Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Summary

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Abstract

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.

The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Introduction

Modeling human disease in cell culture and animal models has provided invaluable tools for the discovery, modification, and validation of pharmacologic therapeutics, allowing them to advance from candidate drug to approved therapy. Although a combination of in vitro and non-human in vivo models has long been a critical component of the drug development pipeline they frequently fail to predict the clinical performance of novel drug candidates¹. There is a clear need for the development of technologies that bridge the gap between simplistic human cellular monocultures and clinical trials. Recent technological advances in self-organized three-dime....

Protocol

1. Thawing, passaging, and expanding iPSCs/ESCs

NOTE: For all cell culturing steps, use best practices to maintain a sterile cell culture.

1. Coat a 6-well cell culture plate with basement membrane matrix medium.
   1. To prepare 1x of this medium, follow product specifications or dilute 75 µL cold matrix medium with 9 mL of DMEM/F12. Add 1.5 mL of freshly prepared 1x medium per well in a 6-well plate. Incubate at 37 °C for 30 min.
   2. Aspirate off the basement .......

Discussion

Differentiation protocol variations
Since the invention of self-forming optic cups by Yoshiki Sasai's group²⁰, many labs have developed protocols to generate retinal organoids that can vary at almost every step^5,18,19,21. An exhaustive list of protocols can be found in Capowski et al.²². The differentiation protocol we p.......

Materials

Name	Company	Catalog Number	Comments
0.5M EDTA	Invitrogen	15575020
125mL Erlenmeyer Flasks	VWR	89095-258
1-deoxysphinganine	Avanti	860493
B27 Supplement, minus vitamin A	Gibco	12587010
Beaver 6900 Mini-Blade	Beaver-Visitec	BEAVER6900
D-(+)-Sucrose	VWR	97061-432
DAPI	Thermo-fisher	D1306
Dispase II, powder	Gibco	17105041
DMEM, high glucose, pyruvate	Gibco	11995073
DMEM/F12	Gibco	11330
Donkey anti-rabbit Ig-G, Alexa Fluor plus 555	Thermo-fisher	A32794
donkey serum	Sigma	D9663-10ML
FBS, Heat Inactivated	Corning	45001-108
Fenofibrate	Sigma	F6020
Glutamax	Gibco	35050061
Heparin	Stemcell Technologies	7980
In Situ Cell Death Detection Kit, Fluorescin	Sigma	11684795910
Matrigel, growth factor reduced	Corning	356230
MEM Non-Essential Amino Acids Solution	Gibco	11140050
mTeSR 1	Stemcell Technologies	85850
N2 Supplement	Gibco	17502048
Penicillin-Streptomycin	Gibco	15140122
Pierce 16% Formaldehyde	Thermo-fisher	28906
Rabbit anti-Recoverin antibody	Millipore	AB5585
Sodium Citrate	Sigma	W302600
Steriflip Sterile Disposable Vacuum Filter Units	MilliporeSigma	SE1M179M6
Taurine	Sigma	T0625
Tissue Plus- O.C.T. compound	Fisher Scientific	23-730-571
Tissue-Tek Cryomold	EMS	62534-10
Triton X-100	Sigma	X100
Tween-20	Sigma	P1379
Ultra-Low Attachment 6 well Plates	Corning	29443-030
Ultra-Low Attachment 75cm2 U-Flask	Corning	3814
Vacuum Filtration System	VWR	10040-436
Vectashield-mounting medium	vector Labs	H-1000
wax pen-ImmEdge	vector Labs	H-4000
Y-27632 Dihydrochloride (Rock inhibitor)	Sigma	Y0503