Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Summary

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Abstract

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6⁺ and ChX10⁺ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX⁺ and RCVRN⁺ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

Introduction

The retina is a light-sensitive tissue present at the back of the vertebrate eye that converts light signals into nerve impulses by a biochemical phenomenon known as the photo-transduction pathway. The initial nerve impulses generated in the photoreceptor cells of the retina get transduced to other retinal interneurons and retinal ganglion cells (RGCs) and reach the visual cortex of the brain, which helps in image perception and visual response.

According to the World Health Organization (WHO), an estimated 1.5 million children are blind, of which 1 million are in Asia. Inherited Retinal Dystrophy (IRD) is a major blinding disease that affe....

Protocol

All experiments involving hiPSCs were carried out aseptically, in adherence to the standard laboratory practices, ethical and biosafety guidelines, and with the approvals of regulatory bodies such as the Institutional Ethics Committee (IEC), Institutional Committee for Stem Cell Research (IC-SCR), and Institutional Bio-Safety Committee (IBSC).

1. Preparation of iPSC culture and retinal differentiation medium and reagents

1. iPSC culture and maintenance medium
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Discussion

hiPSCs are a powerful tool to study organ and tissue development in vitro. Recapitulating the disease phenotype by differentiating healthy versus disease-specific hiPSCs toward the retinal lineage can help in gaining newer insights into the pathophysiology of different forms of inherited retinal dystrophies. Several protocols have been described and adopted for the in vitro differentiation of PSCs into retinal cell types. Most of them involve the use of culture medium containing complex cocktails of rec.......

Materials

Name	Company	Catalog Number	Comments
0.22 µm Syringe filters	TPP	99722
15 mL centrifuge tube	TPP	91015
50 mL centrifuge tube	TPP	91050
6 well plates	TPP	92006
Anti-Chx10 Antibody; Mouse monoclonal	Santa Cruz	SC365519	1:50 dilution
Anti-CRX antibody; Rabbit monoclonal	Abcam	ab140603	1:300 dilution
Anti-MiTF antibody, Mouse monoclonal	Abcam	ab3201	1:250 dilution
Anti-Recoverin Antibody; Rabbit polyclonal	Millipore	AB5585	1:300 dilution
B-27 Supplement (50x), serum free	Thermo Fisher	17504044
Basic Fibroblast growth factor (bFGF)	Sigma Aldrich	F0291
Centrifuge 5810R	Eppendorf
Coplin Jar (50 mL)	Tarson
Corning Matrigel hESC-Qualified Matrix	Corning	354277
CryoTubes	Thermo Fisher	V7884
DMEM/F-12, GlutaMAX supplement (basal medium)	Thermo Fisher	10565-018
DreamTaq DNA polymerase	Thermo Fisher	EP0709
Dulbeco’s Phosphate Buffered Saline	Thermo Fisher	14190144
Essential 8 medium kit	Thermo Fisher	A1517001
Ethylene diamine tetraaceticacid disodium salt dihydrate (EDTA)	Sigma Aldrich	E5134
Falcon Not TC-treated Treated Petri Dish, 60 mm	Corning	351007
Fetal Bovine Serum, qualified, United States	Gibco	26140079
GelDocXR+ with Image lab software	BIO-RAD		Agarose Gel documentation system
GlutaMAX Supplement	Thermo Fisher	35050061
Goat anti-Mouse IgG (H+L), Alexa Fluor 488	Invitrogen	A11001	1:300 dilution
Goat anti-Mouse IgG (H+L), Alexa Fluor 546	Invitrogen	A11030	1:300 dilution
Goat anti-Rabbit IgG (H+L), Alexa Fluo 546	Invitrogen	A11035	1:300 dilution
Goat anti-Rabbit- IgG (H+L), Alexa Fluor 488	Invitrogen	A11008	1:300 dilution
HistoCore MULTICUT	Leica		For sectioning
KnockOut Serum Replacement	Thermo Fisher	10828028
L-Acsorbic acid	Sigma Aldrich	A92902
MEM Non-Essential Amino Acids Solution (100x)	Thermo Fisher	11140-050
N2 supplement (100x)	Thermo Fisher	17502048
NanoDrop 2000	Thermo Fisher		To quantify RNA
Paraformaldehyde	Qualigens	23995
Pasteur Pipets, 9 inch, Non-Sterile, Unplugged	Corning	7095D-9
Penicillin-Streptomycin	Thermo Fisher	15140-122
Recombinant Anti-Otx2 antibody , Rabbit monoclonal	Abcam	ab183951	1:300 dilution
Recombinant Anti-PAX6 antibody; Rabbit Monoclonal	Abcam	ab195045	1:300 dilution
Recombinant Anti-RPE65 antibody, Rabbit Monoclonal	Abcam	ab231782	1:300 dilution
Recombinant Human Noggin Protein	R&D Systems	6057-NG
SeaKem LE Agarose	Lonza	50004
Serological pipettes 10 mL	TPP	94010
Serological pipettes 5 mL	TPP	94005
Sodium Chloride	Sigma Aldrich	S7653
Sodium Citrate Tribasic dihydrate	Sigma Aldrich	S4641
Starfrost (silane coated) microscopic slides	Knittel
SuperScript III First-Strand Synthesis System	Thermo Fisher	18080051
SuperScript III First-Strand Synthesis System for RT-PCR	Invitrogen	18080051
Triton X-100	Sigma Aldrich	T8787
TRIzol Reagent	Invitrogen	15596026
UltraPure 0.5 M EDTA, pH 8.0	Thermo Fisher	15575020
VECTASHIELD Antifade Mounting Medium with DAPI	Vector laboratories	H-1200
Vitronectin	Thermo Fisher	A27940
Y-27632 dihydrochloride (Rho-kinase inhibitor)	Sigma Aldrich	Y0503
Zeiss LSM 880	Zeiss		Confocal microscope