Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa
Summary
In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.
Abstract
Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.
Introduction
Human retinal diseases, such as retinitis pigmentosa and age-related macular degeneration, are poorly understood due to the lack of appropriate experimental models1,2. Although the mouse retina is very similar to the human retina and is a powerful tool for studying the etiology of retinal degeneration, there are huge species differences between mice and humans3,4. For instance, the nuclear architecture of the photoreceptor cells in mice and humans is different, and the mouse retina does not possess a macula5,6. Induced pluripotent stem cell (iPSC) technology enables us to return the specialized cells of organisms to the initial pluripotent state through the "reprogramming" processes by combinations of transcription factors and/or compounds7,8,9,10. Those iPSCs have nearly unlimited division and proliferation ability and could develop into various types of cells. Recently, iPSC-derived 3D retinal organoids have been developed to model the early events of human retinal development and to delineate the pathophysiology of human retinal diseases11,12,13,14,15. Retinal organoids have many advantages: (1) they can be used to recapitulate in vivo retinal development and disease pathogenesis; (2) they can be used for high-throughput drug screening and preclinical trials of gene therapy; and (3) they can be used as preclinical evaluations of treatment options for retinal degenerative diseases16,17.
One objective of this project was to study the pathogenesis of retinal pigmentosa (RP), a disease remaining incurable because of its extreme heterogeneity18. To date, over 90 genes have been identified to be associated with RP19,20. The RPGR gene, which is considered one of the most prevalent causative genes of RP15, accounts for approximately 16% of all RP4,21,22. iPSCs carrying a frameshift mutation in the RPGR gene have been successfully generated and differentiated into organized and stratified 3D retinal organoids14. By utilizing these organoids, abnormal photoreceptor layer morphology and the dislocation of opsins in photoreceptors were observed.
Altogether, a step-by-step and approachable protocol is described in detail here on how to generate patient-derived 3D retinal organoids23,24. Those organoids successfully recapitulated some clinical phenotypes of the disease. This provides an encouraging model to study retinal development and disease mechanisms, for therapeutic screening, and to evaluate future preclinical gene therapy.
Protocol
Representative Results
Discussion
Retinal organoids are 3D, laminated structures derived from hiPSCs or embryonic stem cells (ESCs) and feature as a very promising model to mimic the spatial and temporal patterns of human retinal development31,32. The ROs consist of various types of retinal cells, including photoreceptors, bipolar cells, ganglion cells, amacrine cells, horizontal cells, and Müller glia33. 2D culture cannot precisely mimic the orientation and developme…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank M.S. Yan-ping Li and Zhuo-lin Liu for their technical support and helpful comments regarding the manuscript. This work was partly supported by the National Natural Science Foundation of China (82171470, 31871497, 81970838, Z20J00122), Beijing Municipal Natural Science Foundation (Z200014, 82125007), and National Key R&D Program of China (2017YFA0105300).
Materials
| 96 V-bottomed conical wells | Sumitomo Bakelite | MS-9096VZ | |
| A-83–01 | R&D Systems | 2939/10 | |
| Adhesion microscope slides | CITOtest | 188105 | |
| Agarose | Gene Tech | 111760 | |
| Amaxa Nucleofector 2b Device | Lonza | AAB-1001 | Transfection system |
| B-27 | Thermo Fisher Scientific | 17504044 | |
| bFGF | R&D Systems | 3718-FB | |
| Blebbistatin | Nuwacell Biotechnologies | RP01008 | |
| Blood collection tube | BD Vacutainer EDTA | 366643 | |
| CHIR99021 | TOCRIS | 4423/10 | |
| Cover slides | CITOGLAS | 10212440C | |
| cTarget hPSC Medium | Nuwacell Biotechnologies | RP01020 | |
| DAPI | Invitrogen | D-1306 | |
| DMEM/Ham’s F12 | Gibco | 10565-042 | |
| Donkey anti-mouse 488 | Invitrogen | A-21202 | |
| Donkey anti-rabbit 594 | Invitrogen | A-21207 | |
| EDTA | Nuwacell Biotechnologies | RP01007 | |
| Embedding medium | FluorSaveTM Reagent | 345789 | |
| EX-CYTE growth enhancement medium | Sigma | 811292 | Growth enhancement medium |
| Fetal bovine serum | Gibco | 04-002-1A | |
| Ficoll | Sigma-Aldrich | 26873-85-8 | Density gradient medium |
| FLT3L | Peprotech | 300-19 | |
| GlutaMAX | Life Technologies | 35050-061 | L-glutamine supplement |
| HA-100 | STEMCELL Technologies | 72482 | |
| Ham’s F12 | Gibco | 11765-054 | |
| hLIF | Thermo Fisher Scientific | AF-250-NA | |
| Homogenizer | EDEN lab | D-130 | |
| IL-3 | Peprotech | 213-13 | |
| IL-6 | Peprotech | 200-06 | |
| Iscove’s Modified Dulbecco Medium | Gibco | 12440053 | |
| KnockOut Serum Replacement – Multi-Species | Gibco | A3181502 | Serum replacement media |
| L/M-opsin | Millipore | ab5405 | |
| Monothioglycerol | Sigma | M6145 | |
| N-2 supplement | Thermo Fisher Scientific | 17502048 | |
| Nanodrop Spectrophotometer | Thermo Fisher Scientific | ND2000 | Spectrophotometer |
| ncEpic 125x Supplement | Nuwacell Biotechnologies | RP01001-02 | 125x Supplement |
| ncEpic Basal Medium | Nuwacell Biotechnologies | RP01001-01 | Basal hpsc medium |
| ncLaminin511 human recombinant protein | Nuwacell Biotechnologies | RP01025 | |
| PD0325901 | STEMCELL Technologies | 72182 | |
| Penicillin-streptomycin | Gibco | 15140-122 | |
| Recombinant human BMP4 | R&D Systems | 314-BP | |
| Retinoic acid | Sigma | R2625 | |
| Rhodopsin | Sigma | O4886 | |
| RNeasy Mini Kit | Qiagen | 74104 | |
| RNeasy Mini Kit | Qiagen | 74104 | |
| sIL6-R | Thermo Fisher Scientific | RP-75602 | |
| StemSpan SFEM medium | STEMCELL Technologies | 09600 | |
| Taurine | Sigma | T8691 | |
| Trizol reagent | Invitrogen | 15596026 | |
| Vitronectin | Nuwacell Biotechnologies | RP01002 | |
| V-Lance knife | Alcon Surgical | 8065912001 |
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