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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Published: April 21st, 2021

DOI:

10.3791/62298

1Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Science Key Laboratory

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Pluripotent stem cells (PSCs) are characterized by their self-renewal and ability to differentiate into all kinds of somatic cells. Thus, organoids derived from PSCs have become an important resource in regenerative medicine research. Retinal degeneration is characterized by the loss of photoreceptors (rods and cones) and retinal pigment epithelium. Retinal cell replacement could be an encouraging treatment for this disease. However, it is not feasible to obtain human retinas for disease research and therapy. Therefore, retinal organoids (ROs) derived from PSCs, which effectively and successfully recapitulate multi-layered native retinal cells, are beneficial for basi....

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This study was approved by the institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. H9 hESCs were obtained from the WiCell Research Institute and genetically engineered to tdTomato-tagged cell line.

1. Generation of human ROs

  1. Culture the hESCs under feeder-free conditions.
    1. Coat one well of a 6-well plate with 1 mL of 0.1 mg/mL reagent A (Table of Materials) at 37 °C for at least half an hour following the manufact.......

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The schematic illustration depicts the differentiation protocol to improve precursor cells with COCO (Figure 1). From PSC to ROs, numerous details could cause result variations. It is recommended to record every step and even the catalog number and lot number of every medium to track the entire procedure.

Herein, we provide bright field images for days 6, 12, 18, and 45 (Figure 2). On day 6, the organoids are usually around 600 µ.......

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Retinal organoid differentiation is a desirable method for the generation of ample functional retinal cells. The RO is a composite of different retinal cells, such as ganglion cells, bipolar cells, and photoreceptors, generated by pluripotent stems cells toward the neural retina4,5,8,9. Although confluent ROs could be harvested, it is time-consuming, which may require long culturing periods (up.......

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We thank members of 502 laboratory for their technical supports and helpful comments regarding the manuscript. This work was partly supported by the Beijing Municipal Natural Science Foundation (Z200014) and National Key R&D Program of China (2017YFA0105300).

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Name Company Catalog Number Comments
2-mercaptoethanol Life Technologies 21985-023
COCO R&D Systems 3047-CC-050 DAN Domain family of BMP antagonists
DMEM/F-12 Gibco 10565-042
DMSO Sigma D2650
DPBS Gibco C141905005BT
EDTA Thermo 15575020
Fetal Bovine Serum (FBS), Qualified for Human Embryonic Stem Cells Biological Industry 04-002-1A
GMEM Gibco 11710-035
KnockOut Serum Replacement-Multi-Species Gibco A3181502
MEM Non-essential Amino Acid Solution (100X) sigma M7145
Pen Strep Gibco 15140-122
Primesurface 96 V-plate Sbio MS9096SZ Cell aggregation in 1.2.7
Pyruvate Sigma S8636
Reagent A BD 356231 Matrigel in 1.1.1
Reagent B StemCell 5990 mTeSR- E8 , PSCs basal medium in 1.1.2
Reagent C Gibco 12563-011 TrypLE Express in 1.2
Reagent D Roche 11284932001 DNase I , in 1.2
Retinoic acid Sigma R2625-100MG
SAG Enzo Life Science ALX-270-426-M001
Supplement 1 Life Technologies 17502-048 N-2 Supplement (100X), Liquid, supplemet in medum III
Taurine Sigma T-8691-25G
Trypsin-EDTA (0.25%), phenol red Gibco 25200056 organoids dissociation in 2.1.3
Wnt Antagonist I, IWR-1-endo - Calbiochem Sigma 681669 Wnt inhibitor
Y-27632 2HCl Selleck S1049

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  10. Zhang, C. J., Ma, Y., Jin, Z. B. The road to restore vision with photoreceptor regeneration. Experimental Eye Research. 202, 108283 (2020).
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