Defining Islet Heterogeneity Using Single Islet Transcriptomics

使用单胰岛转录组学定义胰岛异质性

• 批准号: 8812982
• 负责人: MARTHA CAMPBELL-THOMPSON
• 金额: $ 159.77万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-19 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8812982
• 关键词: Address; Animal Model; Animals; Antibodies; Area; Autoantibodies; Beta Cell; Biological; CD3 Antigens; Cell Death; Cell Survival; Cells; Cellular Stress; Collaborations; Complex; Critical Pathways; Crude Extracts; Cryoultramicrotomy; Data; Development; Diabetes Mellitus; Diabetes autoantibodies; Disease; Disease Marker; Fluorescence Microscopy; Freezing; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Head; Heterogeneity; Human; Immune; Immune System and Related Disorders; Immunofluorescence Immunologic; Individual; Inflammation; Insulin; Insulin-Dependent Diabetes Mellitus; Lasers; Leukocytes; Measures; Metabolic stress; Methods; Microdissection; Microscope; Mission; Molecular; Molecular Profiling; Organ; Organ Donor; Pancreas; Paraffin Embedding; Pathway interactions; Pattern; Phenotype; Process; Process Measure; Protocols documentation; RNA; Reporting; Research; Research Personnel; Serum; Staging; Staining method; Stains; Stress; Surveys; System; T-Lymphocyte; Tail; Technology; Therapeutic; Time; Tissues; Viral Markers; Virus Diseases; base; cell injury; diabetic; insight; interest; islet; laser capture microdissection; new technology; non-diabetic; novel; novel strategies; novel therapeutics; prevent; public health relevance; research and development; tool; transcriptomics; type I diabetic

DESCRIPTION (provided by applicant): Our studies are focused on examination of single islet gene expression profiles (transcriptomes) to determine pathways critical to human beta cell death and survival. In preliminary studies we have developed new approaches and protocols that allow laser-capture of individual islets from cryosections of human pancreas, extract the RNA, and obtain comprehensive transcriptomes. Our proposal to the HIRN-CBDS consortium is based on using and expanding these proven protocols to gain deeper insights into islet heterogeneity and differences in gene expression patterns of individual islets from individuals with and without diabetes and with or without serum autoantibodies (AAB). Based on preliminary data in islets from non-diabetic individuals with or without AAB, we hypothesize that islets develop a gene expression signature of "cellular stress" before the early stages of insuliti. A full characterization of early abnormalities in islet gene expression signatures could point to novel mechanisms of disease and new therapeutic strategies that target islet abnormalities to delay or prevent beta cell death. The use of comprehensive gene expression profiling of thousands of expressed genes will allow a very detailed look at specific pathways and processes. Our proposal has the following specific aims: Specific Aim 1. To characterize transcriptomes of individual islets in a survey of islets from different regions of the pancreas an from organ donors with or without autoantibodies (AAB) and with or without diabetes. This will provide an unbiased survey of islet gene expression and its heterogeneity. Specific Aim 2. To characterize transcriptomes of individual islets selected by rapid immuno-laser capture microdissection for the presence or absence of insulin-positive beta cells and presence or absence of CD3 T-cells. In this aim we will obtain expression profiles from islets that have been selected based on multiple fluorescent stains for beta cells and insulitis. We will develop protocols for using rapid immunofluorescence that provide high quality RNA and then directly microdissect islets, across serial sections, based on their staining patterns. Specific Aim 3. To characterize transcriptomes of individual islets selected for staining patterns of markers of viral presence and metabolic stress. In this aim we will use our newly acquired ability to conduct multiplexed immunofluorescence to define islets with novel complex phenotypes indicative of stress due to viral infections or metabolic stress. In this aim phenotypes will be highly complex and we will stain one tissue section and then capture individual islets of interest from adjacent serial tissue sections. Mechanistic studies using isolated islets will follow from all specific aim as new pathways of cellular destruction or survival are discovered.

