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: 10708834
• 负责人: Taylor Matte
• 金额: $ 4.77万
• 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/10708834
• 关键词: Address; Adopted; Affect; Alveolar; Alveolar Cell; Binding; Binding Sites; Biological Assay; Birth; Brain; CRISPR correction; CRISPR interference; Cell Differentiation process; Cell Line; Cells; Coupling; Data; Defect; Development; Disease; Distal; Epithelial Cells; Epithelium; Flow Cytometry; Gene Expression; Generations; Genes; Genetic; Genomics; Heterozygote; 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; Pluripotent Stem Cells; Preparation; Proteins; Protocols documentation; Regenerative Medicine; Research Personnel; Resolution; Respiratory Insufficiency; Role; Specific qualifier value; Stomach; Study models; Syndrome; System; TAC1 gene; Technology; Testing; Thyroid Diseases; Thyroid Gland; Time; Tissue Sample; Tissues; Up-Regulation; Work; alveolar type II cell; 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; mutation correction; progenitor; programs; pulmonary function; single cell mRNA sequencing; stem cell differentiation; stem cell model; stem cells; surfactant; transcription factor; transcriptome; transcriptomic profiling; 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同源盒1(NKX2-1)是一种在肺组织中起重要作用的转录因子 发育，所有肺上皮细胞来源于NKX2-1+祖细胞池。Nkx2-1 基因敲除的小鼠患有发育不良的肺，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问题，这是一种广为人知的疾病 为脑-肺-甲状腺综合征。众所周知，NKX2-1‘S的重要性源于它的能力 影响下游靶基因的表达，包括表面活性物质等基因 蛋白质和分泌球蛋白，它们对肺功能有重大贡献。尽管它已知 NKX2-1的重要性，S在早期人类发育中的作用还没有完全确定。 对早期人类发育的研究因以下困难而受到严重阻碍 获取人类胎儿组织以及基因操作胎儿衍生细胞的问题。一个 最近开发的技术，使研究人员能够调查早期人类发育 是诱导多能干细胞(IPSC)系统，它允许生成和后续 干细胞分化为组织类型的选择。我们的实验室和其他实验室已经开发出 从IPSCs产生呼吸道和肺泡细胞的方案，积极概述发育 一路走来。我们的团队还获得了技术方面的专业知识，以表征和 操纵我们的细胞，包括单细胞转录和CRISPR干扰(CRISPRi)。 在这个项目中，我们寻求协同结合这个科学工具箱的组件，以 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的下游靶点是上下文 依赖于细胞，并因细胞类型和发育时间点而异。我们试图做到这一点 通过基因组结合分析确定NKX2-1结合位点 发展。此外，我们还将对NKX2-1突变体进行单细胞mRNA测序 与具有该突变的同基因细胞系相比，该突变被纠正。使用这两个 方法我们希望找出跨越发育的NKX2-1‘S结合位点，并观察 NKX2-1突变带来的分子后果。我们还试图调整 NKX2-1下游靶点的表达，包括SOX2、TP63和NKX2-1本身 进一步推断NKX2-1通过基因调控网络发挥作用。我们基于IPSC的模型 允许前所未有的基因操作和高分辨率的结合 转录学，以进一步了解关键的转录因子如何影响发育。