Single-Cell Transcriptomic Analysis of Human Retina

项目摘要 视觉是人类最重要的感官，占大脑功能的25%。它需要一个 眼睛各部分之间的协调。在所有的部分中，视网膜是最重要的正常 一个图像的感知。它是衬在眼睛后部表面上的精确分层结构，包括 数以百万计的细胞聚集在一起形成一个紧密的网络。视神经连接视网膜和视网膜。 个脑袋视网膜不仅接收光，而且还处理光，并将下游信号传输到视网膜。 中脑和丘脑当视网膜患病时，不幸的结果是失明， 最可怕的残疾影响视网膜的疾病是复杂的，因为不同数量的细胞 涉及的细胞类型和总数。评估病理表型是否影响 不同的细胞群体与高度特异性的细胞类型。虽然视网膜疾病诊断的进展 虽然视网膜疾病的治疗进展迅速，但针对原发性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，但视觉社区仍然落后于精确度的进步 医学发生在其他专业。取得的进展不大，部分原因是对以下方面的认识不全面： 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 严重阻碍了实验室结果向人类健康的转化。因此，迫切需要 收集和分析人眼视网膜细胞，以促进我们对人类视网膜疾病的了解 并评估小鼠和人之间的细胞类型保守性。最近的技术突破， 单细胞RNA-seq（scRNA-seq）使测量单细胞中的基因表达成为可能，为基因测序铺平了道路。 探索细胞异质性的方法。与亚拉巴马眼库合作，我们将深入采样 人类视网膜细胞，充分表征细胞多样性，并阐明基因组研究结果的功能作用- 视网膜疾病的广泛关联研究。我们提出以下目标。目标1将生成scRNA-seq 来自20名健康成年人供体的眼睛的数据，并产生去噪的基因表达数据， 下游分析。目标2将表征人类视网膜和支持组织中的细胞多样性， 通过免疫组织化学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 和等位基因特异性基因在每种细胞类型中的表达， 正常人的眼睛。这些开创性的研究利用新颖的方法和跨学科的专业知识， 表征人视网膜和支持组织中细胞类型特异性基因表达。通过详细 表征的细胞图谱在四个地理区域的人眼，我们的研究将提供新的 深入了解细胞类型特异性功能，可以为视网膜疾病的精确治疗靶向提供动力。

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.