Generation and characterisation of transplantable hiPSC-derived photoreceptors for treatment of retinal degeneration

由视网膜中的非再生感光光感受器的变性和丧失引起的视力损伤和失明影响数百万患者，目前还没有建立治愈性疗法。健康光感受器的移植代表了一种有前途的治疗方法。在BMBF资助的联盟“ReSight”中，已经建立了一个用于诱导多能干细胞（iPSC）衍生的人类光感受器的稳健和可再现的生成和分离以及将其移植到视网膜变性的临床前小鼠模型中的平台。使用光感受器特异性报告iPSC系，产生含有高达60%荧光标记的光感受器的视网膜类器官，并富集FACS用于移植研究。从Crx-mCherry-iPSC系产生的视网膜类器官含有至少三种主要细胞类型-感光细胞前体，视杆细胞和视锥细胞-在不同发育阶段具有变化的比例。考虑到这种标记细胞群的异质性，单细胞水平的表达谱分析对于评估不同年龄的视网膜类器官衍生移植物的确切细胞组成及其随时间在不同疾病环境中的分化和成熟是必要的。事实上，供体人类光感受器在小鼠受体中存活长达9个月，表达光感受器特异性标记，接触宿主双极细胞并与宿主Müller胶质细胞相互作用。随着移植后时间的延长，人类光感受器进一步成熟，形成带状突触、富含突触的内节和含有朝向视网膜色素上皮的外节的膜盘。然而，组织学分析显示移植后供体光感受器的结构和成熟存在明显差异，这与（i）移植物的不同发育阶段有关（供体感光细胞从较年轻与较年长的视网膜类器官分离），（ii）在受体内的时间，（iii）选择的视网膜变性模型（中度与完全变性），和（iv）在同一宿主视网膜内整合与非相互作用的移植物区域。因此，scRNAseq的分析将提供对人类光感受器移植特征的详细了解，这对于定义和优化可利用的供体细胞群是不可或缺的，这是成功的光感受器移植和临床应用的必要前提。

Vision impairment and blindness caused by the degeneration and loss of the non-regenerating light-sensing photoreceptors in the retina affects millions of patients with no curative therapy currently established. Transplantation of healthy photoreceptors represents a promising treatment approach. Within the BMBF-funded consortium ‘ReSight’ a platform for the robust and reproducible generation and isolation of induced pluripotent stem cell (iPSC)-derived human photoreceptors and their transplantation into pre-clinical mouse models of retinal degeneration has been established. Using photoreceptor-specific reporter iPSC lines, retinal organoids containing up to 60% fluorescently-labelled photoreceptors are generated and FACS enriched for transplantation studies. Retinal organoids generated from the Crx-mCherry-iPSC line contain at least three principle cell-types - photoreceptor precursors, rods and cones - with changing proportions at different developmental stages. Given the heterogeneity of this reporter-labeled cell population, expression profiling at the single cell level will be necessary to assess the exact cell composition of retinal organoid-derived transplants of different ages and their differentiation and maturation within different disease environments over time. Indeed, donor human photoreceptors survive up to 9 months in mouse recipients, express photoreceptor-specific markers, contact host bipolar cells and interact with host Müller glia. With prolonged time after transplantation, human photoreceptors further mature, forming ribbon synapses, mitochondria-rich inner segments and membrane-discs containing outer segments oriented towards the retinal pigment epithelium. However, histological analysis shows obvious differences in regard to structure and maturation of donor photoreceptors after transplantation, correlating with (i) different developmental stages of the transplants (donor photoreceptor isolation from younger vs. older retinal organoids), (ii) time within the recipient, (iii) chosen retinal degeneration model (moderate vs. complete degeneration), and (iv) integrating vs. non-interacting graft areas within the same host retina. Analysis by scRNAseq will therefore provide detailed insights into human photoreceptor transplant characteristics, indispensable for defining and optimizing utilizable donor cell populations as an essential prerequisite for successful photoreceptor transplantation and translation towards clinical application.