Optimization and validation of single-nucleus RNA sequencing for non-human primate BPD lungs

非人灵长类 BPD 肺单核 RNA 测序的优化和验证

基本信息

批准号：
10570177
负责人：
SULE CATALTEPE
金额：
$ 21.98万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-10 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10570177
关键词：
Affect Alveolar Animal Model Animals Archives Biological Biomedical Research Bronchopulmonary Dysplasia Cell Nucleus Cells Cessation of life Chronic Collaborations Complex Complication Data Set Department chair Development Disease Elements Ensure Epidemiology Evidence based treatment Extremely Low Birth Weight Infant Formalin Foundations Freezing Funding Gene Expression Profile Gene Expression Regulation Genomic approach Genomics Gestational Age Goals Grant Health Health Sciences Immunofluorescence Immunologic Immunohistochemistry Impairment In Situ Hybridization Infant Interruption Knowledge Laboratories Letters Lung Maps Modeling Molecular Molecular Abnormality National Heart, Lung, and Blood Institute Neonatal Neurodevelopmental Impairment Newborn Infant Oxygen Therapy Care Papio Paraffin Embedding Pathogenesis Pathology Perinatal Care Personal Communication Pregnancy Premature Birth Primates Protocols documentation Quality Control RNA Research Research Personnel Research Proposals Resolution Resources Sampling Specimen Steroids Structure of parenchyma of lung Techniques Technology Texas Time Tissues United States Universities Validation Vascularization antenatal biobank cell type cohort cost high risk hospital readmission improved insight lung development new therapeutic target nonhuman primate novel pediatric department power analysis premature lungs preservation programs respiratory morbidity single nucleus RNA-sequencing single-cell RNA sequencing supplemental oxygen surfactant replacement transcriptome transcriptome sequencing validation studies ventilation

项目摘要

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth, affecting approximately 18,000 infants annually in the United States. BPD causes interrupted alveolarization and vascularization in the lung, resulting in life-long chronic respiratory morbidity. Currently, there is a limited understanding of the underlying molecular pathogenesis and a shortage of evidence-based treatments for BPD. One of the most relevant animal models of BPD was generated in preterm baboons through an NHLBI-funded U01 program. As one of the past investigators of this program, we have an archive of frozen and paraffin-embedded lung tissue specimens from this model. Recent advances in single-cell genomic approaches have transformed our ability to interrogate cell types and their functional states from complex tissues. We propose that optimization and application of single-nucleus RNA-sequencing (snRNA-Seq) to our rare biorepository of non-human primate (NHP) BPD lung specimens would provide a unique opportunity to gain novel insights into the molecular pathogenesis of BPD at a single-cell resolution. Towards this goal, we have established a collaboration with Dr. Alex K. Shalek (MIT, Ragon Institute, Broad Institute), an expert and innovator in single-cell genomics, and developed the current research proposal to leverage our banked NHP BPD lung tissue specimens. The specific aims of this proposal are to: 1) optimize and validate protocols for snRNA-seq of frozen NHP lung specimens, and 2) apply these protocols to generate single-nucleus transcriptomes of developing lungs from prematurely delivered NHP infants and lungs with evolving and established BPD. To accomplish these aims, our laboratories will optimize and validate a workflow for single-nucleus isolation from control and BPD lung specimens. Power analysis strategies will be applied to determine the number of nuclei to be sequenced from each sample to achieve statistical significance across different comparisons. We will apply vigorous quality controls (QCs) to ensure the utility of our single-nucleus transcriptional profiles. Validation studies will employ bulk RNA-Seq on frozen lung samples as well as RNA in situ hybridization (RNA-ISH), immunohistochemistry and immunofluorescence on FFPE lung specimens. Final protocols will be applied to a cohort of control and BPD lung tissues to generate transcriptome maps of the NHP lungs during normal development and in early- and late- stage BPD at 3 different time points, thus providing a longitudinal assessment of molecular alterations associated with BPD. Overall, these studies will allow us to leverage a unique and well-preserved archive of newborn NHP lung specimens and generate an essential resource to inform us on temporal and spatial gene regulation at a single cell resolution during both normal lung development and BPD progression.

支气管肺发育不良（BPD）是早产最常见的并发症，影响约美国每年有 18,000 名婴儿。 BPD 导致肺泡化和血管化中断肺，导致终生慢性呼吸道疾病。目前，人们对此的了解还很有限。潜在的分子发病机制和缺乏基于证据的 BPD 治疗方法。最有之一通过 NHLBI 资助的 U01 项目，在早产狒狒中建立了 BPD 的相关动物模型。作为作为该计划的过去研究人员之一，我们拥有冷冻和石蜡包埋的肺组织的档案该模型的样本。单细胞基因组方法的最新进展改变了我们的能力询问复杂组织中的细胞类型及其功能状态。我们建议优化和单核 RNA 测序 (snRNA-Seq) 在我们稀有的非人类灵长类动物生物样本库中的应用 (NHP) BPD 肺标本将为获得分子生物学新见解提供独特的机会。单细胞分辨率下 BPD 的发病机制。为了实现这一目标，我们与 Dr. Alex K. Shalek（麻省理工学院、Ragon 研究所、Broad 研究所），单细胞基因组学专家和创新者，制定了当前的研究计划，以利用我们储存的 NHP BPD 肺组织标本。具体的该提案的目的是：1）优化和验证冷冻 NHP 肺标本的 snRNA-seq 方案， 2）应用这些协议来生成过早发育的肺部的单核转录组分娩的 NHP 婴儿和肺部患有不断发展和确定的 BPD。为了实现这些目标，我们的实验室将优化和验证从对照和 BPD 肺样本中分离单核的工作流程。力量将应用分析策略来确定每个样本中待测序的细胞核数量在不同的比较中达到统计显着性。我们将采用严格的质量控制 (QC) 确保我们的单核转录谱的实用性。验证研究将采用批量 RNA 测序冷冻肺样本以及 RNA 原位杂交 (RNA-ISH)、免疫组织化学和 FFPE 肺样本的免疫荧光。最终方案将应用于一组对照和 BPD 肺组织生成正常发育期间以及早期和晚期 NHP 肺的转录组图谱在 3 个不同时间点进行 BPD 阶段，从而提供相关分子改变的纵向评估与边缘性人格障碍。总的来说，这些研究将使我们能够利用独特且保存完好的新生儿 NHP 档案肺部标本并生成重要资源，让我们了解基因在时间和空间上的调控正常肺部发育和 BPD 进展期间的单细胞分辨率。