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 2

Sorted iPSC-derived NKX2-1GFP+ cells exhibit lung progenitor potential and ability to form epithelial spheroids in 3D culture.

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Sorted iPSC-derived NKX2-1GFP+ cells exhibit lung progenitor potential a...
(A) Schematic overview of organoid generation with representative phase contrast and GFP fluorescence microscopy images (day 25–28) as well as immunostaining (day 36) for NKX2-1 and EPCAM proteins. Cell nuclei are counterstained with DAPI. Each panel shows outgrowth in 3D culture of structures arising from iPSC-derived cells that were either unsorted or sorted on day 15 as GFP+ versus GFP– populations (C17). Scale bars: 100 μm (left and center columns) and 20 μm (right column). (B) Time-lapse microscopy (merged GFP fluorescence and phase contrast) of unsorted organoids over 25 hours. Arrows indicate epithelial organoids undergoing induction of the GFP reporter in real time (C17). See also Supplemental Video file. (C) FACS quantification on day 36 of the percentage of cells expressing GFP in the outgrowth wells shown in A (C17). Data indicate individual biological replicates (squares and triangles) with mean ± SD, n = 6 biological replicates. ****P ≤ 0.0001 by Student’s t test. Fold change [RT-qPCR; 2(–ΔΔCT)] in mRNA expression on day 36 compared with day 0 for each GFP+ versus GFP– outgrowth compared with fetal lung control tissue (n = 3 biological replicates) (C17). (D) Immunostaining of GFP+ outgrowth organoids on day 36 for MUC1, SFTPB, and TP63 (C17). Nuclei are counterstained with DAPI. Scale bars: 10 μm.