Single-Cell Transcriptomic Profiling of Pluripotent Stem Cell-Derived SCGB3A2+ Airway Epithelium

Abstract

SummaryLung 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 lineages 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 phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, 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.

Single-cell time-series mapping of cell fate trajectories reveals an expanded developmental potential for human PSC-derived distal lung progenitors

“…For this small panel we observed that expression of surfactant-encoding and lamellar bodyrelated genes, SFTPB, SFTPC, SFTPA2, ABCA3, CLDN18 and LAMP3, increased over time to a maximum at days 25-29 and deceased thereafter ( Figure 2C), while NKX2-1 remained constant. As expected, lung epithelial markers were relatively depleted in the outgrowth of GFP negative controls, consistent with our prior reports that all PSC-derived human lung epithelial lineages derive via the gateway of an NKX2-1+ primordial progenitor stage Jacob et al, 2017;McCauley et al, 2018;McCauley et al, 2017;Serra et al, 2017). TPS identified an inflection point when using the optimal 6 time points (days 15 17, 21, 25, 29 and 31).…”

“…As a negative control we included a 7 th sample, the day 15 non lung population, isolated based on GFP exclusion (NKX2-1 GFP negative sorted). Flow cytometry monitoring of NKX2-1 and SFTPC locus activity at each of the 6 time points of alveolar differentiation ( Figure 3B) indicated the expected emergence of SFTPC tdTomato expression over time in some cells, peaking on day 29 of differentiation, but a loss of NKX2-1 GFP expression in other cells over time, predicting potential loss of lung cell fate in a subset of the population, consistent with our prior reports (McCauley et al, 2018).…”

“…We and others have published in vitro PSC directed differentiation protocols which reduce the duration of this analogous in vivo developmental window to 2 weeks in vitro as PSC-derived lung endodermal precursors (NKX2-1+ primordial lung progenitors) are differentiated in culture into lung alveolar epithelial type 2 cells (iAEC2s), the life-long facultative progenitors of the alveolar epithelium (Gotoh et al, 2014;Huang et al, 2014;Jacob et al, 2017). We have further shown that despite sorting to purity for the earliest known lung progenitors, identified by NKX2-1 expression, during directed differentiation the resulting cells are plastic, transcriptomically heterogenous, and tend to drift over time into a variety of both lung and non-lung molecular phenotypes (McCauley et al, 2018).…”

Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults

“…The remaining cells include macrophages (light blue), epithelial cells (circled), and leukocytes such as lymphocytes and dendritic cells (lower left). Conventional markers for select cell types, namely KLK7, MUC5AC, and FOXJ1 for epithelial cells (McCauley et al, 2018), CSF3R for neutrophils (Ancuta et al, 2009), CSF1R for macrophages 20 (Lavin et al, 2015), TLR9 for plasmacytoid dendritic cells, and TRAC and TRDC for T lymphocytes, further delineated cell heterogeneity inside each broad cluster (Figure 1B-C).…”

“…scRNA-Seq permits simultaneously map human and viral transcripts in a single cell (Martin-Gayo et al, 2018;Russell et al, 2019b;Zanini et al, 2018aZanini et al, , 2018b, and has been applied to various host-virus interaction scenarios (Cristinelli and Ciuffi, 2018), including human immunodeficiency virus (HIV) (Bradley et al, 2018;Kazer et al, 2019) and hepatitis C virus (HCV) infection (McWilliam Leitch and McLauchlan, 2013). To date, scRNA-Seq in the context of influenza virus infection has 80 been applied to the A549 cell line and mouse models (Kudo et al, 2019;Russell et al, 2018Russell et al, , 2019aSteuerman et al, 2018;Vera et al, 2019). However, given that such studies cannot recapitulate the natural course of influenza infection, an investigation of the response to influenza virus infection and viral replication efficiency in its natural setting is warranted.…”