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 models^1,2. 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 humans^3,4. For instance, the nuclear architecture of the photoreceptor cells in mice and humans is different, and the mouse retina does not possess a macula^5,

Protocol

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.......

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 development^31,32. 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.......

Materials

Name	Company	Catalog Number	Comments
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