Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Summary

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

Abstract

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.

Introduction

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....

Protocol

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 manufacturer's instructions. Thaw an aliquot of 1x10⁶ hESCs.
      NOTE: Prepare 3 mL of pre-warmed reagent B (Table of Materials

Results

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 µm in .......

Discussion

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 retina^4,5,8,9. Although confluent ROs could be harvested, it is time-consuming, which may require long culturing periods (up.......

Materials

List of materials used in this article

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