A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
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.
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.
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....
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
Differentiation of hiPSCs into eye lineages is achieved by culturing the cells in different cocktails of culture medium containing supplements and growth factors in sequential steps at different time points, as described in Figure 1. The hiPSC cultures are maintained in Essential 8 medium, the pluripotent stem cell maintenance medium. Once they reach 70%-80% confluency (Figure 2A), the medium is replaced with Differentiation Induction Medium (DIM) on day 0 (refe.......
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.......
The authors acknowledge the scientific and technical support from Dr. Chitra Kannabiran, Geneticist; Dr. Subhadra Jalali, Retinal Consultant; Dr. Milind Naik, Oculoplastic Surgeon; and Dr. Swathi Kaliki, Ocular Oncologist at the LV Prasad Eye Institute, Hyderabad toward the generation of normal and patient-specific iPSC lines. The authors acknowledge the R&D grants from the Science and Engineering Research Board, Department of Science and Technology (IM), (SB/SO/HS/177/2013), Department of Biotechnology (IM), (BT/PR32404/MED/30/2136/2019) ,and Senior Research Fellowships from ICMR (S.M., D.P.), UGC (T.A.), and CSIR (V.K.P.), Government of India.
....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 DAPIÂ | Vector laboratories | H-1200Â | |
Vitronectin | Thermo Fisher | A27940 | |
Y-27632 dihydrochloride (Rho-kinase inhibitor) | Sigma Aldrich | Y0503 | |
Zeiss LSM 880 | Zeiss | Confocal microscope |
Explore More Articles
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved