Investigating cone photoreceptor migration using stem cell derived retinal organoids

遗传性视网膜营养不良，以杆状和锥状光感受器的进行性变性为特征，导致不可逆的失明。总的来说，它们代表了最常见的遗传性人类视觉障碍形式，估计全世界的患病率为每3000人中有1人。虽然杆状体在昏暗的光线条件下能够视觉，但人类的高敏锐度和彩色视觉依赖于锥状光感受器。尽管锥体光感受器是一个罕见的种群，仅占视网膜细胞总数的2-4%，但直接或通过失去杆状细胞衍生的营养信号而影响它们的疾病，对受影响者的生活产生了丧失能力的影响。不幸的是，目前的治疗方法非常有限，只能延缓视力下降的发生，而不能阻止或逆转视力下降。因此，有很大的动力来开发新的和更有效的治疗方案。以多能干细胞（hPSC）为来源的视网膜细胞治疗旨在替代失去的光感受器，是未来最有希望的治疗选择之一。特别是，hPSC衍生的视网膜类器官在开发新的治疗干预措施方面具有很大的前景，包括用于细胞治疗、疾病建模和药物筛选的供体细胞生产。然而，尽管近年来光感受器供体细胞的生产方法取得了显著进展，但细胞移植的效果仍然很差，在视网膜亚移植后，很少有细胞融入剩余的视网膜回路。这在很大程度上是由于缺乏对光感受器发育的强大理解以及将其用于治疗应用的手段。在这种情况下，控制人类视锥光感受器迁移的信号网络的细节，细胞治疗努力旨在为临床环境利用的过程，仍有待阐明。在这里，我们提出了一项研究计划，该计划结合干细胞和基因组编辑技术进行功能性高含量筛选，以阐明参与控制锥体迁移的信号级联的组成部分，并确定操纵这一过程的药理学工具。为了实现这一目标，我们将利用一个锥体特异性的人类多能干细胞报告系来生产3D视网膜类器官，并在不同的迁移阶段跟踪和分离锥体。首先，为了促进我们对迁移之前、期间和之后的差异转录景观的理解，我们将建立纯化的视锥光感受器的基因表达特征。其次，使用大规模的CRISPR介导的基因功能缺失方法，我们将确定g蛋白信号机制的所有表达组分在细胞周期退出后发生的顶基锥迁移的典型过程中的参与。最后，利用筛选中发现的靶基因，我们将识别和测试能够激活它们的化合物，从而刺激锥虫的迁移行为。我们期望这项工作将极大地促进我们对人类视锥光感受器发育的理解，同时也揭示了指导复杂视网膜结构整体组装的共同原则。此外，这里确定的药理学试剂可能促进移植供体细胞在细胞替代治疗环境中的迁移和突触发生。

Inherited retinal dystrophies, featuring the progressive degeneration of the rod and cone photoreceptors, lead to irreversible blindness. Collectively, they represent the most frequent inherited forms of human visual handicap, with an estimated prevalence of 1 in 3000 worldwide. While rods enable vision in dim lighting conditions, human high acuity and colour vision depends on cone photoreceptors. Even though cone photoreceptors are a rare population, forming only 2-4% of total retinal cells, diseases affecting them, either directly or through the loss of rod derived trophic signalling, have an incapacitating impact on the lives of those affected. Unfortunately, current treatments are very limited and can only delay the onset of sight-loss, but not stop or reverse it. Thus, there is a great impetus to develop new and more effective treatment options. Retinal cell therapy, aiming to replace lost photoreceptors, using pluripotent stem cell (hPSC) derived sources, is one of the most promising future treatment options. In particular, hPSC derived retinal organoids hold great promise for the development of novel therapeutic interventions, including donor cell production for cell therapy, disease modelling and drug screens. However, while production methods for photoreceptor donor cells have advanced significantly in recent years, cell transplantation outcomes remain poor with few cells integrating into the remaining retinal circuitry after sub-retinal transplantation. This is in large part due to a lack of a robust understanding of photoreceptor development and the means to leverage it for therapeutic applications. In this context, the details of the signalling network controlling human cone photoreceptor migration, the process cell therapy efforts aim to harness for the clinical setting, remain to be elucidated. Here, we propose a research programme, which combines stem cell and genome editing technology to conduct functional high-content screens to elucidate components of the signalling cascade involved in controlling cone migration and identify pharmacological tools to manipulate this process. To achieve this, we will take advantage of a cone specific human pluripotent stem cell reporter line to produce 3D retinal organoids and track, as well as isolate cones at different phases of migration. First, with the goal of advancing our understanding of the differential transcriptional landscape before, during and after migration, we will establish the gene expression signatures of purified cone photoreceptors. Second, using a large scale, CRISPR mediated, genetic loss-of function approach, we will determine the involvement of all expressed components of the G-protein signalling machinery in the stereotypical process of apico-basal cone migration that occurs following exit from cell cycle. Lastly, using target genes identified in the screen, we will identify and test chemical compounds, which can activate them and thus stimulate migratory behaviour of cones. We expect that this work will greatly advance our understanding of human cone photoreceptor development, but also reveal common principles guiding the assembly of the complex retinal architecture as a whole. Furthermore, the pharmacological reagents identified here may facilitate migration and synaptogenesis of transplanted donor cells in the cell replacement therapy setting.