Lowy Medical Research Institute; The Scripps Research Institute;
Lowy Medical Research Institute; The Scripps Research Institute.
J Vis Exp. 2021 Mar 4(169). doi: 10.3791/62269.
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.
类器官为研究疾病机制和治疗方法提供了一个很有前景的平台，它直接在人类组织背景下，结合细胞培养的多功能性和高通量来进行研究。成熟的人类视网膜类器官被用于筛选潜在的药物治疗方法，以治疗与年龄相关的视网膜退行性疾病—— 2 型黄斑毛细血管扩张症（MacTel）。我们最近发现，MacTel 可能是由一种非典型脂质物质——脱氧鞘脂（deoxySL）水平升高引起的。这些脂质对视网膜有毒性，可能导致 MacTel 患者中发生的光感受器损失。为了筛选药物预防脱氧 SL 光感受器毒性的能力，我们从非 MacTel 诱导多能干细胞（iPSC）系生成了人类视网膜类器官，并将其成熟到有丝分裂后期，此时它们会发育出视网膜的所有神经元谱系衍生细胞，包括功能成熟的光感受器。将脱氧 SL 代谢物处理于视网膜类器官，并用免疫组织化学法在光感受器层中测量细胞凋亡。使用这种毒性模型，筛选了能预防脱氧 SL 诱导的光感受器死亡的药物。通过靶向候选药物的方法，我们确定了一种常用的降胆固醇和甘油三酯药物——非诺贝特（fenofibrate），也可以预防视网膜细胞中的脱氧 SL 毒性。该毒性筛选成功鉴定出一种可预防光感受器死亡的 FDA 批准药物。鉴于该模型与疾病高度相关，因此这是一个可直接实施的发现。该平台可以轻松修改，以测试任何数量的代谢应激源和潜在的药理学干预措施，用于未来的视网膜疾病治疗发现。
