High Resolution Spatial Transcriptomics using seq-FISH+

使用 seq-FISH 进行高分辨率空间转录组学

• 批准号: 10703380
• 负责人: John E Harris
• 金额: $ 36.4万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-12 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703380
• 关键词: Address; Affect; Antibodies; Atlases; Autoimmunity; Biological Process; Biopsy; Bulla; Cell Communication; Cells; Center for Translational Science Activities; Clone Cells; Communication; Complement; Complex; Data; Dendritic Cells; Dermal; Epidermis; Equilibrium; Fluorescent in Situ Hybridization; Freezing; Gene Targeting; Genes; Genetic Transcription; Goals; Image; Immune; In Situ; Individual; Inflammation; Inflammatory; Knowledge; Label; Learning; Lesion; Ligands; Location; Maps; Messenger RNA; Microscope; Modification; Normal tissue morphology; Organ; Pathogenesis; Pigments; Population; Positioning Attribute; Privatization; Process; Punch Biopsy; RNA; Receptor Cell; Research; Resolution; Signal Transduction; Skin; Spatial Distribution; Structure; T cell clonality; T cell receptor repertoire sequencing; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Tissues; Transcript; Vitiligo; White Spots; cell type; cellular imaging; design; health disparity; immune cell infiltrate; improved; in silico; innovation; insight; interest; keratinocyte; melanocyte; multiple omics; receptor; receptor expression; single-cell RNA sequencing; skin disorder; transcriptome; transcriptomics; treatment strategy

Much has been learned about mechanisms that drive autoimmunity by studying vitiligo, a dis- ease of the skin that results from destruction of pigment-producing melanocytes. This results in disfiguring white spots that are particularly devastating for those with darker skin, which thus leads to health disparities for the most vulnerable of our population. We generated an extensive set of preliminary data using single cell RNA sequencing (scRNA-Seq) that revealed hun- dreds of conversations that are unique to vitiligo lesions involving every cell type of the epider- mis. These data indicate that cells and their signaling networks within affected and unaffected vitiligo skin are in fact more complex than previously appreciated. Yet because scRNA-Seq re- quires disruption of the tissue, the position of each cell and their communications within the skin remains unknown. We hypothesize that cells connected in silico by matching induced ligand-receptor expression are in close physical proximity in situ, and that T cell clones marked by identical T cell receptor (TCR) sequences associate in physical contact with melanocytes. The objective of the High- Resolution Spatial Transcriptomics Research Core using sequential fluorescence in situ hybridi- zation (seqFISH+) is to use our highly innovative platform to provide spatial context to T cell clonality, cellular positioning, and cell-cell communications revealed by the complementary, syn- ergistic Center Projects 1+2. Our seqFISH+ platform provides a cutting-edge, innovative ap- proach to map RNA expression in tissues with high accuracy and sub-diffraction-limit resolution, allowing for identification of cell classes, expressed genes, and spatial organization in tissues. This approach will be applied in the following specific aims: 1) Identify the position and distribu- tion of T cell clones marked by unique TCR sequences within tissue; and 2) Reveal the spatial organization of communicating cells by receptor-ligand mapping directly within vitiligo lesions. We will use punch biopsies taken from the same vitiligo lesions that have been analyzed by sin- gle cell techniques in each Project. We will design seqFISH+ probes that complement TCR se- quences and ligand-receptor pairs for hybridization, which will indicate the spatial distribution of T cell clonality and cell-cell communications through ligand-receptor pairing. This Research core combines a highly innovative conceptual approach with cutting-edge technology. The prelimi- nary data, investigative team, and innovative plan provides a robust and unique opportunity for discovery of the fundamental mechanisms by which immune cells target self-tissues, which may lead to improved treatment strategies.

通过研究白癜风，已经了解了很多关于驱动自身免疫的机制， 由于产生色素的黑色素细胞被破坏而导致的皮肤松弛。这导致 毁容的白色斑点，特别是对那些具有较深的皮肤， 导致我们人口中最弱势群体的健康差距。我们生成了大量 使用单细胞RNA测序（scRNA-Seq）的一组初步数据显示匈奴 一系列的对话是独特的白癜风病变涉及每一种细胞类型的表皮- 小姐这些数据表明，受影响和未受影响的细胞及其信号网络 白癜风的皮肤实际上比以前认识到的更复杂。然而，由于scRNA-Seq重新- 需要破坏组织，每个细胞的位置和它们在皮肤内的通讯 仍然未知。 我们假设，通过计算机连接的细胞通过匹配诱导配体-受体表达 在原位物理上非常接近，并且由相同的T细胞受体标记的T细胞克隆 (TCR)序列与黑素细胞物理接触。高的目标-- 分辨率空间转录组学研究核心使用顺序荧光原位杂交- zization（seqFISH+）是使用我们高度创新的平台，为T细胞提供空间背景， 克隆性，细胞定位，和细胞间通信所揭示的互补，同步， 工程中心项目1+2。我们的seqFISH+平台提供了一个尖端的，创新的应用程序， 以高精度和亚衍射极限分辨率绘制组织中RNA表达的方法， 允许鉴定细胞类别、表达的基因和组织中的空间组织。 这种方法将应用于以下具体目标：1）确定位置和分布- 组织内由独特TCR序列标记的T细胞克隆的分离;以及2）揭示了组织内由独特TCR序列标记的T细胞克隆的空间分布。 通过受体-配体直接定位在白癜风病变内的通讯细胞的组织。 我们将使用从相同的白癜风病变，已通过单克隆抗体分析的打孔活检， 在每个项目中的网格技术。我们将设计与TCR互补的seqFISH+探针， 序列和配体-受体对进行杂交，这将指示 通过配体-受体配对的T细胞克隆性和细胞间通讯。本研究核心 将高度创新的概念方法与尖端技术相结合。关于Prelimi- 无数据、调查团队和创新计划为以下方面提供了强大而独特的机会： 发现免疫细胞靶向自身组织的基本机制， 从而改进治疗策略。