Salahudeen Ameen A, Choi Shannon S, Rustagi Arjun, Zhu Junjie, de la O Sean M, Flynn Ryan A, Margalef-Català Mar, Santos António J M, Ju Jihang, Batish Arpit, van Unen Vincent, Usui Tatsuya, Zheng Grace X Y, Edwards Caitlin E, Wagar Lisa E, Luca Vincent, Anchang Benedict, Nagendran Monica, Nguyen Khanh, Hart Daniel J, Terry Jessica M, Belgrader Phillip, Ziraldo Solongo B, Mikkelsen Tarjei S, Harbury Pehr B, Glenn Jeffrey S, Garcia K Christopher, Davis Mark M, Baric Ralph S, Sabatti Chiara, Amieva Manuel R, Blish Catherine A, Desai Tushar J, Kuo Calvin J
bioRxiv. 2020 Jul 27:2020.07.27.212076. doi: 10.1101/2020.07.27.212076.
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid cells contained a distinct mitotic population whose proliferation segregated to a subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A subset of FACS-purified ITGA6 ITGB4 basal cells from human lung or derivative organoids. In vivo, TNFRSF12A cells comprised ~10% of KRT5 basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 apical-out organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.
肺远端包含终末细支气管和肺泡，可促进气体交换，并受包括间质性肺疾病、癌症和SARS-CoV-2相关的新冠肺炎在内的疾病影响。由于缺乏三维体外人肺远端培养系统，对这些局部病变的研究受到了阻碍。此外，人肺远端干细胞的鉴定因细胞静止、与小鼠的解剖差异以及缺乏谱系追踪和克隆培养而受到影响。在此，我们开发了强大的无饲养层、化学成分明确的人肺远端祖细胞培养方法，将其培养成类器官，这些类器官克隆自单个成人肺II型肺泡上皮细胞（AT2）或基底细胞。AT2类器官表现出向AT1转分化的潜力，而基底细胞类器官逐渐形成由分化的杯状细胞和纤毛细胞排列的管腔。未观察到仅由杯状细胞组成的类器官。通过单细胞RNA测序（scRNA-seq），肺泡类器官由增殖性AT2细胞组成；然而，基底类器官细胞包含一个独特的有丝分裂群体，其增殖分离到一个亚群。克隆性类器官生长在来自人肺或衍生类器官的FACS纯化的ITGA6 ITGB4基底细胞的TNFRSF12A亚群中显著富集。在体内，TNFRSF12A细胞约占KRT5基底细胞的10%，并聚集在终末细支气管内。为了模拟新冠肺炎肺远端疾病，我们使基底和肺泡类器官的极性翻转，将分化的杯状细胞和纤毛细胞从类器官管腔快速重新定位到外表面，从而在向外的顶端表面展示SARS-CoV-2受体ACE2。因此，基底和AT2顶端向外的类器官被SARS-CoV-2感染，确定杯状细胞为一个新的靶细胞群体。这种长期的、无饲养层的人肺远端肺泡和基底干细胞类器官培养，结合单细胞分析，确定了未被怀疑的基底细胞功能异质性，并例证了在缓慢增殖的人体组织中祖细胞的鉴定。此外，我们的研究建立了一种简便的体外类器官模型，用于研究包括新冠肺炎相关肺炎在内的人肺远端感染性疾病。
