In this review, we present a state-of-the art of preclinical strategies conducted in animal models for photoreceptor replacement from stem cell-derived photoreceptors and we discuss the important obstacles to overcome in the future.

TITLE: Nouvelle approche thérapeutique pour les rétinites pigmentaires - La transplantation de photorécepteurs dérivés de cellules souches. ABSTRACT: Dans les maladies dégénératives de la rétine affectant les photorécepteurs, la transplantation de cellules permettant la restauration de la vision est aujourd'hui envisagée. La dernière décennie a vu des progrès remarquables dans la génération de cellules de rétine à partir de cellules souches pluripotentes humaines avec, en particulier, le développement de systèmes de culture en trois dimensions (3D) permettant la génération d'organoïdes de rétine. Dans cette revue, nous faisons un état des lieux sur les stratégies précliniques menées dans des modèles animaux pour le remplacement des photorécepteurs par des photorécepteurs dérivés de cellules souches et présentons les obstacles importants qui restent à être surmontés.

Pikachurin Is Partially Involved in the Synaptic Connection Between Donor and Host Cells in Late-Stage rd1 Mice Following Conspecific Photoreceptor Transplantation.

Photoreceptor transplantation can rescue the retinal function of late-stage rd1 mice. Many studies have used synaptic markers to suggest that there are synaptic connections after transplantation, but how donor and host cells are connected remains unknown. Many molecules are needed for triad ribbon synapse formation in wild-type mice. Among them, pikachurin is an important extracellular matrix protein that bridges the pre- and postsynaptic components. To investigate the mechanism of the synaptic connection between donor photoreceptor and host retina, we studied the expression of pikachurin in late-stage rd1 mice before and after transplantation. The results showed that the full-length form of pikachurin could still be detected in the degenerated retina. After photoreceptors were transplanted to the subretinal space of rd1 or wild-type mice, pikachurin was detected in the cytoplasm of most donor photoreceptor cells. Pikachurin puncta may represent the cleaved form of the protein and may indicate synapse generation, but it was barely observed in the donor mass of wild-type mice (3.83 ± 3.17 puncta per 100 donor cells). In contrast, pikachurin puncta could be found in the graft of the rd1 mouse retina, but the number was low (21.35 ± 9.48 puncta per 100 donor cells). In addition, 54.12 ± 8.45% of bassoon puncta were paired with pikachurin puncta and 45.5 ± 6.33% were not, indicating that there were fewer pikachurin puncta than bassoon. These results suggest that pikachurin is involved in only a portion of the synaptic connection between the donor photoreceptor and host retina.

Growth Factors as Tools in Photoreceptor Cell Regeneration and Vision Recovery.

Photoreceptor loss is a major cause of blindness around the world. Stem cell therapy offers a new strategy in retina degenerative disease. Retinal progenitors can be derived from embryonic stem cells (ESC) in vitro, but cannot be processed to a mature state. In addition, the adult recipient retina presents a very different environment than the photoreceptor precursor donor. It seems that modulation of the recipient environment by ectopic development regulated growth factors for transplanted cells could generate efficient putative photoreceptors. The purpose of this review article was to investigate the signaling pathway of growth factors including: insulin-like growth factors (IGFs), fibroblast growth factors (FGF), Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Taurin and Retinoic acid (RA) involved in the differentiation of neuroretina cell, like; photoreceptor and retinal progenitor cells. Given the results available in the related literature, the differentiation efficacy of ESCs toward the photoreceptor and retinal neurons and the important role of growth factors in activating signaling pathways such as Akt, Ras/Raf1/ and ERKs also inhibit the ASK1/JNK apoptosis pathway. Manipulating differentiated culture, growth factors can influence photoreceptor transplantation efficiency in retinal degenerative disease.

Treatment with Stem Cells from Human Exfoliated Deciduous Teeth and Their Derived Conditioned Medium Improves Retinal Visual Function and Delays the Degeneration of Photoreceptors.

Retinitis pigmentosa (RP) is a hereditary disease characterized by degeneration and the loss of photoreceptors. Stem cell-based therapy has emerged as a promising strategy for treating RP. Stem cells from exfoliated deciduous teeth (SHEDs), a type of mesenchymal stem cell from human exfoliated deciduous teeth, have the potential to differentiate into photoreceptor-like cells under specific induction in vitro. It has been confirmed that through paracrine secreta, SHEDs exert neurotrophic, angiogenic, immunoregulatory, and antiapoptotic functions in injured tissues. This study was designed to determine whether retinal-differentiated SHEDs and the conditioned medium derived from SHEDs (SHEDs-CM) have therapeutic effects in a mouse model of RP. The results showed that both SHEDs and SHEDs-CM improved electroretinogram responses, ameliorated photoreceptor degeneration, and maintained the structure of the outer segments of photoreceptors. The therapeutic effects were related to antiapoptotic activity of SHEDs and SHEDs-CM. Thus, SHEDs may be a promising stem cell source for treating retinal degeneration.

