Sources and Regulation of Epithelial Stem/Progenitor Cells in Alveolar Regeneration

项目摘要/摘要 严重急性损伤后正常肺功能的重述暗示着天生的再生能力 肺部。然而，肺上皮干/祖细胞的来源和相对再生能力仍然存在 不清楚，尤其是在人类的肺部。根据损伤的类型和严重程度，几个不同的前体 被激活，并通过增殖和分化来响应，以帮助接近完全恢复。无论是呼吸道还是 肺泡干/祖细胞被激活，并有助于严重创伤后的肺泡修复，如 流感或博莱霉素。为此，我们实验室最近的研究发现了一种呼吸道上皮祖细胞 主要组织相容性复合体(MHC)I类蛋白H2-K1水平升高的细胞。尽管 与成熟的杆状细胞不同，H2-K1高分化细胞的转录组与成熟的杆状细胞高度相似 细胞在损伤后选择性增殖，并有助于改善原位损伤小鼠的氧合 移植。然而，这些祖细胞早期和选择性激活的机制仍然存在。 未知。此外，远端肺再生还有其他几个方面尚不清楚。酋长 其中之一是我们在小鼠肺中观察到的远端上皮祖细胞层级是否保持不变 在人类的远端肺中。人肺远端有较高比例的基底细胞和较多的 异质性分泌细胞群比小鼠呼吸道上皮细胞多。此外，人类2型 肺泡上皮细胞(AEC2)在体外具有显著的增殖和再生能力。因此， 还有一个尚未得到满足的需求，那就是了解人类远端呼吸道和肺泡的特性和调节 祖先。为此，我们的初步数据显示，人类远端的分泌亚群是 类似于小鼠H2-K1的高杆状祖细胞，在体外可以产生AEC2。相反，我们有 发现了一种新的和意想不到的能力，成熟的人类AEC2分化为呼吸道谱系 体外和体内，这表明至少有两个来源的远端上皮再生在人类。因此，它是 对明确小鼠和人类远端上皮祖细胞的身份和特征至关重要 用于牙槽修复。本提案试图通过三个目标来回答这些问题：1)确定 H_2-K_1高祖细胞在肺泡修复中的激活机制。2)鉴定远端分泌细胞 作为人肺远端损伤后肺泡细胞的来源。3)确定一个子种群是否 成熟的人AEC2细胞具有可逆的双向分化为肺泡基底细胞的潜能。这些 AIMS将利用单细胞信使核糖核酸和开放染色质测序，对体外和体内操作提出建议 信号通路和祖细胞的原位移植以阐明上皮干/祖细胞 远端小鼠和人肺的细胞层级。这些研究将为审问几个 新的信号通路最终帮助我们努力操纵再生机制来实现 更好的疾病结局。

PROJECT SUMMARY/ABSTRACT Recapitulation of normal lung function following a severe acute injury implies an inherent regenerative ability of the lung. However, sources and relative regenerative capacities of lung epithelial stem/progenitor cells remain unclear, especially in the human lung. Depending on the injury type and severity, several distinct progenitors are activated and respond by proliferating and differentiating to aid in near complete recovery. Both airway and alveolar stem/progenitor cells are activated and contribute to alveolar repair following severe injuries such as influenza or bleomycin. To this end, recent studies from our lab have uncovered an airway epithelial progenitor cell marked by elevated levels of Major Histocompatibility Complex (MHC) Class I protein, H2-K1. Despite having a transcriptome highly similar to the mature club cells, the H2-K1high progenitors, unlike mature club cells, selectively proliferate post injury and aid in improved oxygenation in injured mice after orthotopic transplantation. However, mechanisms underlying early and selective activation of these progenitors remain unknown. In addition, there are several more aspects of distal lung regeneration that are yet unclear. The chief among them is whether the distal epithelial progenitor hierarchy that we observe in mouse lungs is maintained in distal human lungs. The distal human lung airways have a higher proportion of basal cells and have more heterogeneous secretory cell populations than the mouse airway epithelium. Furthermore, the human type 2 alveolar epithelial cells (AEC2s) have remarkable in vitro proliferative and regenerative capacity. Therefore, there is an unmet need to understand the identity and regulation of distal human airway and alveolar progenitors. To this end, our preliminary data show that a distal human secretory subpopulation that is analogous to the mouse H2-K1high club-like progenitors can give rise to AEC2s in vitro. Conversely, we have uncovered a novel and unexpected ability of mature human AEC2s to differentiate towards airway lineages in vitro and in vivo, suggesting at least two sources of distal epithelial regeneration in the human. Therefore, it is critical to clarify the identity and characteristics of both distal mouse and human epithelial progenitors primed for alveolar repair. This proposal seeks to answer these questions through three aims: 1) Determine the mechanisms underlying activation of H2-K1high progenitors in alveolar repair. 2) To identify distal secretory cells as a source of alveolar cells post injury in the distal human lung. 3) Determine whether a subpopulation of mature human AEC2s has reversible bi-directional potential to differentiate into alveolar basal cells. These aims will utilize single cell mRNA and open chromatin sequencing, in vitro and in vivo manipulation of proposed signaling pathways, and orthotopic transplantation of progenitor cells to clarify the epithelial stem/progenitor cell hierarchy in distal mouse and human lungs. These studies will lay the foundation for interrogating several novel signaling pathways to ultimately aid in our efforts to manipulate regenerative mechanisms to achieve better disease outcomes.