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

项目摘要 世界卫生组织估计，超过6500万人患有中度到重度慢性疾病 阻塞性肺病，一种以气流不畅和呼吸受限为特征的疾病1.能力 再生受损的肺组织将显著改善这些人的生活质量，同时 减少世界范围内肺部疾病的流行和负担。解决这个问题的有希望的方法 是使用人类多能干细胞来产生肺和呼吸道祖细胞。的确，专门化 已经开发出将干细胞转化为最终内胚层的方案，内胚层是一种肺前体细胞类型10-19。 这些方案使用小分子来调节肺发育的关键调节因子的表达。 包括wnt、转化生长因子β、骨形态发生蛋白和成纤维细胞生长因子；然而，这些协议受限于不能生成 确定的内胚层细胞11，15的同质群体。这个问题需要更好的机制 了解单个细胞如何从其多能细胞状态转变为最终的内胚层。 具体地说，迫切需要了解给定细胞中的基因调控网络是如何控制其 形态发生、增殖和分化的决定。因此，随着长期目标的增加， 肺前体细胞的同质性，这项研究的目标是确定如何 人类胚胎干细胞转录的异质性影响其对最终决定的承诺 内胚层。我假设起始细胞群体中的异质性产生了另一种轨迹 最终的内胚层(或其他细胞类型)，由于相互抑制，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 将首次使用单细胞RNA测序26来确定人类多能干细胞的转录异质性 细胞分化为最终的内胚层。然后我将量化基因随时间变化的情况 使用RNA速度表达每个细胞，这是一种使用剪接和非剪接转录本的计算方法 用于估计未来基因表达状态的计数28-29。使用这些单细胞测量，然后我将 建立调控DE分化的基因调控网络的机制模型，并验证 使用已知的基因-基因交互作用的模型。模型模拟将：(1)确认驱动的主要基因调控因子 分化；(2)确定在分化前和分化过程中控制异质性的新的基因网络；以及 (3)揭示控制分化和其他正在进行的细胞的基因调控网络之间的串扰 增殖和新陈代谢等过程。拟议的实验和计算研究 提供一个通用框架，系统地识别控制分化的基因调控机制 最终的内胚层和有助于开发更有效和更均匀的 再生细胞疗法的分化/转分化方案。

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.