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

基本信息

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

项目摘要

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.

被告知您患有无法治疗的视力障碍可能很难接受，但这是全世界 2.85 亿人必须承受的负担。 26% 的整体失明是由视网膜功能障碍引起的，视网膜是位于眼睛后部的最内部的光敏感组织，对于光传感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遗传的错误基因的影响以及损害的积累和各种疾病的影响而导致的。我们预防和治疗视力丧失的能力与我们对“视网膜如何形成”以及何时以及可能出现问题的了解密切相关。我们的视网膜主要在出生前发育；因此，这一时期可供研究的组织非常有限。我的团队在弥合这一差距方面处于独特的地位，通过与人类发育生物学资源中心的密切和良好的合作，能够接触到人类视网膜，该资源中心在母亲同意的情况下从流产的胚胎和胎儿中收集样本。我们还具有在实验室中创建称为“视网膜类器官”的三维结构的优势，它类似于人类视网膜在发育过程中的形成，并包含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形成的方式和时间，以及在有缺陷时导致视力丧失的基因的作用。视网膜是一个复杂的组织，由七种细胞类型组成：这些细胞在视网膜发育过程中的不同时间点出现。我们的发育是从一组祖细胞开始的，这些祖细胞本身是异质的，具有不同的子集，表明随着时间的推移，它们会在简明的过程中产生不同的细胞类型。因此，依靠细胞混合物研究的传统研究方法很难精确定位产生构成人类视网膜的所有细胞类型的祖细胞。在这里，我们建议使用一种称为单细胞分析的重要新技术，它使我们能够查看群体中每个细胞中哪些基因被打开。单细胞水平的基因表达是精确分类细胞的非常可靠的工具，使我们能够识别在显微镜下观察其形状或位置时不易察觉的细胞类型。我们将以此为第一步，探索发育中的视网膜和我们实验室生成的视网膜类器官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，我们将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相匹配，预测它们的最终命运，并评估实验室生成的细胞的接近程度。视网膜类器官模仿人类视网膜的发育。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。这种方法以“伪时间”组织细胞，预测细胞命运决定的顺序和模式，使我们能够预测在树的分支点周围占据特殊位置的基因。第三，我们将应用一种新方法，使我们能够将单个细胞的基因表达谱与其在视网膜中的位置相关联，从而创建视网膜发育时的空间图。该空间图将使我们能够验证在分支点附近发现的关键基因的表达，这对于理解祖细胞最终成为视网膜细胞的决定可能很重要。最后，我们将评估分支点周围表达的基因是否通过操纵其表达在控制细胞命运决策中发挥积极作用。这些信息将提供给所有科学家和临床医生，以帮助他们了解视网膜发育和疾病。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

人类视网膜类器官为毒理学研究提供了合适的工具：使用影响视网膜的药物和化合物进行综合验证。

DOI：
http://dx.10.1093/stcltm/szab010
发表时间：
2022
期刊：
Stem cells translational medicine
影响因子：
6
作者：
Dorgau B
通讯作者：
Dorgau B

Light-responsive microRNA molecules in human retinal organoids are differentially regulated by distinct wavelengths of light

人视网膜类器官中的光响应 microRNA 分子受到不同波长的光的差异调节

DOI：
http://dx.10.1016/j.isci.2023.107237
发表时间：
2023
期刊：
iScience
影响因子：
5.8
作者：
Celiker C
通讯作者：
Celiker C

Single-cell RNA sequencing reveals transcriptional changes of human choroidal and retinal pigment epithelium cells during fetal development, in healthy adult and intermediate age-related macular degeneration

单细胞 RNA 测序揭示了胎儿发育过程中、健康成人和中年年龄相关性黄斑变性中人类脉络膜和视网膜色素上皮细胞的转录变化

DOI：
10.1093/hmg/ddad007
发表时间：
2023-05-05
期刊：
Human Molecular Genetics
影响因子：
3.5
作者：
J. Collin;Megan S R Hasoon;Darin Zerti;Sarah Hammadi;B. Dorgau;L. Clarke;David H Steel;Rafiqul Hussain;J. Coxhead;S. Lisgo;R. Queen;M. Lako
通讯作者：
M. Lako

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