Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.
Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.
Millions of people around the world are impacted by retinal degeneration (RD) with resulting visual impairment or blindness associated with loss of the light-sensing photoreceptors (PRs). Age-related macular degeneration (AMD) is a major cause of blindness resulting from a combination of genetic risk factors and environmental/lifestyle factors. In addition, over 200 genes and loci have been found to cause inherited RD (IRD)1. Retinitis pigmentosa (RP), the commonest IRD, is genetically heterogenous with more than 3,000 genetic mutations in approximately 70 genes being reported2,3,4. Leber Congenital Amaurosis (LCA), which causes blindness in childhood is also genetically heterogenous5,6. Gene augmentation therapy has been developed and is in clinical trials for treating a small number of IRDs3,7. However, a separate therapy must be developed for the treatment of each distinct genetic form of IRD and thereby only treating a small subset of patients. Furthermore, gene augmentation relies on the presence of a population of rescuable photoreceptors and is, therefore, not applicable for advanced degeneration.
There is, therefore, an urgent and yet unmet clinical need for the development of therapies addressing and treating advanced RDs and profound to terminal blindness. Over the last 2 decades neuroprosthetic implants have been developed and tested in large animal models, such as the cat, prior to human use8,9,10,11,12,13,14. Likewise, in the past 20 years retinal replacement therapies utilizing sheets of embryonic or even mature mammalian retina grafted subretinally have been developed15,16,17,18,19,20,21,22 and even tested successfully in RD patients23,24,25. Both approaches utilize the idea of introducing new sensors (photovoltaic silicon photodiodes in the case of neuroprosthetic devices26,27, and healthy mutation-free photoreceptors organized in sheets, in the case of retinal sheet implantation) into retina with degenerated PRs. Recent studies have investigated the use of stem cells-based approaches such as transplantation of human pluripotent stem cell (hPSC)-derived retinal progenitors28,29, hPSC-photoreceptors30, and hPSC-retinal organoids31,32,33. Retinal organoids enable the formation of retinal tissue in a dish and derivation of photoreceptor sheets with thousands of mutation-free PRs, which resemble the photoreceptor layer in the developing human fetal retina34,35,36,37,38,39,40. Transplanting hPSC-derived retinal tissue (organoids) into the subretinal space of patients with RD conditions is one of the new and promising investigational cell therapy approaches, being pursued by a number of teams31,32,41,42. Compared to transplantation of the cell suspension (of young photoreceptors or retinal progenitors), transplanted sheets of fetal photoreceptors were demonstrated to result in vision improvements in clinical trials23,24.
The protocol presented here describes, in detail, a transplantation procedure for subretinal delivery of the whole retinal organoids (rather than organoid rims33,41) as a potentially better way to introduce intact retinal sheets with PRs, to increase graft survival and improve the sheet preservation. Though procedures for introducing a flat piece of human retina and also RPE patches have been developed43,44,45, transplantation of larger 3D grafts has not been investigated. Stem cell-derived retinal organoids provide an inexhaustible source of photoreceptor sheets for developing vision restoration technologies, are free of ethical restriction, and are considered an excellent source of human retinal tissue for therapies focused on treating advanced RD and terminal blindness46. Development of surgical methods for precise subretinal implantation of retinal organoids with minimal injury to the host retinal niche (neural retina, retinal pigment epithelium and retinal and choroidal vasculature) is one of the critical steps for advancing such therapy toward clinical applications31,32. Large animal models such as cats, dogs, pigs, and monkeys have proven to be good models for investigating surgical delivery methods as well as to demonstrate the safety of implanted sheets of tissue (retinal pigment epithelium (RPE) cells) and investigate the use of organoids41,44,45,47,48,49,50. The large animal eye has a similar globe size to human as well as similar anatomy including the presence of a region of high photoreceptor density, including cones (the area centralis), resembling the human macula6,51,52.
In this manuscript, a technique for the implantation of hPSC-derived retinal tissue (organoids) into the subretinal space of feline large animal models (both wild-type and CrxRdy/+ cats) is described, which, together with promising efficacy results32,53 builds a foundation for further development of such investigational therapy toward clinical applications to treat RD conditions.
