Single-Cell Transcriptomic Analysis of Human Retina

项目总结 视觉是人类最重要的感官，占大脑功能的25%。它需要一个 协调好眼睛各部分之间的协调。在所有的部分中，视网膜是最重要的 对图像的感知。它是一种精确的分层结构，衬里在眼睛后面的表面，包括 数以百万计的细胞聚集在一起，形成一个紧密的网络。视神经将视网膜与 大脑。视网膜不仅接收光，而且还处理光，并将下行信号传输到 中脑和丘脑。当视网膜发生病变时，不幸的结果是失明，这是 最令人畏惧的残疾。影响视网膜的疾病很复杂，因为细胞的数量多种多样。 涉及的单元格的类型和总数。评估病理表型是否会影响 多样化的细胞群体与高度特定的细胞类型。虽然视网膜疾病诊断的进展 虽然进展很快，但针对原发遗传缺陷的视网膜疾病的治疗进展缓慢。 尽管在遗传学方面取得了重大成功，但视觉群体在精确度方面落后于进步。 发生在其他专科的医学。进展不大，部分原因是对 人类视网膜生物学。人类和常用动物模型之间的解剖学差异 严重阻碍了从实验室到人类健康的成果转化。因此，迫切需要 收集和分析人眼视网膜细胞，加深对人类视网膜疾病的认识 并对小鼠和人之间的细胞类型保守性进行了评估。最近在技术上的突破 单细胞rna-seq(scrna-seq)使测量单细胞中的基因表达成为可能，为 探索细胞异质性的方法。与阿拉巴马州眼科银行合作，我们将深入采样 人类视网膜细胞，充分描述细胞多样性，并阐明从基因组中发现的功能作用- 视网膜疾病的广泛关联研究。我们提出了以下目标。AIM 1将产生scRNA-seq 来自20名健康成年人类捐赠者的眼睛数据，并产生去噪基因表达数据 下游分析。目标2将描述人类视网膜和支持组织中的细胞多样性，以及 通过免疫组织化学方法验证新的细胞类型特异性标记基因。目标3将推断细胞类型组成 通过集成scRNA-seq和批量RNA-seq数据 正常的人类眼睛。这些开创性的研究利用新的方法和跨学科的专业知识 研究人类视网膜和支持组织中细胞类型特异性基因的表达。按详细说明 对人眼四个地理区域细胞图谱的刻画，我们的研究将提供新的 对细胞类型特定功能的洞察，可以推动视网膜疾病的精确治疗靶向。

PROJECT SUMMARY Vision, the most important of the human senses, occupies 25% of the brain function. It requires an orchestrated coordination between all parts of the eye. Of all the parts, the retina is the most vital for normal perception of an image. It is a precisely layered structure lining the surface of the back of the eye, comprising many millions of cells packed together in a tightly knit network. The optic nerve connects the retina with the brain. The retina not only receives light, but also processes it, and transmits downstream signals to the midbrain and the thalamus. When the retina becomes diseased, the unfortunate result is blindness, which is the most feared disability. Diseases that affect the retina are complex because of the diverse number of cell types and total number of cells involved. It remains challenging to assess if pathological phenotypes affect diverse cell populations versus highly specific cell types. While advances in retinal disease diagnostics have progressed rapidly, treatments for retinal diseases directed at primary genetic defects have progressed slowly. Despite major successes in genetics, the vision community is lagging behind the advances in precision medicine occurring in other specialties. Modest progress is due in part to an incomplete understanding of human retinal biology. Anatomical differences between humans and commonly used animal models have severely hindered the translation of results from laboratory to human health. Therefore, there is an urgent need to collect and analyze retinal cells from human eyes to advance our understanding of human retinal diseases and assess the cell type conservation between mouse and human. Recent technologic breakthroughs in single-cell RNA-seq (scRNA-seq) have made it possible to measure gene expression in single cells, paving the way for exploring cellular heterogeneity. Collaborating with the Alabama Eye Bank, we will deeply sample human retinal cells, fully characterize cell diversity, and elucidate the functional roles of findings from genome- wide association studies for retinal diseases. We propose the following aims. Aim 1 will generate scRNA-seq data from eyes of 20 healthy adult human donors, and produce de-noised gene expression data for downstream analyses. Aim 2 will characterize cell diversity in human retina and supporting tissues, and validate novel cell type-specific marker genes by immunohistochemistry. Aim 3 will infer cell type compositions and allele-specific gene expression in each cell type by integrating scRNA-seq and bulk RNA-seq data from normal human eyes. These pioneering studies leverage novel methods and interdisciplinary expertise to characterize cell type-specific gene expression in human retina and supporting tissues. By detailed characterization of the cell atlases in four geographical areas in human eye, our study will provide novel insights into cell-type specific functions that can power precision therapeutic targeting of retinal diseases.