描述（由申请人提供）：我们的研究集中在检查单个胰岛基因表达谱（转录组），以确定对人β细胞死亡和存活至关重要的途径。在初步研究中，我们已经开发出新的方法和协议，允许激光捕获的个人胰岛从冷冻切片的人胰腺，提取RNA，并获得全面的转录组。我们向HIRN-CBDS联盟提出的建议是基于使用和扩展这些经过验证的方案，以更深入地了解胰岛异质性和来自患有和不患有糖尿病以及有或没有血清自身抗体（AAB）的个体的单个胰岛基因表达模式的差异。基于有或没有AAB的非糖尿病个体的胰岛中的初步数据，我们假设胰岛在胰岛炎的早期阶段之前产生了"细胞应激"的基因表达特征。胰岛基因表达特征早期异常的全面表征可能指向新的疾病机制和新的治疗策略，靶向胰岛异常以延迟或预防β细胞死亡。使用数千个表达基因的综合基因表达谱将允许非常详细地查看特定的途径和过程。我们的建议有以下具体目标：具体目标1。在对来自胰腺不同区域的胰岛以及来自有或无自身抗体（AAB）和有或无糖尿病的器官供体的胰岛的调查中，表征单个胰岛的转录组。这将提供一个公正的调查胰岛基因表达及其异质性。具体目标2。表征通过快速免疫激光捕获显微切割选择的单个胰岛的转录组，以确定是否存在胰岛素阳性β细胞和是否存在CD3 T细胞。在这个目标中，我们将获得表达谱的胰岛，已选定的基础上，多种荧光染色的β细胞和胰岛炎。我们将开发使用快速免疫荧光的方案，提供高质量的RNA，然后直接显微解剖胰岛，在连续切片，基于他们的染色模式。具体目标3。表征选择用于病毒标志物染色模式的单个胰岛的转录组， 存在和代谢应激。在这一目标中，我们将使用我们新获得的进行多重免疫荧光的能力来定义具有新的复杂表型的胰岛，该表型指示由于病毒感染或代谢应激而引起的应激。在这个目标中，表型将是高度复杂的，我们将对一个组织切片进行染色，然后从相邻的连续组织切片中捕获感兴趣的单个胰岛。随着细胞破坏或存活的新途径的发现，使用分离的胰岛的机制研究将遵循所有特定的目标。

项目成果

Alpha cell receptor for advanced glycation end products associate with glucagon expression in type 1 diabetes.

• DOI: 10.1038/s41598-023-39243-x
• 发表时间: 2023-08-09
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Leung, Sherman S.;Lenchik, Nataliya;Mathews, Clayton;Pugliese, Alberto;McCarthy, Domenica A.;Le Bagge, Selena;Ewing, Adam;Harris, Mark;Radford, Kristen J.;Borg, Danielle J.;Gerling, Ivan;Forbes, Josephine M.
• 通讯作者: Forbes, Josephine M.

Genetic predisposition in the 2'-5'A pathway in the development of type 1 diabetes: potential contribution to dysregulation of innate antiviral immunity.

• DOI: 10.1007/s00125-021-05469-5
• 发表时间: 2021-08
• 期刊: Diabetologia
• 影响因子: 8.2
• 作者: Pedersen K;Haupt-Jorgensen M;Krogvold L;Kaur S;Gerling IC;Pociot F;Dahl-Jørgensen K;Buschard K
• 通讯作者: Buschard K

The IGFBP3/TMEM219 pathway regulates beta cell homeostasis.

• DOI: 10.1038/s41467-022-28360-2
• 发表时间: 2022-02-03
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: D'Addio F;Maestroni A;Assi E;Ben Nasr M;Amabile G;Usuelli V;Loretelli C;Bertuzzi F;Antonioli B;Cardarelli F;El Essawy B;Solini A;Gerling IC;Bianchi C;Becchi G;Mazzucchelli S;Corradi D;Fadini GP;Foschi D;Markmann JF;Orsi E;Škrha J Jr;Camboni MG;Abdi R;James Shapiro AM;Folli F;Ludvigsson J;Del Prato S;Zuccotti G;Fiorina P
• 通讯作者: Fiorina P

Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes.

• DOI: 10.2337/db15-0779
• 发表时间: 2016-03
• 期刊: Diabetes
• 影响因子: 7.7
• 作者: Campbell-Thompson M;Fu A;Kaddis JS;Wasserfall C;Schatz DA;Pugliese A;Atkinson MA
• 通讯作者: Atkinson MA

Abnormal islet sphingolipid metabolism in type 1 diabetes.

• DOI: 10.1007/s00125-018-4614-2
• 发表时间: 2018-07
• 期刊: Diabetologia
• 影响因子: 8.2
• 作者: Holm LJ;Krogvold L;Hasselby JP;Kaur S;Claessens LA;Russell MA;Mathews CE;Hanssen KF;Morgan NG;Koeleman BPC;Roep BO;Gerling IC;Pociot F;Dahl-Jørgensen K;Buschard K
• 通讯作者: Buschard K