Procedures were conducted in compliance with the Association for Research in Vision and Ophthalmology (ARVO) statement for Use of Animals in Ophthalmic and Vision Research. They were also approved by the Michigan State University Institutional Animal Care and Use Committee. Wild-type and CrxRdy/+ cats from a colony of cats maintained at Michigan State University were used in this study. Animals were housed under 12 h : 12 h light-dark cycles and fed a commercial complete cat diet.
1. Pre-implantation procedures and surgical set-up
2. Preparation of the organoids for subretinal implantation (Figure 1)
3. Subretinal organoids implantation
4. Post-implantation procedures, post-operative treatment, and assessment
This procedure enables the successful and reproducible implantation of hPSC-derived retinal organoids in the subretinal space of a large eye animal model (demonstrated here using 2 examples: wild-type cats with healthy photoreceptors (PRs) and CrxRdy/+ cats with degenerating PRs and retina). Using the steps indicated in Figure 1 prepare and load the hPSC-derived retinal organoids into the borosilicate glass cannula of the injection device so that the organoids are not damaged. This can be confirmed by direct visualization during the loading of the organoids (step 2.10) and during the surgery (step 3.16) (Figure 2A,B) as well as by fundus imaging at the end of surgery (step 3.23, Figure 2C). The presence of the organoids in the subretinal space using this technique is confirmed post-operatively by ophthalmic examination and fundus imaging (Figure 3A), which records the position and appearance of the organoids. Knowing the position of the transplant is very important when processing the globes for frozen histology and immunohistochemistry and substantially reduces workload, as sectioning a large eye at 12–14 µm (thickness of a cryosection) takes time. Prior to euthanasia, confocal scanning laser ophthalmoscopy (cSLO) and spectral domain – optical coherence tomography (SD-OCT) imaging are also performed to assess the position of the organoids in the subretinal space (Figure 3A-D). These techniques demonstrate the persistence of retinal organoids in the subretinal space (between the neural retina and RPE) of the recipient eye (Figure 3E). Following euthanasia (done humanely following AVMA recommendations) the histology and immunohistochemistry (IHC) is routinely performed (see details in previously published paper31). The histology and IHC demonstrate the survival of xenogeneic grafts (hPSC-derived retinal organoids) in the subretinal space of a large eye when the animals were immunosuppressed (as previously described31), see Figure 4.
Figure 1: Schematic of the steps for organoids preparation prior to the implantation.
Please click here to view a larger version of this figure.
Figure 2: Surgical subretinal implantations of organoids. (A) Direct visualization of organoids being delivered into the subretinal space through a glass cannula without being damaged, (B) Direct visualization of organoids in the subretinal bleb, (C) Wide-angle fundus color image of the subretinally implanted organoids immediately after surgery. The bleb edges are indicated by the black arrowheads and the retinotomy site by the black stars.
Please click here to view a larger version of this figure.
Figure 3: Post-operative assessment of subretinally implanted organoids 3 months post-implantation in a CrxRdy/+ cat. (A) Fundus color image of the subretinally implanted organoids, (B) cSLO fundus image of the subretinally implanted organoids, (C) 3D volume scan reconstruction of the area containing the organoids, (D) cSLO image of the area containing subretinal organoids, (E) SD-OCT high-resolution, cross-section image of the subretinally implanted organoids. The retinotomy site is indicated by the black stars.
Please click here to view a larger version of this figure.
Figure 4: Human retinal organoid-derived photoreceptor sheets (PR marker RCVRN) in subretinal space of CrxRdy/+ cat, 3 months after grafting. *Synaptic boutons (hSYP=Synaptophysin) in cat inner nuclear layer (INL).
Please click here to view a larger version of this figure.
Implantation of hPSC-derived retinal tissue (retinal organoids) into the subretinal space is a promising experimental approach for restoring vision for late-stage retinal degenerative diseases caused by PR cell death (profound or terminal blindness). The presented approach builds on an earlier developed and successfully tested experimental therapy based on subretinal grafting of a piece of human fetal retinal tissue23,24,25. It presents the use of an alternative, replenishable and ethically acceptable retinal tissue source derived from hPSCs. Demonstrating surgical feasibility and ocular safety of therapy in large eye animal models51,55 is needed for advancement of this promising approach toward clinical applications. This manuscript provides a detailed method for subretinal implantation of hPSC-3D retinal tissue (retinal organoids) in a large animal model with both normal and degenerating retina (model of CrxRdy/+ cats model of LCA). Though procedures for introducing a flat piece of human retina and also RPE patches have been developed43,44,45, transplantation of larger 3D grafts (needed to restore vision in conditions with advanced RD) have not been investigated. The protocol described here in detail is for a transplantation procedure for subretinal delivery of the whole retinal organoids (rather than organoid rims33,41) also carrying some RPE as a potentially better way to introducing intact retinal sheets with PRs, to increase graft survival and improve the sheet preservation. Note that different portions of this protocol are well established. For example, vitrectomy is widely used by vitreoretinal surgeons during retinal reattachment surgery56,57,58,59,60. Subretinal injections are becoming more commonly used, for example, in gene augmentation therapy3,7,61,62,63,64. There are limited descriptions of creation of an adequate retinotomy and the injection of relatively large organoids into the subretinal space.