Organoid-derived C-Kit+/SSEA4- human retinal progenitor cells promote a protective retinal microenvironment during transplantation in rodents.

Stem cell therapy may replace lost photoreceptors and preserve residual photoreceptors during retinal degeneration (RD). Unfortunately, the degenerative microenvironment compromises the fate of grafted cells, demanding supplementary strategies for microenvironment regulation. Donor cells with both proper regeneration capability and intrinsic ability to improve microenvironment are highly desired. Here, we use cell surface markers (C-Kit+/SSEA4-) to effectively eliminate tumorigenic embryonic cells and enrich retinal progenitor cells (RPCs) from human embryonic stem cell (hESC)-derived retinal organoids, which, following subretinal transplantation into RD models of rats and mice, significantly improve vision and preserve the retinal structure. We characterize the pattern of integration and materials transfer following transplantation, which likely contribute to the rescued photoreceptors. Moreover, C-Kit+/SSEA4- cells suppress microglial activation, gliosis and the production of inflammatory mediators, thereby providing a healthier host microenvironment for the grafted cells and delaying RD. Therefore, C-Kit+/SSEA4- cells from hESC-derived retinal organoids are a promising therapeutic cell source.

Mechanisms Underlying the Visual Benefit of Cell Transplantation for the Treatment of Retinal Degenerations.

The transplantation of retinal cells has been studied in animals to establish proof of its potential benefit for the treatment of blinding diseases. Photoreceptor precursors have been grafted in animal models of Mendelian-inherited retinal degenerations, and retinal pigmented epithelial cells have been used to restore visual function in animal models of age-related macular degeneration (AMD) and recently in patients. Cell therapy over corrective gene therapy in inherited retinal degeneration can overcome the genetic heterogeneity by providing one treatment for all genetic forms of the diseases. In AMD, the existence of multiple risk alleles precludes a priori the use of corrective gene therapy. Mechanistically, the experiments of photoreceptor precursor transplantation reveal the importance of cytoplasmic material exchange between the grafted cells and the host cells for functional rescue, an unsuspected mechanism and novel concept. For transplantation of retinal pigmented epithelial cells, the mechanisms behind the therapeutic benefit are only partially understood, and clinical trials are ongoing. The fascinating studies that describe the development of methodologies to produce cells to be grafted and demonstrate the functional benefit for vision are reviewed.

Transplantation of photoreceptor precursor cells into the retina of an adult Drosophila.

Blindness caused by the disconnection between photoreceptor cells and the brain can be cured by restoring this connection through the transplantation of retinal precursor neurons. However, even after transplanting these cells, it is still unclear how to guide the axons over the long distance from the retina to the brain. To establish a method of guiding the axons of transplanted neurons, we used the Drosophila visual system. By testing different conditions, including the dissociation and preincubation length, we have successfully established a method to transplant photoreceptor precursor cells isolated from the developing eye discs of third-instar larvae into the adult retina. Moreover, we overexpressed N-cadherin (CadN) in the transplant, since it is known to be broadly expressed in the optic lobe well after developmental stages, continuing through adult stages. We found that promoting the cell adhesive properties using CadN enhances the axonal length of the grafted photoreceptor neurons and therefore is useful for future transplantation. We tested the overexpression of a CadN::Frazzled chimeric receptor and found that there was no difference in axonal length from our wild-type transplants, suggesting that the intracellular domain of CadN is necessary for axonal elongation. Altogether, using the Drosophila visual system, we have established an excellent platform for exploring the molecules required for proper axon extension of transplanted neuronal cells. Future studies building from this platform will be useful for regenerative therapy of the human nervous system based on transplantation.

Otx2-Genetically Modified Retinal Pigment Epithelial Cells Rescue Photoreceptors after Transplantation.

Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.

Photoreceptor replacement therapy: challenges presented by the diseased recipient retinal environment.

Vision loss caused by the death of photoreceptors is the leading cause of irreversible blindness in the developed world. Rapid advances in stem cell biology and techniques in cell transplantation have made photoreceptor replacement by transplantation a very plausible therapeutic strategy. These advances include the demonstration of restoration of vision following photoreceptor transplantation and the generation of transplantable populations of donor cells from stem cells. In this review, we present a brief overview of the recent progress in photoreceptor transplantation. We then consider in more detail some of the challenges presented by the degenerating retinal environment that must play host to these transplanted cells, how these may influence transplanted photoreceptor cell integration and survival, and some of the progress in developing strategies to circumnavigate these issues.

Cell transplantation strategies for retinal repair.

