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

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

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 6

Single-cell RNA sequencing of sorted and unsorted iPSC-derived cells reveals NKX2-1+ lung and NKX2-1– non-lung lineages and suggests markers for their identification.

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Single-cell RNA sequencing of sorted and unsorted iPSC-derived cells rev...
(A) Schematic of the single-cell capture and global RNA sequencing of sorted C17 NKX2-1GFP+ and unsorted BU3 iPSCs on day 15 of lung-directed differentiation. (B) PCA of the top 150 most variant genes of all sequenced cells reveals 4 cell clusters, color coded to match the clusters shown in panel C. (C) Heatmap of gene expression (global Z score) with unsupervised hierarchical clustering of all 153 cells (x axis) and the top 150 most variant genes (y axis). Dendrograms as well as colored boxes indicate 4 cell clusters (CC1–4; matching colors shown in panel B). y-axis dendrograms and thick black lines indicate 3 gene clusters (GC1–3). Key genes indicated on right. CC1 = orange, CC2 = blue, CC3 = red, CC4 = green. (D) Top 10 genes correlated with NKX2-1 expression. (E) Unsupervised cell clustering using Monocle’s pseudotime spanning tree analysis reveals 7 cell states. Individual cells are labeled in subsequent panels by NKX2-1 level, genetic background (iPSC clone), or cell cycle (mitosis), respectively. (F) Pseudotime plots of expression levels of NKX2-1, CD47, SOX9, NFIB, FGB, and APOA2 with cells colored based on the 7 states determined in E. (G) Immunostaining of RUES2-derived day 15 cells for NKX2-1 (red) and SOX9 (green) nuclear proteins. Nuclei counterstained with DAPI. Scale bars: 25 μm.