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Representative Results





Developmental Biology

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Published: June 16th, 2022



1Beijing Tongren Hospital, Beijing Institute of Ophthalmology, Beijing Ophthalmology & Visual Science Key Laboratory, Beijing Tongren Eye Center, Capital Medical University
* These authors contributed equally

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.

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.

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,

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The protocol follows the guidelines of Capital Medical University's human research ethics committee.

1. Cell culture and generation of iPSCs

  1. Choose RPGR patients for this study. Here, three patients, one familial carrier and three healthy controls, were used. Patient 1 possessed a mutation c.1685_1686delAT in exon 14 of the RPGR gene, patient 2 harbored a mutation c.2234_2235delGA in exon 15 of the RPGR gene, and patient 3 had a mutation c.2403_2404delAG in exo.......

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The schematic illustration describes the differentiation procedures to generate healthy and patient iPSC-derived retinal organoids (Figure 1). From iPSC to ROs, variations can be produced owing to several factors. The status of the iPSC is the determinant step of the RO generation. In addition, it is highly recommended that researchers should record every step, catalog, and lot number of all media so that the entire experiments are trackable. In Figure 2A, the i.......

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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.......

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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).


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NameCompanyCatalog NumberComments
96 V-bottomed conical wellsSumitomo BakeliteMS-9096VZ
A-83–01R&D Systems2939/10
Adhesion microscope slidesCITOtest188105
AgaroseGene Tech111760
Amaxa Nucleofector 2b DeviceLonzaAAB-1001Transfection system
B-27Thermo Fisher Scientific17504044
bFGFR&D Systems3718-FB
BlebbistatinNuwacell BiotechnologiesRP01008
Blood collection tubeBD Vacutainer EDTA366643
Cover slidesCITOGLAS10212440C
cTarget hPSC MediumNuwacell BiotechnologiesRP01020
DMEM/Ham’s F12Gibco10565-042
Donkey anti-mouse 488InvitrogenA-21202
Donkey anti-rabbit 594InvitrogenA-21207
EDTANuwacell BiotechnologiesRP01007
Embedding mediumFluorSaveTM Reagent345789
EX-CYTE growth enhancement mediumSigma811292Growth enhancement medium
Fetal bovine serumGibco04-002-1A
FicollSigma-Aldrich26873-85-8Density gradient medium
GlutaMAXLife Technologies35050-061L-glutamine supplement
HA-100STEMCELL Technologies72482
Ham’s F12Gibco11765-054
hLIFThermo Fisher ScientificAF-250-NA
HomogenizerEDEN labD-130
Iscove’s Modified Dulbecco MediumGibco12440053
KnockOut Serum Replacement - Multi-SpeciesGibcoA3181502Serum replacement media
N-2 supplementThermo Fisher Scientific17502048
Nanodrop SpectrophotometerThermo Fisher ScientificND2000Spectrophotometer
ncEpic 125x SupplementNuwacell BiotechnologiesRP01001-02125x Supplement
ncEpic Basal MediumNuwacell BiotechnologiesRP01001-01Basal hpsc medium
ncLaminin511 human recombinant proteinNuwacell BiotechnologiesRP01025
PD0325901STEMCELL Technologies72182
Recombinant human BMP4R&D Systems314-BP
Retinoic acidSigmaR2625
RNeasy Mini KitQiagen74104
RNeasy Mini KitQiagen74104
sIL6-RThermo Fisher ScientificRP-75602
StemSpan SFEM mediumSTEMCELL Technologies09600
Trizol reagentInvitrogen15596026
VitronectinNuwacell BiotechnologiesRP01002
V-Lance knifeAlcon Surgical8065912001

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