Investigating cone photoreceptor migration using stem cell derived retinal organoids

遗传性视网膜营养不良，以视杆和视锥感光细胞进行性退化为特征，导致不可逆性失明。总体而言，它们代表了人类最常见的遗传性视力障碍形式，据估计，全世界的患病率为1/3000。虽然视杆能在昏暗的光线条件下实现视觉，但人类的高敏锐度和色觉依赖于视锥感光细胞。尽管视锥感光细胞是一种罕见的群体，仅占视网膜细胞总数的2%-4%，但影响它们的疾病，无论是直接影响它们，还是通过丧失视杆来源的营养信号，都会对受影响者的生活造成丧失能力的影响。不幸的是，目前的治疗方法非常有限，只能延缓失明的发生，而不能阻止或逆转失明。因此，开发新的、更有效的治疗方案是一种巨大的推动力。视网膜细胞疗法旨在利用多能干细胞(HPSC)来源来替代丢失的光感受器，是未来最有希望的治疗选择之一。特别是，hPSC衍生的视网膜有机类化合物为开发新的治疗干预措施带来了巨大的希望，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有了显著的进步，但细胞移植的结果仍然很差，在视网膜下移植后，很少有细胞整合到剩余的视网膜回路中。这在很大程度上是因为对光感受器的发展和利用它进行治疗应用的手段缺乏强有力的了解。在这种背景下，控制人类视锥细胞光感受器迁移的信号网络的细节仍有待阐明，这是细胞治疗努力旨在为临床环境所利用的过程。在这里，我们提出了一项研究计划，将干细胞和基因组编辑技术结合起来，进行功能性的高含量筛选，以阐明参与控制视锥迁移的信号级联的组件，并确定操纵这一过程的药理学工具。为了实现这一点，我们将利用视锥细胞特异性的人类多能干细胞报告系来产生3D视网膜器官并跟踪，以及在不同的迁移阶段分离视锥细胞。首先，为了促进我们对迁移前、迁移中和迁移后的差异转录格局的理解，我们将建立纯化的视锥体光受体的基因表达特征。其次，使用CRISPR介导的大规模遗传功能丧失方法，我们将确定G蛋白信号机制的所有表达组件是否参与了退出细胞周期后发生的根尖-基锥迁移的典型过程。最后，使用筛选出的目标基因，我们将识别和测试化合物，这些化合物可以激活它们，从而刺激锥体的迁移行为。我们预计，这项工作将极大地促进我们对人类视锥感光细胞发育的理解，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，在细胞替代治疗环境中，这里确定的药理试剂可能会促进移植的供体细胞的迁移和突触生成。

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.