Stem Cell Reports Article Single-Cell Transcriptomic Profiling of Pluripotent Stem Cell-Derived SCGB3A2+ Airway Epithelium Katherine B. McCauley, 1,2 Konstantinos-Dionysios Alysandratos, 1,2 Anjali Jacob, 1,2 Finn Hawkins, 1,2 Ignacio S. Caballero, 1 Marall Vedaie, 1,2 Wenli Yang, 3 Katherine J. Slovik, 3 Michael Morley, 3 Gianni Carraro, 4 Seunghyi Kook, 5 Susan H. Guttentag, 5 Barry R. Stripp, 4 Edward E. Morrisey, 3 and Darrell N. Kotton 1,2, * 1 Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118, USA 2 The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA 3 Penn Center for Pulmonary Biology and Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 4 Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA 5 Department of Pediatrics, Monroe Carell Jr. Children’s Hospital, Vanderbilt University, Nashville, TN 37232, USA *Correspondence: dkotton@bu.edu https://doi.org/10.1016/j.stemcr.2018.03.013 SUMMARY Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lin- eages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to pheno- typic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell pro- gram, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway. INTRODUCTION The mature respiratory epithelium comprises two major compartments, the distal alveoli and the proximal airway, both of which include numerous functional cell types. The various cell types of the airways, including goblet, secretory, and ciliated cells, form a mucociliary escalator that protects the lung from pathogens and other inhaled agents. The lung secretory (or club) cell lineage represents an abundant and heterogeneous cell type localized to the conducting airways. These cells provide critical protective functions to the lung by participating in immune modula- tion, oxidative stress reduction, and xenobiotic meta- bolism. Club cells are also capable of differentiation to other proximal cell types, are a major known facultative progenitor for ciliated and goblet cells, and can reconstitute the airway epithelium after injury (Boers et al., 1999; Mango et al., 1998; Rawlins et al., 2009). Differentiated lung club cells are marked by the expres- sion of club cell secretory protein (Cc10/Ccsp/Scgb1a1). In addition to Scgb1a1, secretoglobin family members, including Scgb3a1 and Scgb3a2, are expressed in heteroge- neous populations of secretory cells and are important to the function of these cells in immune homeostasis and mediation of oxidant-induced stress (Mango et al., 1998; Reynolds et al., 2002). In particular, expression of Scgb3a2 has been reported in proximal epithelial progenitors in lung development prior to maturation of the Scgb1a1+ club cell lineage, and is the earliest known marker of differ- entiation to this fate (Guha et al., 2012). The in vitro directed differentiation of pluripotent stem cells (PSCs) via sequential regulation of developmental signaling pathways has been established as a model to study early stages of human development that are other- wise difficult to examine in vivo. In addition, lung epithelia generated from patient-specific induced PSCs (iPSCs) using this approach (Huang et al., 2013) have the potential to provide primary-like cells for disease modeling and drug testing. We have previously described a protocol for gener- ating functional proximalized airway spheres expressing markers of multiple airway epithelial cell types, including basal and secretory lineages. These cells can be further differentiated to multiciliated cells in the context of Notch inhibition or air-liquid interface culture (McCauley et al., 2017). While the expression of lineage-specific markers, including secretory cell markers SCGB1A1 and SCGB3A2, as well as basal cell markers TP63 and KRT5, suggest that PSC-derived airway spheres contain defined lung lineages, questions remain about the identity, heterogeneity, and long-term phenotypic stability of these cells, as well as the establishment and segregation of these separate line- ages during airway sphere formation and how these findings relate to in vivo murine biology. The PSC model system has suggested that manipulation of key signaling pathways can regulate the sequence of lung endodermal Stem Cell Reports j Vol. 10 j 1579–1595 j May 8, 2018 j ª 2018 The Author(s). 1579 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).