A single cell sequencing approach to determine the heterogeneity, dynamics and cell fate decisions of retinal progenitor cells in vivo and in vitro

一种单细胞测序方法，用于确定体内和体外视网膜祖细胞的异质性、动态和细胞命运决定

• 批准号: BB/T004460/1
• 负责人: Majlinda Lako
• 金额: $ 116.85万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT004460%2F1
• 关键词: single; cell; sequencing; approach; determine

Being told that you have a visual impairment that can't be treated can be difficult to accept but this is the burden that 285 million people worldwide must bear. 26% of global blindness is caused by dysfunction of the retina, which is the innermost, light sensitive tissue that lines the back of the eye and is vital for light sensing and image processing. Dysfunction of retina and subsequent vision loss can occur through the effect of faulty genes we inherit from our parents as well as the accumulation of damage and the effect of various diseases throughout our lives. Our ability to prevent and treat vision loss is closely linked to our knowledge of "how our retinas form" and when and what is likely to go wrong. Our retinas develop mostly before birth; hence the availability of tissue to study from this time period is very limited. My group is in a unique position to bridge this gap, having access to human retinas through close and well established collaborations with the Human Developmental Biology Resource, which collects samples from aborted embryos and fetuses with the mother's consent. We also have the advantage of creating in the lab three-dimensional structures called "retinal organoids", which resemble the formation of human retina during development and contain the key retinal cell types. Our aim is to use both of these unique resources to understand how and when the retina forms and the role of genes that cause loss of vision when faulty.Retina is a complex tissue and is composed of seven cell types: these emerge at different points during our development from a pool of progenitor cells, which in itself is heterogeneous with various subsets suggested to give rise to the different cell types in a concise progression through time. For this reason, it has been difficult to pinpoint the progenitor cells which give rise to all the cell types that make up the human retina with the traditional research methods that rely on studies of cell mixtures. Here we propose to use an important new technology called single cell analysis which allows us to look at which genes are turned on in each cell in the population. Gene expression at the single cell level is a very reliable tool for the precise categorisation of cells and allows us to identify types of cells that are not noticeable when looking under the microscope at their shape or position. We will use this as a first step to explore the molecular differences of individual retinal cells in both developing retinas and the retinal organoids generated in our lab. The use of advanced data analysis techniques will then allow us to build a catalogue of cell types and the genes that characterise them, to match the progenitors to the various cell types across development, to predict their ultimate fate and to assess how closely the lab generated retinal organoids mimic the development of human retina. Second, we will use the single cell sequencing data to reconstruct a lineage tree using bioinformatics tools. This approach organises cells in 'pseudo-time', predicting the order and mode in which cell fate decisions are made, enabling us to predict genes that occupy special positions around branch points of the tree. Third, we will apply a new approach, which allows us to correlate the gene expression profile of individual cells with their location in the retina, thus creating a spatial map of our retinas as they develop. This spatial map will allow us to validate the expression of key genes that are found near the branch points which may be important to understand the decision that progenitor cells make towards their final trip to become retinal cells. Finally, we will assess whether genes expressed around branch points play an active role in controlling cell fate decisions by manipulating their expression. The information will be available to all scientists and clinicians to help their understanding of retinal development and disease.

