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.
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Method Article
Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model
* These authors contributed equally
In This Article
Summary
Abstract
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.
Introduction
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,<....
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Protocol
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
- Select wild-type or CrxRdy/+ cats dependi....
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Results
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 ar.......
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Discussion
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 .......
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Disclosures
Ratnesh K. Singh, Ph.D., Francois Binette, Ph.D., and Igor O. Nasonkin Ph.D. are employees of Lineage Cell Therapeutics, Inc. The authors declare no conflict of interest.
Acknowledgements
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 grate....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.22 µm pore syringe filter with PES membrane | Cameo | NA | can be found through 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 through 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 through various suppliers |
| Buprenorphine 0.3 mg/mL | Par Pharmaceutical | NA | can be found through various suppliers |
| cSLO + SD-OCT | Heidelberg Engineering | Spectralis HRA+ OCT | |
| Cyclosporine | Novartis | NA | can be found through various suppliers |
| Dexamethasone 2mg/mL (Azium) | Vetone | NA | can be found through various suppliers |
| Doxycyline 25mg/5mL | Cipla | NA | can be found through various suppliers |
| Fatal Plus solution (pentobarbital solution) | Vortech | NA | can be found through various suppliers |
| Gentamicin 20mg/2mL | Hospira | NA | can be found through 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 through 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 through various suppliers |
| Paraformaldehyde 16% solution | EMS | 15719 | |
| Phenylephrine Hydrochloride 10% Ophthalmic Solution | Akorn | NA | can be found through various suppliers |
| Prednisolone 15mg/5mL | Akorn | NA | can be found through various suppliers |
| Propofol 500mg/50mL (10 mg/mL) (PropoFlo28) | Zoetis | NA | can be found through various suppliers |
| RetCam II video fundus camera | Clarity Medical Systems | NA | can be found through various suppliers |
| Triamcinolone 400mg/10 mL (Kenalog-40) | Bristol -Myers Squibb Company | NA | can be found through various suppliers |
| Tropicamide 1% ophthalmic solution | Akorn | NA | can be found through various suppliers |
| Vitrectomy 23G port | Alcon | Accurus systems | |
| Vitrectomy machine | Alcon | Accurus systems | |
| Vitreo-retinal vertical 80° scissors with squeeze handle | Frimen | FT170206T |
References
- Veleri, S., et al. Biology and therapy of inherited retinal degenerative disease: insights from mouse models. Disease Models and Mechanisms. 8 (2), 109-129 (2015).
- Dias, M. F., et al.
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