Inferring Gene Regulatory Networks Governing Definitive Endoderm Differentiation from Single Cell RNA Velocity Measurements

从单细胞 RNA 速度测量推断控制定形内胚层分化的基因调控网络

• 批准号: 10544286
• 负责人: Jolene Sarah Ranek
• 金额: $ 3.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-02 至 2024-04-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544286
• 关键词: Air Movements; Algorithms; Automobile Driving; Back; Breathing; CRISPR interference; Cell Lineage; Cell Therapy; Cell physiology; Cells; Chronic Obstructive Pulmonary Disease; Computing Methodologies; Consensus; Coupling; Data; Decision Making; Development; Differential Equation; Disease; Disease model; Encapsulated; Endoderm; Endoderm Cell; Event; Fellowship; Fibroblast Growth Factor; Foundations; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Heterogeneity; Individual; Knowledge; Lung; Lung diseases; Measurement; Metabolism; Methods; Modeling; Molecular; Morphogenesis; Natural regeneration; Pathway interactions; Persons; Pluripotent Stem Cells; Population; Prevalence; Process; Protocols documentation; Quality of life; RNA; RNA Splicing; Regulator Genes; Research; Role; SOX17 gene; Structure of parenchyma of lung; Testing; Time; Tissue-Specific Gene Expression; Training; Transcript; Transforming Growth Factor beta; Undifferentiated; Variant; World Health Organization; cell fate specification; cell type; computational pipelines; computer studies; differentiation protocol; directed differentiation; experimental study; gene interaction; gene network; gene regulatory network; human embryonic stem cell; human pluripotent stem cell; improved; insight; lung development; lung injury; mathematical model; models and simulation; novel; pluripotency; precursor cell; regenerative; self-renewal; single-cell RNA sequencing; skills; small molecule; stem cell biology; stem cells; temporal measurement; transcription factor; transdifferentiation

Project Summary The World Health Organization estimates that over 65 million people suffer from moderate to severe chronic obstructive pulmonary disease, a condition characterized by poor airflow and restricted breathing 1. The ability to regenerate damaged lung tissue would dramatically improve the quality of life for these individuals while reducing the prevalence and burden of pulmonary diseases worldwide. A promising approach to this problem is to use human pluripotent stem cells to produce lung and airway progenitor cells. Indeed, specialized protocols have been developed to convert stem cells into definitive endoderm, a lung precursor cell type 10-19. These protocols use small molecules to modulate the expression of key regulators of lung development including WNT, TGFβ, BMP, and FGF; however, these protocols are limited by the inability to generate a homogeneous population of definitive endoderm cells 11,15. This problem necessitates a better mechanistic understanding of how individual cells transition from their pluripotent cell state into definitive endoderm. Specifically, there is a critical need to understand how the gene regulatory networks in a given cell control its morphogenesis, proliferation, and differentiation decisions. Therefore, with the long-term goal of increasing homogeneity in lung precursor cells, the research objective of this fellowship is to determine how transcriptional heterogeneity in human embryonic stem cells influences their commitment to definitive endoderm. I hypothesize that heterogeneity in the starting population of cells generates alternate trajectories to definitive endoderm (or other cell types) and that these differences increase over time due to mutual inhibition between specific pairs of transcription factors (e.g., OCT4/SOX17, NANOG/GATA6). To test this hypothesis, I will first use single-cell RNA sequencing26 to define the transcriptional heterogeneity in human pluripotent stem cells during differentiation to definitive endoderm. I will then quantify the time-dependent changes in gene expression for each cell using RNA velocity, a computational method that uses spliced and unspliced transcript counts to estimate future gene expression states 28-29. Using these single-cell measurements, I will then develop a mechanistic model of the gene regulatory networks governing differentiation to DE and validate the model using known gene-gene interactions. Model simulations will: (1) confirm major gene regulators that drive differentiation; (2) identify novel gene networks that control heterogeneity before and during differentiation; and (3) reveal crosstalk among gene regulatory networks governing differentiation and other ongoing cellular processes such as proliferation and metabolism. The proposed experimental and computational studies provide a general framework to systematically identify gene regulatory mechanisms controlling differentiation to definitive endoderm and aid in the development of more efficient and homogeneous differentiation/transdifferentiation protocols for regenerative cellular therapies.

项目摘要 世界卫生组织估计，超过6500万人患有中等至重度的慢性病 阻塞性肺部疾病，这种疾病的特征是气流不良和呼吸受限1。 再生受损的肺组织将显着改善这些人的生活质量 减少全球肺部疾病的患病率和燃烧。解决这个问题的一种有希望的方法 是使用人多能干细胞产生肺和气道祖细胞。确实，专业 已经开发了方案将干细胞转化为确定的内胚层，一种肺前体细胞型10-19。 这些方案使用小分子调节肺发育的关键调节剂的表达 包括Wnt，TGFβ，BMP和FGF；但是，这些协议受到无法生成a的限制 确定的内胚层细胞的同质种群11,15。这个问题是更好的机械 了解单个细胞如何从多能细胞状态转变为确定的内胚层。 具体而言，迫切需要了解给定细胞中的基因调节网络如何控制其 形态发生，增殖和分化决策。因此，以长期目标的增加 肺前体细胞中的同质性，该研究金的研究目标是确定如何 人胚胎干细胞中的转录异质性影响其对确定性的承诺 内胚层。我假设细胞起始群体中的异质性产生了替代轨迹 确定的内胚层（或其他细胞类型），这些差异随着时间的推移而增加 在特定的转录因子对之间（例如Oct4/Sox17，Nanog/Gata6）。为了检验这一假设，我 将首先使用单细胞RNA测序26来定义人多能茎中的转录异质性 分化与确定内胚层的细胞。然后，我将量化基因的时间依赖性变化 使用RNA速度来表达每个单元格，一种使用剪接和未剪接的转录本的计算方法 估计未来基因表达的计数28-29。使用这些单细胞测量值，我将 开发一个管理分化的基因调节网络的机械模型，以验证并验证 使用已知的基因 - 基因相互作用的模型。模型模拟将：（1）确认驱动的主要基因调节剂 分化； （2）确定在分化之前和期间控制异质性的新型基因网络；和 （3）揭示了控制分化和其他持续细胞的基因调节网络之间的串扰 诸如增殖和新陈代谢之类的过程。提出的实验和计算研究 提供一个通用框架，以系统地识别控制分化为分化的基因调节机制 确定的内胚层，并有助于开发更有效和均匀的 再生性细胞疗法的分化/跨分化方案。