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Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells
Finn Hawkins, … , Brian R. Davis, Darrell N. Kotton
Finn Hawkins, … , Brian R. Davis, Darrell N. Kotton
Published May 2, 2017
Citation Information: J Clin Invest. 2017;127(6):2277-2294. https://doi.org/10.1172/JCI89950.
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Research Article Pulmonology Article has an altmetric score of 83

Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells

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Abstract

It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1–expressing (NKX2-1+) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.

Authors

Finn Hawkins, Philipp Kramer, Anjali Jacob, Ian Driver, Dylan C. Thomas, Katherine B. McCauley, Nicholas Skvir, Ana M. Crane, Anita A. Kurmann, Anthony N. Hollenberg, Sinead Nguyen, Brandon G. Wong, Ahmad S. Khalil, Sarah X.L. Huang, Susan Guttentag, Jason R. Rock, John M. Shannon, Brian R. Davis, Darrell N. Kotton

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Figure 4

Time series global transcriptomic profiling of human iPSCs undergoing lung-directed differentiation.

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Time series global transcriptomic profiling of human iPSCs undergoing lu...
(A) Schematic overview of the lung-directed differentiation with time points and populations analyzed by microarrays. (B) Principal component analysis (PCA) of global transcriptomes of the biological triplicates from time points shown in A. (C) Unsupervised hierarchical clustering by dendrogram of the samples shown in A, based on the top ~1,000 transcripts differentially expressed as determined by ANOVA across all 27 samples. (D) Heatmap of the top 20 transcription factors differentially expressed in each direction (10 up and 10 down) between neural NKX2-1GFP+ and day 15 lung NKX2-1GFP+ (15+) populations (ranked by fold change; filtered by FDR < 0.01). (E) Heatmap of the top 10 genes differentially expressed between day 15 lung NKX2-1GFP+ (15+) and day 15 lung NKX2-1GFP– (15–; ranked by fold change; filtered by FDR < 0.01). (F) Heatmap of the expression of known markers of neuroectoderm, endoderm, and lung epithelium (separated into progenitor stage and distal/alveolar vs. proximal/airway epithelium). Scale = row-normalized log2 expression. All differentiation samples were from C17 iPSCs.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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