被告知你患有无法治疗的视力障碍可能很难接受，但这是全球2.85亿人必须承受的负担。26%的全球失明是由视网膜功能障碍引起的，视网膜是眼睛后部最内侧的感光组织，对光线感知和图像处理至关重要。视网膜功能障碍和随后的视力丧失可能是由于我们从父母那里遗传的缺陷基因的影响，以及我们一生中各种疾病的累积和损害的影响。我们预防和治疗视力丧失的能力与我们对“我们的视网膜是如何形成的”以及什么时候和什么可能出错的知识密切相关。我们的视网膜大多在出生前发育；因此，从这一时期可供研究的组织非常有限。我的团队处于一个独特的位置来弥合这一差距，通过与人类发育生物学资源密切而可靠的合作获得人类视网膜，该资源在征得母亲同意的情况下从流产的胚胎和胎儿中收集样本。我们还有一个优势，那就是在实验室里创造出被称为“视网膜类器官”的三维结构，它类似于人类视网膜在发育过程中的形成，包含了关键的视网膜细胞类型。我们的目标是利用这两种独特的资源来了解视网膜是如何以及何时形成的，以及在故障时导致失明的基因的作用。视网膜是一个复杂的组织，由七种细胞类型组成：在我们发育过程中，这些细胞类型出现在我们发育的不同时间点，祖细胞池本身是异质的，不同的子集被认为可以随着时间的推移简明地产生不同的细胞类型。出于这个原因，用依赖于细胞混合物研究的传统研究方法，很难准确地确定哪些祖细胞产生了组成人类视网膜的所有细胞类型。在这里，我们建议使用一种名为单细胞分析的重要新技术，它允许我们查看种群中每个细胞中哪些基因处于开启状态。单细胞水平的基因表达是对细胞进行精确分类的非常可靠的工具，使我们能够识别在显微镜下观察其形状或位置时不太明显的细胞类型。我们将以此作为第一步，探索发育中的视网膜和我们实验室产生的视网膜器质中单个视网膜细胞的分子差异。然后，使用先进的数据分析技术将使我们能够建立细胞类型和表征它们的基因的目录，将祖细胞与发育过程中的各种细胞类型进行匹配，预测它们的最终命运，并评估实验室产生的视网膜有机体与人类视网膜发育的相似性。其次，我们将使用单细胞测序数据利用生物信息学工具重建谱系树。这种方法在“伪时间”中组织细胞，预测细胞命运决定的顺序和模式，使我们能够预测占据树分支点周围特殊位置的基因。第三，我们将应用一种新的方法，允许我们将单个细胞的基因表达谱与它们在视网膜中的位置相关联，从而创建视网膜发育过程的空间地图。这张空间图将允许我们验证在分支点附近发现的关键基因的表达，这可能对于理解祖细胞在最终成为视网膜细胞的过程中做出的决定可能是重要的。最后，我们将评估分支点周围表达的基因是否通过操纵它们的表达在控制细胞命运决定方面发挥积极作用。这些信息将提供给所有的科学家和临床医生，以帮助他们了解视网膜的发育和疾病。

项目成果

Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.

• DOI: 10.1111/jcmm.17670
• 发表时间: 2023-02
• 期刊: Journal of cellular and molecular medicine
• 影响因子: 5.3

Dissecting the Transcriptional and Chromatin Accessibility Heterogeneity of Proliferating Cone Precursors in Human Retinoblastoma Tumors by Single Cell Sequencing-Opening Pathways to New Therapeutic Strategies?

• DOI: 10.1167/iovs.62.6.18
• 发表时间: 2021-05-03
• 期刊: Investigative ophthalmology & visual science
• 影响因子: 4.4
• 作者: Collin J;Queen R;Zerti D;Steel DH;Bowen C;Parulekar M;Lako M
• 通讯作者: Lako M

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: A Comprehensive Validation Using Drugs and Compounds Affecting the Retina.

• DOI: 10.1093/stcltm/szab010
• 发表时间: 2022-03-17
• 期刊: Stem cells translational medicine
• 影响因子: 6
• 作者: Dorgau B;Georgiou M;Chaudhary A;Moya-Molina M;Collin J;Queen R;Hilgen G;Davey T;Hewitt P;Schmitt M;Kustermann S;Pognan F;Steel DH;Sernagor E;Armstrong L;Lako M
• 通讯作者: Lako M

Deciphering the spatio-temporal transcriptional and chromatin accessibility of human retinal organoid development at the single cell level

在单细胞水平上破译人类视网膜类器官发育的时空转录和染色质可及性

• DOI: 10.1101/2023.07.19.549507
• 发表时间: 2023
• 作者: Dorgau B

Spatial transcriptomics of human pluripotent stem cell derived retinal organoids offers new insight in retinal development

人类多能干细胞来源的视网膜类器官的空间转录组学为视网膜发育提供了新的见解

• 发表时间: 2022
• 期刊: INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
• 影响因子: 4.4
• 作者: Dorgau Birthe