Characterization of a New Epithelial Cell in the Developing Human Lung

项目摘要 肺芽经历了一个称为分枝形态发生的过程，它建立了复杂的、树枝状的 肺的网络1-7。在分枝过程中，在分枝的顶端有一群专门的肺祖细胞， 芽尖祖细胞(BTP)可分化为各种类型的肺上皮细胞，包括近端呼吸道的细胞 (气管、支气管)和远端肺(肺泡)细胞1，8-16。 我们最近对10到21周的人胎肺进行了单细胞RNA测序(ScRNAseq) 16，并鉴定了一种新的上皮细胞群体，其特征是独特的基因表达谱： SCGB3A2 HI/SFTPB HI/CFTRHI，我们称之为胎儿呼吸道分泌细胞(Fas)。我们已经证明了 在培养中生长的人BTP可产生Fas细胞，并已在原位小气道中得到验证。 我的初步数据表明，Fas细胞是暂时的，在发育过程中数量不断减少。此外，我们的 数据表明，Fas细胞是人类独有的，在小鼠中不存在。因为对此一无所知 新发现的种群，这一建议的目标是审问功能、分化 Fas细胞的潜能和调控。 我的初步数据表明：(A)Fas细胞可能是一种发育的前体状态，当BTP (B)Fas细胞的发育受Notch信号调节，a 细胞命运决定的关键调控因子17，18。根据我的初步数据，我 假设Fas细胞是一个过渡性祖细胞群体，它能产生所有类型的呼吸道细胞和 Notch信号调节从BTP到Fas到承诺的呼吸道细胞类型的转变。使用一种新的电池 条形码谱系追踪方法在人肺器官模型中，我的目的是1)测试Fas细胞的功能， 尤其是当它们分化为近端呼吸道细胞时。我的目标也是研究缺口信号的作用 在Fas细胞中，使用功能获得和功能丧失的慢病毒结构、缺口阻断抗体和 在类有机物模型中激活或抑制缺口信号的药物抑制剂。

PROJECT ABSTRACT The lung buds undergo a process known as branching morphogenesis, which establishes the complex, arborized network of the lung1–7. During branching, a specialized population of lung progenitors at the tips of the branches, bud tip progenitors (BTPs), give rise to all epithelial cell types of the lung including cells of the proximal airway (trachea, bronchi) and cells of the distal lung (alveoli)1,8–16. We recently performed single-cell RNA sequencing (scRNAseq) on the human fetal lung from 10 to 21 weeks gestation16, and identified a novel epithelial cell population characterized by a unique gene expression profile: SCGB3A2HI/SFTPB HI/CFTRHI which we refer to as Fetal Airway Secretory (FAS) cells. We have shown that human BTPs grown in culture can give rise to FAS cells, and they have been verified in the small airways in situ. My preliminary data suggest that FAS cells are transient, dwindling in number across development. Further, our data suggests that FAS cells are uniquely human, and do not exist in mice. Since nothing is known about this newly identified population, the goal of this proposal is to interrogate the function, differentiation potential, and regulation of FAS cells. My preliminary data suggests that: (a) FAS cells may be a developmental precursor state that exists while BTPs are en route to becoming proximal airway and (b) FAS cell development is regulated by NOTCH signaling, a critical regulator of cell fate decision making in the developing lung17,18. Based on my preliminary data, I hypothesize that FAS cells are a transitional progenitor population that give rise to all airway cell types and NOTCH signaling regulates the transition from BTP-to-FAS to committed airway cell types. Using a novel cell barcoding lineage tracing approach in a human lung organoid model, I aim to 1) test the function of FAS cells, specifically if they differentiate into proximal airway cells. I also aim to 2) investigate the role of NOTCH signaling in FAS cells using gain-of-function and loss-of-function lentiviral constructs, NOTCH blocking antibodies, and pharmacologic inhibitors that activate or inhibit NOTCH signaling in an organoid model.