Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung Progenitors

Abstract

Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life but are difficult to isolate from patients. Here, we engineer AEC2s from human pluripotent stem cells (PSCs) in vitro and use timeseries single-cell RNA sequencing with lentiviral barcoding to profile the kinetics of their differentiation in comparison to primary fetal and adult AEC2 benchmarks. We observe bifurcating cell-fate trajectories as primordial lung progenitors differentiate in vitro, with some progeny reaching their AEC2 fate target, while others diverge to alternative non-lung endodermal fates. We develop a Continuous State Hidden Markov model to identify the timing and type of signals, such as overexuberant Wnt responses, that induce some early multipotent NKX2-1 + progenitors to lose lung fate. Finally, we find that this initial developmental plasticity is regulatable and subsides over time, ultimately resulting in PSC-derived AEC2s that exhibit a stable phenotype and nearly limitless selfrenewal capacity.

Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors

“…To understand the suitability of lung cancer cell lines compared to iAT2s as an in vitro model of the alveolar epithelium, we evaluated the extent to which cancer cell lines and iAT2s transcriptomically resemble primary AT2s. We compared expression of key AT2 genes in RNA sequencing data from primary human adult AT2s and iAT2s (Jacob 2017, Hurley 2020 to RNA sequencing data from 150 lung cancer cell lines (Klijn 2015). The cell lines Calu-3, A549 and H1299, described in SARS-CoV-2 infection studies, were included (Hoffmann 2020, Letko 2020, Matsuyama 2020, Zhang Science 2020.…”

“…Expression levels of the putative host factors required for SARS-CoV-2 cellular entry, ACE2 and TMPRSS2, are potential indicators of cell susceptibility to viral infection (Hoffman 2020) and therefore model suitability. To assess whether iAT2s express ACE2 and TMPRSS2, we compared RUES2-derived iAT2s cultured as 3D epithelial spheres to freshly sorted adult HTII-280+ primary AT2s (n=3 separate adult lungs) that were profiled head to head in our previously published RNA-Seq dataset without the potentially confounding batch effects that may be introduced when merging datasets procured in separate experiments (Jacob 2017, Hurley 2020.…”

“…Libraries were sequenced using a NextSeq 500. Where indicated in the text and figures, previously published datasets were also employed (Jacob 2017, Hurley 2020, profiling iAT2s derived from the RUES2 human embryonic stem cells line.…”

Variability within rare cell states enables multiple paths towards drug resistance

“…Barcoding, in which cells are labeled by unique and sometimes mutable nucleic acid sequences (Alemany et al, 2018;Frieda et al, 2016;Kalhor et al, 2018;McKenna et al, 2016;Raj et al, 2018) , allows one to track cells by sequencing. When combined with single cell RNA sequencing methods, one can connect cellular lineages to transcriptomic profiles (Al'Khafaji et al, 2019;Biddy et al, 2018;Hurley et al, 2020;Weinreb et al, 2020) . These techniques, however, are difficult to combine with assays that measure molecular features outside of gene expression levels, which may be important to distinguish key subpopulations (Weinreb et al, 2020;Wu et al, 2020) .…”

Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids

“…In the last several years, a series of protocols have been reported to direct hPSC differentiation to various lung lineages [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . We differentiated hPSCs to lung organoids using a previously reported stepwise strategy 4,15 , including the progressive differentiation first into definitive endoderm (DE), followed by specification to anterior foregut endoderm (AFE), AFE/lung progenitor cells (LPs), and finally lung organoids (Extended Data Fig.…”