Gene regulatory networks in early lung epithelial cell fate decisions

早期肺上皮细胞命运决定中的基因调控网络

• 批准号: 10587615
• 负责人: Laertis Ikonomou
• 金额: $ 64.57万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587615
• 关键词: ATAC-seq; Address; Adult; Affect; Air; Alveolar; Animal Model; Anterior; Autologous; Binding; Binding Sites; Biological Assay; CRISPR interference; CRISPR-mediated transcriptional activation; Cell Line; Cell Lineage; Cell Transplantation; Cells; ChIP-seq; Chromatin; Chronic Obstructive Pulmonary Disease; Clinical; Competence; Complex; Computer Models; Computing Methodologies; Coupled; Cystic Fibrosis; DNA Binding; Data; Dependence; Derivation procedure; Development; Disease; Disease model; Distal; Embryo; Embryonic Development; Endoderm; Epigenetic Process; Epithelial Cells; Epithelium; Ethical Issues; FOXP2 gene; Flow Cytometry; Genetic; Genetic Models; Genetic Transcription; Goals; Heterogeneity; Human; Immunohistochemistry; In Vitro; Knowledge; Lead; Link; Liquid substance; Lung; Lung diseases; Maps; Mediator; Modeling; Morphology; Mus; Nucleic Acid Regulatory Sequences; Organism; Pathway interactions; Patients; Pattern; Pluripotent Stem Cells; Population; Primitive foregut structure; Primordium; Process; Prosencephalon; Regenerative Medicine; Regulation; Reporter; Respiratory System; Role; Signal Transduction; Sorting; Specific qualifier value; System; Techniques; Testing; Thyroid Gland; Time; Tissues; Transplantation; WNT Signaling Pathway; Work; airway epithelium; alpha 1-Antitrypsin Deficiency; alveolar epithelium; apoAI regulatory protein-1; beta catenin; cell type; clinical application; clinically relevant; differentiation protocol; directed differentiation; embryo tissue; epigenomics; gain of function; gene function; gene regulatory network; genetic manipulation; genome wide association study; homeodomain; human pluripotent stem cell; immunocytochemistry; in vitro Model; in vivo; individualized medicine; induced pluripotent stem cell; induced pluripotent stem cell technology; insight; knock-down; loss of function; lung development; mouse genetics; mouse model; multimodality; mutant; novel; overexpression; preimplantation; progenitor; programs; pulmonary function; self-renewal; single-cell RNA sequencing; spatiotemporal; stem cell differentiation; stem cell therapy; stem cells; tool; transcription factor; transcriptome sequencing; transcriptomics; zygote

PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to develop stem-cell-based, autologous therapies for diseases affecting the lung epithelium. Different potential approaches for the use of stem cells for lung disease treatment include enhancement of endogenous stem cell differentiation or in vitro differentiation of stem cells to lung lineages followed by cell transplantation. Both approaches require that the identity and pathways of differentiation of lung stem or progenitor cells be known and well characterized. The discovery of induced pluripotent stem cells (iPSCs) has opened new possibilities for regenerative medicine as these cells are easy to derive, are not fraught with ethical issues and offer the possibility of patient-specific therapies. A major roadblock in the effective application of the iPSC technology to lung regenerative medicine is the incomplete understanding of the cell fate decision repertoire that characterizes various progenitor cell types during lung differentiation. This is particularly relevant to the primordial lung progenitors, the rare cells that appear at the moment of lung specification and give rise to all lung epithelial cell types. We hypothesize that this hurdle is directly related to paucity of information on the gene regulatory network (GRN), i.e. the specific combination of transcriptional regulators and their interactions, that defines the identity of primordial lung progenitors. Understanding and manipulating the GRN of the primordial lung progenitors is the overall objective of this proposal. In Aim 1 putative new regulators of primordial lung fate will be studied. We will map their expression during in vivo lung specification and early lung epithelial differentiation. The function of these genes in lung development, will be evaluated by their deletion within the anterior foregut endoderm. In Aim 2, similar studies will be undertaken in vitro using CRISPR interference in a human NKX2-1 reporter line. We will introduce perturbations of putative regulator expression (either overexpression or knock-down) during in vitro lung specification and evaluate the competency of the resulting lung progenitors to give rise to distal and proximal lung epithelial cell types. In Aim 3, the epigenomic landscape of early epithelial lung development will be characterized. We will combine single-cell RNA-Seq with ATAC-Seq, a technique that reveals chromatin accessibility, to identify the potential regulatory regions and provide insights in early lung cell-fate decisions. ChIP-Seq will also be used to reveal the global DNA binding sites of our putative regulators and the Wnt signaling effector, β-catenin. Finally, in Aim 3 we will use computational methods to construct a proof-of-principle model of the putative GRNs governing lung primordial identity and the integration of lung specification signals, such as Wnt, with the core regulatory program. At the conclusion of our studies, we will have defined the lung primordium GRN and gained insights in the reconfiguration of this GRN during early lung development.

项目摘要/摘要 该项目的长期目标是开发基于干细胞的自体疗法，用于影响影响该疾病的疾病 肺上皮。使用干细胞进行肺部疾病治疗的不同潜在方法包括 增强内源性干细胞分化或干细胞对肺谱系的体外分化 然后进行细胞移植。两种方法都要求肺分化的身份和途径 茎或祖细胞已知并且表征良好。发现诱导的多能干细胞 （IPSC）为再生医学开辟了新的可能性，因为这些细胞很容易得出 有道德问题，并提供特定于患者的疗法的可能性。有效的主要障碍 IPSC技术在肺部再生医学上的应用是对细胞脂肪的不完全理解 肺部分化过程中各种祖细胞类型的决策库。尤其是 与原始肺祖细胞相关，肺祖细胞出现在肺部规范时刻和 引起所有肺上皮细胞类型。我们假设这个障碍与信息的匮乏直接相关 在基因调节网络（GRN）上，即转录调节器及其其特定组合 相互作用，定义了原始肺祖细胞的身份。理解和操纵 原始肺祖细胞是该提议的总体目标。 在AIM 1中，将研究原始肺命运的新调节剂。我们将在IN期间绘制它们的表达 体内肺规范和早期肺上皮分化。这些基因在肺发育中的功能， 将通过前肢内胚层内的缺失评估。在AIM 2中，类似的研究将是 在人类NKX2-1记者系列中使用CRISPR干扰在体外进行体外进行。我们将介绍扰动 在体外肺规范期间的推定调节剂表达（过表达或敲低）和 评估所得肺祖细胞的能力，从而产生远端和近端肺上皮细胞 类型。在AIM 3中，将表征早期上皮肺发育的表观基因组景观。我们将 将单细胞RNA-Seq与ATAC-Seq结合在一起，ATAC-SEQ是一种揭示染色质可及性的技术，以识别 潜在的调节区域，并提供早期肺部污染决策的见解。 chip-seq也将用于 揭示了我们推定的调节剂的全局DNA结合位点和Wnt信号效应子β-catenin。最后， 在AIM 3中，我们将使用计算方法来构建推定GRNS的原理证明模型 管理肺原始身份和肺规范信号（例如Wnt）与核心的整合 监管计划。在我们的研究结束时，我们将定义肺原始GRN并获得 在早期肺部发育过程中重新配置该GRN的见解。