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：
10372630
负责人：
SULE CATALTEPE
金额：
$ 28.43万
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 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10372630
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 Institutes Interruption Knowledge Laboratories Letters Life 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 Small Nuclear RNA 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 innovation insight lung development new therapeutic target nonhuman primate novel pediatric department power analysis premature lungs preservation programs respiratory morbidity 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）是早产的最常见并发症，影响大约每年在美国有18,000名婴儿。 BPD导致牙槽中断和血管化肺，导致终身慢性呼吸道发病。目前，对基本的分子发病机理和BPD的循证治疗短缺。最大的 BPD的相关动物模型是通过NHLBI资助的U01程序在早产狒狒中产生的。作为该程序的过去研究人员之一，我们有一个冷冻和石蜡包裹的肺组织的档案该模型的标本。单细胞基因组方法的最新进展已改变了我们的能力从复杂组织中询问细胞类型及其功能状态。我们提出了优化，单核RNA-sequencing（SnRNA-Seq）应用于我们罕见的非人类灵长类动物（NHP）BPD肺标本将提供一个独特的机会，以获得分子的新见解 BPD在单细胞分辨率下的发病机理。为了实现这一目标，我们与博士建立了合作。 Alex K. Shalek（MIT，Ragon Institute，Broad Institute），单细胞基因组学专家和创新者，开发了当前的研究建议，以利用我们的银行NHP BPD肺组织标本。具体该建议的目的是：1）优化和验证冷冻NHP肺样本的SnRNA-Seq方案， 2）应用这些方案来生成从过早发育肺部发育的单核转录组用不断发展且已建立的BPD运送NHP婴儿和肺。为了实现这些目标，我们的实验室将优化并验证从控制和BPD肺样本中隔离单核的工作流程。力量将采用分析策略来确定从每个样品中测序的核数在不同的比较中获得统计显着性。我们将将有力的质量控制（QC）应用于确保我们的单核转录曲线的效用。验证研究将在冷冻的肺样品以及RNA原位杂交（RNA-ish），免疫组织化学和 FFPE肺标本上的免疫荧光。最终协议将应用于控制和BPD的队列肺组织在正常发育期间以及早期和晚期生成NHP肺的转录组图 BPD阶段在3个不同的时间点，因此提供了对相关的分子改变的纵向评估与BPD。总体而言，这些研究将使我们能够利用新生儿NHP的独特且保存完好的档案肺标本并产生一个必要的资源，以告知我们有关时间和空间基因调节正常肺发育和BPD进展过程中的单细胞分辨率。