Investigating the Role of NKX2-1 in Early Human Lung Development Using an Induced Pluripotent Stem Cell (iPSC) Model

使用诱导多能干细胞 (iPSC) 模型研究 NKX2-1 在人类早期肺发育中的作用

• 批准号: 10537255
• 负责人: Taylor Matte
• 金额: $ 4.68万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537255
• 关键词: Address; Adopted; Affect; Alveolar; Alveolar Cell; Binding; Binding Sites; Biological Assay; Birth; Brain; CRISPR interference; Cell Differentiation process; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coupling; Data; Defect; Development; Disease; Distal; Epithelial; Epithelial Cells; Flow Cytometry; Gene Expression; Generations; Genes; Genetic; Genomics; Homeobox; Human; Human Development; Hypothyroidism; Knockout Mice; Laboratories; Lead; Link; Lung; Lung Adenocarcinoma; Lung diseases; Maintenance; Malignant Neoplasms; Maps; Molecular; Mutation; Natural regeneration; Neurologic; Patients; Pattern; Phenotype; Play; Preparation; Proteins; Protocols documentation; Regenerative Medicine; Research Personnel; Resolution; Respiratory Insufficiency; Role; Stomach; Study models; Syndrome; System; TAC1 gene; Technology; Testing; Thyroid Diseases; Thyroid Gland; Time; Tissue Sample; Tissues; Up-Regulation; Work; alveolar type II cell; base; cell type; deviant; differentiation protocol; directed differentiation; experimental study; fetal; gene function; gene regulatory network; genetic manipulation; human fetus tissue; human tissue; immunocytochemistry; in vivo; induced pluripotent stem cell; lentiviral-mediated; lung development; mRNA sequencing; mutant; progenitor; programs; pulmonary function; single cell mRNA sequencing; stem cell differentiation; stem cell model; stem cells; surfactant; transcription factor; transcriptome; transcriptomics; tumor progression

NK2 Homeobox 1 (NKX2-1) is a critically important transcription factor in lung development, with all lung epithelia being derived from an NKX2-1+ progenitor pool. Nkx2-1 knockout mice have hypoplastic lungs, and humans with NKX2-1 mutations suffer from respiratory insufficiency, hypothyroidism, and neurological problems, a disease known broadly as brain-lung-thyroid syndrome. It is known that NKX2-1’s importance is derived from its ability to influence the expression of downstream target genes, which include genes like surfactant proteins and secretoglobins, which contribute significantly to lung function. Despite its known importance, NKX2-1’s role in early human development is not fully defined. Studies into early human development have been significantly hindered by difficulties in access to human fetal tissue and issues with genetically manipulating fetal derived cells. A recently developed technology that allows researchers to investigate early human development is the induced pluripotent stem cell (iPSC) system, which allows for generation and subsequent differentiation of stem cells to tissue types of choice. Our lab and others have developed protocols to generate airway and alveolar cells from iPSCs, actively recapitulating development along the way. Our group has also gained expertise in technologies to characterize and manipulate our cells, including single cell transcriptomics and CRISPR interference (CRISPRi). In this project, we seek to synergistically combine the components of this scientific toolbox to study how NKX2-1 influences early human development. Using our iPSC system, we seek to test the hypothesis that NKX2-1 plays a central role in lung specification and patterning, and that the downstream targets of NKX2-1 are context dependent and vary based on cell-type and developmental time point. We seek to do this through genomic binding assays to identify NKX2-1 binding loci at several points in development. In addition, we will perform single cell mRNA sequencing with an NKX2-1 mutant cell line in comparison with an isogenic cell line with this mutation corrected. Using these two approaches we hope to identify NKX2-1’s binding loci across development, and to observe the molecular consequences that come with an NKX2-1 mutations. We also seek to modulate expression of downstream targets of NKX2-1, including SOX2, TP63, and NKX2-1 itself to further deduce the gene regulatory network by which NKX2-1 acts. Our iPSC-based model allows for the unprecedented coupling of genetic manipulation and high-resolution transcriptomics to further understand how a critical transcription factor influences development.

NK2 同源框 1 (NKX2-1) 是肺中极其重要的转录因子 发育，所有肺上皮均源自 NKX2-1+ 祖细胞库。 NKX2-1 基因敲除小鼠肺部发育不良，携带 NKX2-1 突变的人类患有 呼吸功能不全、甲状腺功能减退和神经系统问题，这是一种众所周知的疾病 如脑-肺-甲状腺综合征。众所周知，NKX2-1的重要性源自其能力 影响下游靶基因的表达，包括表面活性剂等基因 蛋白质和分泌球蛋白，对肺功能有显着贡献。尽管众所周知 NKX2-1 在人类早期发育中的作用尚未完全确定。 对早期人类发展的研究因困难而受到严重阻碍 获取人类胎儿组织以及对胎儿衍生细胞进行基因操作的问题。一个 最近开发的技术使研究人员能够研究早期人类发展 是诱导多能干细胞 (iPSC) 系统，可产生和后续 干细胞分化为所选组织类型。我们的实验室和其他人已经开发了 从 iPSC 生成气道和肺泡细胞的方案，积极重现发育 一路上。我们的团队还获得了技术方面的专业知识来表征和 操纵我们的细胞，包括单细胞转录组学和 CRISPR 干扰 (CRISPRi)。 在这个项目中，我们寻求协同地结合这个科学工具箱的组件 研究 NKX2-1 如何影响早期人类发育。 使用我们的 iPSC 系统，我们试图检验 NKX2-1 发挥核心作用的假设 NKX2-1 的下游靶点是背景 依赖并根据细胞类型和发育时间点而变化。我们力求做到这一点 通过基因组结合测定来识别 NKX2-1 中多个点的结合位点 发展。此外，我们将使用 NKX2-1 突变体进行单细胞 mRNA 测序 细胞系与纠正该突变的同基因细胞系进行比较。使用这两个 我们希望通过这些方法识别 NKX2-1 在整个发育过程中的结合位点，并观察 NKX2-1 突变带来的分子后果。我们还寻求调节 NKX2-1 下游靶标的表达，包括 SOX2、TP63 和 NKX2-1 本身 进一步推论NKX2-1发挥作用的基因调控网络。我们基于 iPSC 的模型 允许基因操作和高分辨率的前所未有的耦合 转录组学以进一步了解关键转录因子如何影响发育。