Santos-Ferreira Tiago, Völkner Manuela, Borsch Oliver, Haas Jochen, Cimalla Peter, Vasudevan Praveen, Carmeliet Peter, Corbeil Denis, Michalakis Stylianos, Koch Edmund, Karl Mike O, Ader Marius
Technische Universität Dresden CRTD - Center for Regenerative Therapies Dresden, Dresden, Germany.
German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany.
Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3509-20. doi: 10.1167/iovs.16-19087.
Preclinical studies on photoreceptor transplantation provided evidence for restoration of visual function with pluripotent stem cells considered as a potential source for sufficient amounts of donor material. Adequate preclinical models representing retinal disease conditions of potential future patients are needed for translation research. Here we compared transplant integration in mouse models with mild (prominin1-deficient; Prom1-/-) or severe (cone photoreceptor function loss 1/rhodopsin-deficient double-mutant; Cpfl1/Rho-/-) cone-rod degeneration.
For photoreceptor transplant production, we combined the mouse embryonic stem cell retinal organoid system with rhodopsin-driven GFP cell labeling by recombinant adeno-associated virus (AAV). Organoid-derived photoreceptors were enriched by CD73-based magnetic-activated cell sorting (MACS) and transplanted subretinally into wild-type, Prom1-/- and Cpfl1/Rho-/- hosts. The survival, maturation, and synapse formation of donor cells was analyzed by immunohistochemistry.
Retinal organoids yielded high photoreceptor numbers that were further MACS-enriched to 85% purity. Grafted photoreceptors survived in the subretinal space of all mouse models. Some cells integrated into wild-type as well as Prom1-/- mouse retinas and acquired a mature morphology, expressing rod and synaptic markers in close proximity to second-order neurons. In contrast, in the novel Cpfl1/Rho-/- model with complete photoreceptor degeneration, transplants remained confined to the subretinal space, expressed rod-specific but only reduced synaptic markers, and did not acquire mature morphology.
Comparison of photoreceptor grafts in preclinical models with incomplete or complete photoreceptor loss, showed differential transplant success with effective and impaired integration, respectively. Thus, Cpfl1/Rho-/- mice represent a potential benchmark model resembling patients with severe retinal degeneration to optimize photoreceptor replacement therapies.
光感受器移植的临床前研究为利用多能干细胞恢复视觉功能提供了证据，多能干细胞被视为充足供体材料的潜在来源。转化研究需要有能够代表未来潜在患者视网膜疾病状况的充分临床前模型。在此，我们比较了在轻度（prominin1缺陷型；Prom1-/-）或重度（视锥光感受器功能丧失1/视紫红质缺陷型双突变体；Cpfl1/Rho-/-）视锥-视杆细胞变性的小鼠模型中的移植整合情况。
为了生产光感受器移植物，我们将小鼠胚胎干细胞视网膜类器官系统与重组腺相关病毒（AAV）介导的视紫红质驱动的绿色荧光蛋白（GFP）细胞标记相结合。通过基于CD73的磁激活细胞分选（MACS）富集类器官来源的光感受器，并将其视网膜下移植到野生型、Prom1-/-和Cpfl1/Rho-/-宿主中。通过免疫组织化学分析供体细胞的存活、成熟和突触形成情况。
视网膜类器官产生了大量光感受器，经MACS进一步富集后纯度达到85%。移植的光感受器在所有小鼠模型的视网膜下间隙中存活。一些细胞整合到野生型以及Prom1-/-小鼠视网膜中，并获得了成熟的形态，在与二级神经元相邻处表达视杆细胞和突触标记物。相比之下，在具有完全光感受器变性的新型Cpfl1/Rho-/-模型中，移植物仍局限于视网膜下间隙，表达视杆细胞特异性标记物但突触标记物减少，且未获得成熟形态。
在光感受器部分或完全丧失的临床前模型中对光感受器移植进行比较，结果显示移植成功率分别存在有效整合和整合受损的差异。因此，Cpfl1/Rho-/-小鼠代表了一种潜在的基准模型，类似于患有严重视网膜变性的患者，可用于优化光感受器替代疗法。
