Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Summary

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Abstract

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Introduction

It has been documented that at least five retinal degenerative diseases are caused by genetic mutations in the BEST1 gene^{1,2,3,4,5,6,7,8}, with the number of reported mutations already over 200 and still increasing. These BEST1-associated diseases, also known as bestrophinopathies, cause progressive vision loss and even blindness, and there are currently no effective treatments. The prot....

Protocol

1. Differentiation of hPSC to RPE

1. Maintain and passage hPSCs as previously described¹⁸.
   NOTE: All cells (including hPSCs and hPSC-RPEs) are grown in 37 °C at 5% CO₂ throughout the duration of the growth and differentiation protocols.
2. Prior to differentiation, split confluent hPSCs to pre-coated 6-well plates.
   1. To coat plates, thaw basement membrane matrix on ice for about 1 h, suspend liquidized matrix in 4 °C DMEM medium at a 1:50 dilution, add 800 µL coating mixture to each well, and incubate for at least 1 h at 37 °C.
   2. When coating is complete, ....

Results

The most technically challenging step is the manual isolation, which aims to achieve a high purity of a differentiated P₀ hPSC-RPE population. After a successful isolation, >90% cells in the P₀ population will grow and mature to display signature RPE morphologies (Figure 1C). The existence of a minor portion of non-RPE or immature RPE cells in the P₀ population is almost inevitable, but will not interfere with the downstrea.......

Discussion

The most important procedure for the disease-in-a-dish approach is to differentiate hPSCs with a disease-causing mutation to the correct cell lineage, which is RPE for BEST1. Thus, after each differentiation experiment, the resulting hPSC-RPE cells should be carefully validated for their mature status by RPE-specific morphologies and protein markers^16,17,18. To minimize clonal artifact, multiple hiPSC clones from the sa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Knock-Out (KO) DMEM	ThermoFisher	10829018
KO serum replacement	ThermoFisher	10829028
Nonessential amino acids	ThermoFisher	11140050
Glutamine	ThermoFisher	35050061
Penicillin-streptomycin	ThermoFisher	10378016
Nicotinamide	Sigma-Aldrich	N0636
Human activin-A	PeproTech	120-14
MEM (a modification)	Sigma-Aldrich	M4526
Fetal Bovine Serum	VWR	97068-085
N1 supplement	Sigma-Aldrich	N6530
Glutamine-penicillin-streptomycin	Sigma-Aldrich	G1146
Nonessential amino acids	Sigma-Aldrich	M7156
Taurine	Sigma-Aldrich	T0625
Hydrocortisone	Sigma-Aldrich	H0386
Triiodo-thyronin	Sigma-Aldrich	T5516
mTeSR-1 medium	Stemcell Technologies	5850
Matrigel	Corning	356230
Collagenase	Gibco	17104019
Trypsin	VWR	45000-664
M-PER mammalian protein extraction reagent	Pierce	78501
proteinase inhibitor cocktail	Sigma-Aldrich	4693159001
RPE65 antibody	Novus Biologicals	NB100-355
CRALBP antibody	Abcam	ab15051
BEST1 antibody	Novus Biologicals	NB300-164
Beta Actin antibody	ThermoFisher	MA5-15739
Alexa Fluor 488-conjugated donkey anti-mouse IgG	ThermoFisher	A-21202
Goat anti-mouse IgG	ThermoFisher	SA5-35521
Goat anti-Rabbit IgG	LI-COR Biosciences	926-68071
Hoechst 33342	ThermoFisher	62249
HEKA EPC10 patch clamp amplifier	Warner Instruments	895000
Patchmaster	Warner Instruments	895040