Cell transplantation is a novel therapeutic strategy to restore visual responses to the degenerate adult neural retina and represents an exciting area of regenerative neurotherapy. So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. In this review, we discuss the differentiation of photoreceptor cells from both adult and embryonic-derived stem cells and their potential for retinal cell transplantation. We also discuss the strategies used to overcome barriers present in the degenerate neural retina and improve retinal cell integration. Finally, we consider the future translation of retinal cell therapy as a therapeutic strategy to treat retinal degeneration.

The simultaneous treatment of MMP-2 stimulants in retinal transplantation enhances grafted cell migration into the host retina.

The success of functional retinal cell transplantation has been limited by the low efficiency of the transplanted cell integration into the host retina. Given that the extracellular matrix (ECM) is thought to inhibit entry and axonal outgrowth of grafted neural cells into the host retina, modulation of the ECMs in the host environment may overcome this limitation. Here, we demonstrate that matrix metalloprotease-2 (MMP-2) expression is associated with the high migratory potential of adult rat hippocampus-derived neural stem cells compared with retinal progenitor cells. In addition, MMP-2, as well as its reported inducers concanavalin A and 17beta-estradiol, can trigger the migration of retinal progenitor cells into explanted retinas. Inhibitors of MMP-2 suppressed these effects. Intense cell migration is not required for photoreceptor transplantation; however, the environment that allows the transplanted cells to integrate is most important. Migration of the transplanted cells is a good index of the acceptance of grafted cell of the host tissue. Strategies modulating the environment by MMP-2 stimulation may provide an advance in the development of retinal transplantation.

Photoreceptor function of retinal transplants implicated by light-dark shift of S-antigen and rod transducin.

The aim was to demonstrate functional properties of transplanted histologically normal photoreceptors. Subretinal intact-sheet transplants of fetal E17-E20 rat retinas to light-damaged albino rat eyes were fixed in light or dark, 2 to 42 weeks after transplantation, and stained immunohistochemically for certain phototransduction proteins. In light adapted transplants, transducin was predominantly found in inner segments of parallel-organized photoreceptors. Transducin shifted to the outer segments with dark-adaptation. S-antigen distribution was opposite to transducin. Rhodopsin distribution did not change. The shift of signal transduction proteins correlated to the light conditions indicates that normal phototransduction processes were established in photoreceptors of transplanted retinal sheets.

Preparation and transplantation of photoreceptor sheets.

PURPOSE: Photoreceptor (PR) transplantation may be a treatment for blindness secondary to PR degeneration. We studied different technical aspects of PR-sheet preparation. METHODS: Geographic variation in the thickness of the cat PR layer (from the outer segments to the outer plexiform layer) and inner retina (width of the remainder of the retina) was studied. PR sheets (cat and human) were prepared through gelatin embedding and subsequent vibratoming or excimer laser ablation. Cat PR sheets were evaluated after transplantation. RESULTS: The thickness of the cat PR layer and inner retina varied in different regions. The superior central retina, including the area centralis, was thickest (PR layer: 115-123 microm, entire retina: 225-230 microm, in fixed tissue). The peripheral retina was approximately 40% thinner than the center. Fresh retina was approximately 7.9% thicker than the fixed retina. Both vibratomy and excimer laser ablation removed the inner retina, leaving a PR-layer sheet with good morphology. To produce good quality PR sheets with vibratomy, use of different gelatin concentrations (2% to 35%) at various stages of sheet preparation was crucial. To produce PR sheets of uniform thickness with excimer laser ablation, control of fluid on the retinal surface was critical. Twenty-four hours after PR transplantation surgery, donor PR cells were well oriented and in close contact with host retinal pigment epithelial cells. Gelatin supporting the transplant dissolved as early as 100 min after surgery. CONCLUSIONS: We confirmed and expanded the work of previous investigators and showed that cat and human PR sheets can be manufactured using vibratomy or excimer laser ablation. This preparation provides a well oriented and organized PR cell layer after transplantation.

Human photoreceptor transplantation in retinitis pigmentosa. A safety study.

OBJECTIVE: To establish the technical feasibility and safety of photoreceptor transplantation in retinitis pigmentosa. METHODS: A sheet of human photoreceptor cells was harvested from 2 human cadaveric eyes with a vibratome and transplanted into the subretinal spaces of 2 patients with advanced retinitis pigmentosa and visual acuity of no light perception by means of submacular surgery techniques. Preoperative and postoperative electrophysiologic testing, fundus photography, fluorescein angiography, and scanning laser ophthalmoscopy were performed. RESULTS: Twelve months after photoreceptor transplantation, the visual acuity of each patient remained no light perception. The temporal edge of the retinotomy in 1 patient was folded but was not associated with a retinal detachment. The patients were not immunosuppressed, and there was no evidence of rejection of the allogeneic transplant. Cystoid macular edema, uveitis, and macular pucker were not observed. CONCLUSION: A sheet of adult human photoreceptor cells can be harvested from human cadaveric eyes and safely transplanted to the subretinal spaces of patients with retinitis pigmentosa without systemic immunosuppression.