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 7

Cell surface profiling and prospective isolation of iPSC-derived NKX2-1+ primordial lung progenitors by CD47hiC26lo cell sorting.

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Cell surface profiling and prospective isolation of iPSC-derived NKX2-1+...
(A) Day 15 iPSCs after lung-directed differentiation, immunostained for CD47 and NKX2-1 proteins (C17). Nuclei are counterstained with DAPI. Scale bars: 50 μm. (B) Flow cytometry dot plots of 4 cell surface markers identified in a screen of 243 surface markers on day 15 of lung-directed differentiation (C17); CD26 is depleted while CD47, ALCAM, MUC1, and CPM exhibit higher expression in the GFP+ population. (C) Schematic of experimental data for C, D, and E. Flow cytometry dot plots of live day 13 cells (BU3) indicates staining with isotype control antibodies (left panel) or antibodies against CD47 and CD26. Sort gates identify presorted cells (gray box) versus a CD47hiCD26lo population (red box) or a CD47lo population (black box) profiled in D and E. (D) Fold change of NKX2-1 mRNA (left graph, n = 4) in each indicated day 13 population, and SFTPC mRNA (right graph, n = 3) expression in the outgrowth of each indicated population on day 36 compared with day 0 iPSCs by RT-qPCR; 2(–ΔΔCT). Data indicate the mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by Student’s t test. (E) GFP expression quantified in each of the gates shown in C: day 13 presort = 62% NKX2-1GFP+; CD47hiCD26lo = 97% GFP+; CD47lo = 8% GFP+. Lower panel is FACS of day 36 outgrowth of each indicated day 13 sorted population: GFP+, CD47hiCD26lo versus CD47lo. (F) Phase contrast and fluorescence microscopy (GFP) of day 42 organoids derived from day 13 sorted GFP+, CD47hiCD26lo, and CD47lo populations from E. Scale bars: 100 μm. (G) Confocal microscopy of outgrowth organoids (C17), sorted on day 13 based on CD47hiCD26lo and analyzed on day 44 by coimmunostaining for NKX2-1 (green) and pro-SFTPC (purple). The individual panels for this merge image are contained in Supplemental Figure 7E. Scale bars: 25 μm.