Developing a High-throughput Single-cell Analysis Platform for Intra- and Inter-cellular Signalling in Colonic Air Liquid Interface Organoids

开发结肠气液界面类器官细胞内和细胞间信号传导的高通量单细胞分析平台

• 批准号: 2546957
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2546957
• 关键词: Developing; throughput; Single; cell; Analysis

Accurate biomimetic models are essential for our understanding of how healthy and diseasedtissues function. Air liquid interface organoids (ALIs) have recently emerged as powerfulbiomimetic tissue models [1, 2]. Unlike traditional organoids that only contain one cell-type(e.g., epithelial cells), ALI organoids comprise all cell-types from a tissue - includingautologous immune cells. As a result, ALI organoids can be used to study how all cell-types ina tissue communicate. Unfortunately, existing ALI organoid cultures formats arecumbersome, extremely low throughput, and incompatible with many single-cell analysistechnologies [3].We hypothesise that ALI cultures can be reengineered into a miniaturised format to becompatible with high-throughput multiplexed single-cell analysis technologies.In this LIDo PhD project, we will reengineer colonic ALI organoids from a bulky 6-well formatinto a high-throughput 96-well plate format and then use our recently developed custommultiplexed thiol organoid barcoding in situ (TOBis) mass cytometry (MC) technologyplatform [4] to study cell-type-specific intracellular communication signalling networkspresent in colonic tissue. We will further compliment these studies with split-pool ligationbasedtranscriptome sequencing (SPLiT-seq) [5] single-cell RNA-sequencing (scRNA-Seq)ligand and receptor expression pairing to reveal intercellular signalling pathways between allcells in ALI organoids. By integrating MC, scRNA-Seq, and ALI organoids, this project willgenerate a new state-of-the-art technology platform for studying intra- and inter-cellularsignalling in tissues.The project will cover 4 Aims:Aim 1: Establish new miniaturised bioengineered ALI cultures from human colonic tissue forhigh-throughput applications.Aim 2: Concurrent single-cell signalling quantification of epithelial, stromal, and autologousimmune cells in ALI cultures by TOBis MC and SPLiT-Seq.Aim 3: Develop integrated intra- and inter-cellular computational signalling model of colonictissue biology from single-cell data.Aim 4: Experimentally perturb key signalling nodes in ALI organoids to define functional intercellularsignalling in tissues.

精确的仿生模型对于我们理解健康和患病组织的功能至关重要。气液界面类器官（ALI）最近已成为强大的仿生组织模型[1，2]。与仅包含一种细胞类型的传统类器官（例如，在一个实施方案中，当细胞（例如上皮细胞）是类器官时，ALI类器官包含来自组织的所有细胞类型，包括自体免疫细胞。因此，ALI类器官可用于研究组织中所有细胞类型如何交流。不幸的是，现有的ALI类器官培养物的形式是繁琐的，极低的通量，并且与许多单细胞分析技术不兼容[3]。我们假设ALI培养物可以被重新设计成一种简化的形式，以与高通量多重单细胞分析技术兼容。在这个LIDo博士项目中，我们将重组结肠ALI类器官，从庞大的6孔板格式到高通量的96孔板格式，然后使用我们最近开发的定制多重硫醇类器官原位条形码（TOBis）质谱细胞术（MC）技术平台[4]研究结肠组织中存在的细胞类型特异性细胞内通讯信号网络。我们将进一步补充这些研究与分裂池连接为基础的转录组测序（SPLiT-seq）[5]单细胞RNA测序（scRNA-Seq）配体和受体表达配对，以揭示ALI类器官中所有细胞之间的细胞间信号传导途径。通过整合MC，scRNA-Seq和ALI类器官，该项目将产生一个新的最先进的技术平台，用于研究组织中的细胞内和细胞间信号传导。该项目将涵盖4个目标：目标1：建立新的从人类结肠组织中分离的生物工程ALI培养物，用于高通量应用。通过TOBis MC和SPLiT-Seq对ALI培养物中的上皮细胞、基质细胞和自体免疫细胞进行同步单细胞信号传导定量。目的3：从单细胞数据开发集成的细胞内和细胞间的计算信号模型colonictissue biology.Aim 4：实验扰动ALI类器官中的关键信号节点，以定义组织中的功能性细胞间信号。