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

由于视网膜中不可再生的光感光感受器的退化和丧失而导致的视力损害和失明影响着数以百万计的患者，目前还没有确定的治疗方法。移植健康的光感受器是一种很有前途的治疗方法。在BMBF资助的联合体‘ReSight’中，已经建立了一个健壮和可重复的平台，用于产生和分离诱导多能干细胞(IPSC)来源的人光感受器，并将其移植到临床前视网膜退化的小鼠模型中。使用光感受器特异性报告IPSC系，可以产生含有高达60%的荧光标记光感受器的视网膜类器官，并为移植研究富含FAC。CRX-mCherry-iPSC系产生的视网膜类器官至少包含三种主要细胞类型-光感受器前体、视杆和视锥-在不同发育阶段比例不同。鉴于这个报告标记的细胞群体的异质性，在单细胞水平上的表达谱将是必要的，以评估不同年龄的视网膜器官来源的移植的确切细胞组成，以及它们随着时间的推移在不同疾病环境中的分化和成熟。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Müler胶质细胞相互作用。随着移植后时间的延长，人的光感受器进一步成熟，形成带状突触、富含线粒体的内节和含有外节的膜盘，朝向视网膜色素上皮。然而，组织学分析表明，移植后供体光感受器的结构和成熟度有明显的差异，这与(I)移植的不同发育阶段(供体光感受器从年轻的和老年的视网膜器官中分离)，(Ii)在受体体内的时间，(Iii)选择的视网膜变性模型(中度和完全退化)，以及(Iv)在同一宿主视网膜内整合与非相互作用的移植物区域有关。因此，scRNAseq的分析将为人类光感受器移植特性提供详细的见解，这对于确定和优化可利用的供体细胞群是必不可少的，这是成功光感受器移植和转化为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.