Investigating cone photoreceptor migration using stem cell derived retinal organoids

使用干细胞衍生的视网膜类器官研究视锥光感受器迁移

• 批准号: BB/X01309X/1
• 负责人: Jorn Lakowski
• 金额: $ 73.42万
• 依托单位: University of Southampton
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX01309X%2F1
• 关键词: Investigating; cone; photoreceptor; migration; using

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.

遗传性视网膜营养不良以视杆细胞和视锥细胞的进行性退化为特征，导致不可逆的失明。总的来说，它们代表了人类视力障碍的最常见的遗传形式，估计全球患病率为1/3000。虽然杆使视力在昏暗的照明条件下，人类的高敏锐度和色觉取决于锥光感受器。尽管视锥光感受器是一种罕见的群体，仅形成总视网膜细胞的2-4%，但直接或通过杆源性营养信号传导的丧失而影响它们的疾病对受影响者的生活具有失能性影响。不幸的是，目前的治疗方法非常有限，只能延迟视力丧失的发生，而不能阻止或逆转它。因此，有很大的动力来开发新的和更有效的治疗方案。视网膜细胞疗法旨在使用多能干细胞（hPSC）衍生的来源来替代丢失的光感受器，是最有前途的未来治疗选择之一。特别是，hPSC衍生的视网膜类器官对于开发新的治疗干预措施具有很大的希望，包括用于细胞治疗的供体细胞生产，疾病建模和药物筛选。然而，虽然近年来感光体供体细胞的生产方法有了显着进步，但细胞移植结果仍然很差，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，控制人类视锥细胞感光细胞迁移的信号网络的细节，细胞治疗的努力旨在利用临床环境，仍有待阐明。在这里，我们提出了一个研究计划，它结合了干细胞和基因组编辑技术进行功能性高内容筛选，以阐明参与控制视锥细胞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，我们将利用视锥细胞特异性人类多能干细胞报告细胞系来产生3D视网膜类器官并跟踪，以及分离处于不同迁移阶段的视锥细胞。首先，为了推进我们对迁移前、迁移中和迁移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介导的遗传功能丧失方法，我们将确定G蛋白信号传导机制的所有表达组分参与细胞周期退出后发生的顶-基底锥迁移的常规过程。最后，使用筛选中确定的靶基因，我们将确定和测试可以激活它们并从而刺激视锥细胞迁移行为的化合物。我们希望这项工作将大大推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以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。