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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Published: April 12th, 2021

DOI:

10.3791/62435

1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, 510060
* These authors contributed equally

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Retinal degenerative diseases (RDs), such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are characterized by the dysfunction and death of photoreceptor cells and typically lead to irreversible vision loss without effective ways to cure1. The mechanism underlying these diseases is largely unknown partially due to lack of human disease models2. Over the past decades, significant advances have been accomplished in regenerative medicine through stem cell technology. Many researchers, including ourselves, have shown that human pluripotent stem cells (hPSCs), including human embryonic stem cells (hES....

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1. Culture and expansion of hPSCs

  1. HPSC culture
    1. Coat two wells of a 6-well plate with extracellular matrix (ECM, hESC-qualified matrix). Prepare 50 mL of an ECM solution containing 8-12 µg/mL of ECM in Dulbecco's Modified Eagle's Medium (DMEM). In 49 mL of DMEM, add 1 mL of the thawed ECM stock solution (50x). Add 1 mL of the ECM solution to each well of a 6-well plate. Incubate it for 1 h in an incubator at 37 °C and 5% CO2.
    2. Prepare hPS.......

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The retinal induction process in this protocol mimics the development of human fetal retina. To initiate the retinal differentiation, hPSCs were dissociated into small clumps and cultured in suspension to induce the formation of EBs. On D1, the uniformed cell aggregates or EBs formed (Figure 1C). The culture medium was gradually transitioned into NIM. On D5, EBs were plated onto the ECM-coated culture dishes. Cells gradually migrated out of the EBs (Figure 1D). .......

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In this multi-step retinal induction protocol, hPSCs were guided step by step to gain the retinal fate, and self-organized into retinal organoids containing laminated NR and RPE. During the differentiation, hPSCs recapitulated all major steps of human retinal development in vivo, from EF, OV, and RPE, to retinal lamination, generating all subtypes of retinal cells, including retinal ganglion cells, amacrine cells, bipolar cells, rod, and cone photoreceptors, and muller glial cells in a spatial and temporal order.......

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This study was supported by the National Key R&D Program of China (2016YFC1101103, 2017YFA0104101), the Guangzhou Science and Technology Project Fund (201803010078), the Science & Technology Project of Guangdong Province (2017B020230003), the Natural Science Foundation (NSF) of China (81570874, 81970842), Hundred talent program of Sun Yat-sen University (PT1001010), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology.

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Name Company Catalog Number Comments
(−)-Blebbistatin Sigma B0560-5mg ROCK-inhibitor
1 ml tips Kirgen KG1313 1 ml
10 ml pipette Sorfa 3141001 Pipette
100 mm Tissue culture BIOFIL TCD000100 100 mm Petri dish
100 mm Tissue culture Falcon 353003 100 mm Petri dish
15 ml Centrifuge tubes BIOFIL CFT011150 Centrifuge tubes
35 mm Tissue culture dishes Falcon 353001 35 mm Petri dish
5 ml pipette Sorfa 313000 Pipette
50 ml Centrifuge tubes BIOFIL CFT011500 Centrifuge tubes
6 wells tissue culture plates Costar 3516 Culture plates
Anti-AP2α Antibody DSHB 3b5 Primary antibody
ANTIBIOTIC ANTIMYCOTIC 100X Gibco 15240062 Antibiotic-Antimycotic
Anti-ISL1 Antibody Boster BM4446 Primary antibody
Anti-Ki67 Antibody Abcam ab15580 Primary antibody
Anti-L/M opsin Antibody gift from Dr. jeremy / Primary antibody
Anti-PAX6 Antibody DSHB pax6 Primary antibody
Anti-rabbit 555 Invitrogen A31572 Donkey anti-Rabbit IgG (H+L)
Secondary Antibody, Alexa Fluor 555
Anti-Recoverin Antibody Millipore ab5585 Primary antibody
Anti-Rhodopsin Antibody Abcam ab5417 Primary antibody
Anti-sheep 555 Invitrogen A21436 Donkey anti-Sheep IgG (H+L)
Secondary Antibody, Alexa Fluor 555
Anti-SOX9 Antibody Abclonal A19710 Primary antibody
Anti-VSX2 Antibody Millipore ab9016 Primary antibody
B-27 supplement W/O VIT A (50X) Gibco 12587010 Supplement
Cryotube vial Thermo scientific-NUNC 375418 1.8 ml
DAPI DOJINDO D532 4',6-Diamidino-2-phenylindole
dihydrochloride; multiple suppliers
Dimethyl sulphoxide(DMSO) Hybri-max Sigma D2650-100ML Multiple suppliers
DMEM Gibco C11995500BT Medium
DMEM /F12 Gibco C11330500BT Medium
EDTA Invitrogen 15575-020 0.5 M PH 8.0
FBS NATOCOR SFBE Serum
Filter Millipore SLGP033RB 0.22μm, sterile Millex filter
GlutaMax, 100X Gibco 35050061 L-alanyl-L-glutamine
Heparin Sigma H3149 2 mg/ml in PBS to use
Matrigel, 100x Corning 354277 Extracellular matrix (ECM)
MEM Non-Essential Amino Acids Solution (100X) Gibco 11140050 MEM NEAA
mTeSR1 STEM CELL 85850 hPSCs maintenance medium (MM)
N2 supplement Gibco 17502048 Supplement
Phosphate-buffered saline (PBS) buffer GNM GNM10010 Without Ca+,Mg+,PH7.2±0.1 0.1M
Taurine Sigma T0625 Supplement
Ultra-low attachment culture dishes 100mm petri dish, low-attachment Corning CLS3262-20EA Petri dish

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