Published: March 4th, 2021
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).
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.
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....
1. Thawing, passaging, and expanding iPSCs/ESCs
NOTE: For all cell culturing steps, use best practices to maintain a sterile cell culture.
Retinal organoids were generated from a non-MacTel control iPSC line. After organoids reached 26 weeks in culture they were selected and split into experimental groups. Organoids were treated with varying concentrations of deoxySA to determine if deoxySA is toxic to photoreceptors. Four concentrations of deoxySA were tested, from 0 to 1 µM (Figure 2) and organoids were treated for 8 days, with media changes every other day. Cell death in response to deoxySA is concentration-dependent an.......
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 step5,18,19,21. An exhaustive list of protocols can be found in Capowski et al.22. The differentiation protocol we p.......
Supported by the Lowy Medical Research Institute. We would like to thank the Lowy family for their support of the MacTel project. We would like to thank Mari Gantner, Mike Dorrell, and Lea Scheppke for their intellectual input and assistance preparing the manuscript.....
|125mL Erlenmeyer Flasks
|B27 Supplement, minus vitamin A
|Beaver 6900 Mini-Blade
|Dispase II, powder
|DMEM, high glucose, pyruvate
|Donkey anti-rabbit Ig-G, Alexa Fluor plus 555
|FBS, Heat Inactivated
|In Situ Cell Death Detection Kit, Fluorescin
|Matrigel, growth factor reduced
|MEM Non-Essential Amino Acids Solution
|Pierce 16% Formaldehyde
|Rabbit anti-Recoverin antibody
|Steriflip Sterile Disposable Vacuum Filter Units
|Tissue Plus- O.C.T. compound
|Ultra-Low Attachment 6 well Plates
|Ultra-Low Attachment 75cm2 U-Flask
|Vacuum Filtration System
|Y-27632 Dihydrochloride (Rock inhibitor)
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