The critical steps include careful positioning and performance of the sclerotomy to avoid the lens, complete removal of vitreous cortex from the retinal surface over the transplant site, controlled formation of the subretinal bleb, generating retinotomy of the optimal size to accommodate the width of the transplantation cannula, maintaining the defined infusion pressure during different steps, and cannula withdrawal. Choosing the right transplantation cannula with optimized inner and outer diameter (ID and OD) and length, controlling intraocular bleeding, sterility of the whole procedure from organoids to surgical room and instruments, and the duration of surgery (30–45 min/animal) to ensure optimal results. The authors find that the best results are obtained with 23 G vitrectomy due to the thickness/viscosity of the cat vitreous, creating a bleb with an injector with a 41 G cannula, and then, extending the retinotomy at the edge of the bleb using 80° angle retinal scissors. Other important factors include extending the sclerotomy using a 2.85 mm keratome to fit the borosilicate glass cannula (outer diameter OD 1.52 mm; inner diameter, ID 1.12 mm; and length 10.16 cm) and permitting implantation of larger organoids in a large eye with axial length about 20.5 mm (20.91 mm ± 0.53 mm)55. The use of a glass capillary was the current best available for loading and delivering descent size organoids without damaging them during the implantation process. It was found that reducing the infusion pressure from 20–30 mmHg during the vitrectomy to 10 mmHg during the bleb formation step is optimal for generating retinal detachments, needed for creating space for retinal organoids. In addition, viability of hPSC-3D retinal tissue (organoids) during long-distance shipment and choosing the optimized immunosuppression regimen for maintenance of xenogeneic human grafts are critical as recently reported31,54.
The authors found that due to the highly reflective cat tapetum endo-illumination was not required to perform the surgery. Transplantation in an atapetal species (e.g., pig, nonhuman primate) would require a 3-port vitrectomy that includes endo-illumination. Tamponade (e.g., with perfluorocarbon followed by silicone oil) as performed in routine retinal detachment surgery was not performed because pressure on the bleb would risk extruding the organoids into the vitreous. Future developments could include the use of materials currently being investigated for sealing retinal holes. This could be used to prevent any post-implantation loss of organoids into the vitreous. Additionally, Triescence, which is similar to Kenalog-40 but contains preservative-free triamcinolone, could be used as an alternative to visualize the vitreous.
The smaller globe size in younger animals (e.g., 1–2 months) presented more surgical challenges compared to the large eye (adult animals). Nevertheless, using the method described here, it is possible to deliver the subretinal grafts. In the CrxRdy/+ LCA model, larger retinal blebs were found to not always reattach. Keeping the bleb to the minimum size needed to allow the retinal organoids to be injected, reduced this complication. Transplanting sooner in RD retina (before the complete loss of PRs when neural retina becomes markedly thinned) is another guideline, in line with the earlier work with human fetal retina grafts20. Another note – the detailed technique does not depend on placing the retinal sheet in correct orientation because the whole retinal organoids are transplanted. With development of flat hESC-retinal sheets, this will be important. However, it is not the focus of this protocol, which is aimed at delivering intact hESC-retinal tissue sheets and optimizing the subretinal preservation of PR sheets. Once the technique was developed, it was reproducible in both normal and RD retina.
