Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Summary

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Abstract

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

Introduction

The retina is an integral part of the central nervous system (CNS) and has a limited capacity to spontaneously regenerate following a traumatic injury or diseases. Therefore, degenerative pathologies causing definitive retinal cell loss, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and diabetic retinopathy, typically lead to irreversible blindness. Rescuing the degenerated retina is a major challenge for which stem cell-based therapies aiming to replace the damaged or lost cells are one of the most promising approaches^1,2,3. Pluripotent....

Protocol

The protocol described in this paper follows the guidelines of the Institut de la Vision's research ethics committee. The Institut de la Vision has been allowed the manipulation of human specimen according to the current French regulation. Specimen handling follows patient data protection in accordance with the Tenets of Helsinki, and national regulations after the ethical approval of the "Comité de Protection des Personnes (CPP) Ile-de-France V".

1. Preparation of Culture Media and Dishes

1. Culture media
   1. Use iPSC medium, a chemically defined medium dedicated to pluripotent stem cell c....

Results

The first step for human iPSC differentiation cultivated in feeder-free conditions¹⁶ is to shut down self-renewal machinery using Bi medium to encourage a spontaneous differentiation (Figure 1A). Then, at D2, the Bi medium is complemented with an N2 supplement to guide differentiating iPSCs cells towards the neural and retinal lineages. This process leads to the appearance of neuroretinal buds at around D28 (Figure.......

Discussion

This protocol describes how to produce RPE cells and retinal organoids, containing retinal RGCs and photoreceptors, from human pluripotent stem cells in xeno-free and feeder-free conditions. Compatible with the Good Manufacturing Practice (GMP) process, the method cultivated presented here allows a large production of iPSC-derived retinal cells as RPE cells, RGCs, and photoreceptors for the development of stem cell-based therapies and drug discovery approaches for the future treatment of retinal degenerative diseases. Th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vitronectin (VTN-N) Recombinant Human Protein, Truncated	ThermoFisher Scientific	A14700	Coating
CTS Vitronectin (VTN-N) Recombinant Human Protein, Truncated	ThermoFisher Scientific	A27940	Coating
Essential 8 Medium	ThermoFisher Scientific	A1517001	medium
Essential 6 Medium	ThermoFisher Scientific	A1516401	medium
CTS (Cell Therapy Systems) N-2 Supplement	ThermoFisher Scientific	A1370701	supplement CTS
N-2 Supplement (100X)	ThermoFisher Scientific	17502048	supplement
B-27 Supplement (50X), serum free	ThermoFisher Scientific	17504044	supplement
CTS B-27 Supplement, XenoFree	ThermoFisher Scientific	A1486701	supplement CTS
DMEM/F-12	ThermoFisher Scientific	11320074	medium
MEM Non-Essential Amino Acids Solution (100X)	ThermoFisher Scientific	11140035	supplement
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15140122	antibiotic
CellStart CTS	ThermoFisher Scientific	A1014201	Matrix CTS
Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix	ThermoFisher Scientific	A1569601	Matrix
Gentle Cell Dissociation Reagent	Stemcell Technologies	7174	dissociation solution
Cryostem Freezing Media	clinisciences	05-710-1D	Cryopreservation medium
Fibroblast growth factor 2 (FGF2)	Preprotech	100-18B	FGF2
Fibroblast growth factor 2 (FGF2) animal free	Preprotech	AF-100-18B	FGF2 Xeno free
AGANI needle 23G	Terumo	AN*2332R1	Needle
Flask 25 cm² Tissue Culture Treated	Falcon	353109	T-25 cm²
24 well plate Tissue Culture Treated	Costar	3526	24-well plate
6 well plate Tissue Culture Treated	Costar	3516	6-well plate