There are many potential complications to this surgical procedure. Only those skilled in vitreoretinal surgery (e.g., trained veterinary ophthalmologists or vitreoretinal surgeons familiar with the species differences in the cat eye compared to human eye) should undertake the procedure. Possible complications include lens touch during trocar placement, scleral bleeding during sclerotomy enlargement, and subretinal or retinal hemorrhages. Other complications such as the host immune response to xenogeneic human organoid grafts leading to destruction of the graft over time or endophthalmitis are possible; the use of oral immunosuppressant and antibiotic medications help prevent these from occurring. It is also important to note there is a difference between implantation in wildtype and CrxRdy/+ cats. When there is advanced retinal degeneration, the retinal bleb tends to spread wider than in the wildtype, and, in some instances, this can prevent complete retinal reattachment after the surgery. The technique presented here is applicable for implantation of organoids in eyes of large animal models of IRDs. Further refinement would be needed once the transplantation is ready for translation to the clinic.
Based on the authors’ experience with performing complex vitreoretinal surgical procedures in large eye models, the technique presented in this manuscript should be applicable to other large animal models (with the inclusion of endo-illumination in atapetal species) that are used for translating vitreoretinal surgical techniques to the clinic43,44,45.
The authors have nothing to disclose.
This work was funded by NEI Fast-track SBIR grant R44-EY027654-01A1 and SBIR grant 3 R44 EY 027654 – 02 S1 (I.O.N., Lineage Cell Therapeutics; Dr. Petersen-Jones is a co-PI). The authors would like to thank Ms. Janice Querubin (MSU RATTS) for her help with anesthesia and general care for the animals included in this study as well as help with surgical setting and instruments preparation/sterilization. The authors would like to thank Dr. Paige Winkler for the help in receiving the organoids and placing them in media on the day prior to the implantation and for the help on the day of the implantation. The authors are also grateful to Mr. Randy Garchar (LCTX) for diligent shipping of retinal organoids, assembling the shipper, and downloading temperature and G-stress-records after each shipment. This work was performed while author Igor Nasonkin was employed by Biotime (now Lineage).
0.22 µm pore syringe filter with PES membrane | Cameo | NA | can be found by various suppliers |
23G subretinal injector with extendable 41 G cannula | DORC | 1270.EXT | |
250 µL hamilton gas tight luer lock syringe | Hamilton | NA | can be found by various suppliers |
6-0 Silk suture | Ethicon | 707G | |
6-0/7-0 polyglactin suture | Ethicon | J570G | |
Acepromazine maleate 500mg/5mL (Aceproject) | Henry Schein Animal Health | NA | can be found by various suppliers |
Buprenorphine 0.3 mg/mL | Par Pharmaceutical | NA | can be found by various suppliers |
cSLO + SD-OCT | Heidelberg Engineering | Spectralis HRA+ OCT | |
Cyclosporine | Novartis | NA | can be found by various suppliers |
Dexamethasone 2mg/mL (Azium) | Vetone | NA | can be found by various suppliers |
Doxycyline 25mg/5mL | Cipla | NA | can be found by various suppliers |
Fatal Plus solution (pentobarnital solution) | Vortech | NA | can be found by various suppliers |
Gentamicin 20mg/2mL | Hospira | NA | can be found by various suppliers |
Glass capillary (Thin-Wall Single-Barrel Standard Borosilicate (Schott Duran) Glass Tubing | World Precision Instruments | TW150-4 | |
Methylprednisolone actetate 40 mg/mL | Pfizer | NA | can be found by various suppliers |
Microscope | Zeiss | NA | |
OCT medium (Tissue-Tek O.C.T. Compound) | Sakura | 4583 | |
Olympic Vac-Pac Size 23 | Natus | NA | can be found by various suppliers |
Paraformaldehyde 16% solution | EMS | 15719 | |
Phenylephrine Hydrochloride 10% Ophthalmic Solution | Akorn | NA | can be found by various suppliers |
Prednisolone 15mg/5mL | Akorn | NA | can be found by various suppliers |
Propofol 5000mg/50mL (10 mg/mL) (PropoFlo28) | Zoetis | NA | can be found by various suppliers |
RetCam II video fundus camera | Clarity Medical Systems | NA | can be found by various suppliers |
Triamcinolone 400mg/10 mL (Kenalog-40) | Bristol -Myers Squibb Company | NA | can be found by various suppliers |
Tropicamide 1% ophthalmic solution | Akorn | NA | can be found by various suppliers |
Vitrectomy 23G port | Alcon | Accurus systems | |
Vitrectomy machine | Alcon | Accurus systems | |
Vitreo-retinal vertical 80° scissors with squeeze handle | Frimen | FT170206T |