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J Clin Invest. 2017 Jun 1;127(6):2277-2294. doi: 10.1172/JCI89950. Epub 2017 May 2.

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

Author information

Center for Regenerative Medicine, and.
The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.
Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, Texas, USA.
Department of Anatomy, UCSF, San Francisco, California, USA.
Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
Columbia Center for Translational Immunology & Columbia Center for Human Development, Columbia University Medical Center, New York, New York, USA.
Department of Pediatrics, Monroe Carell Jr. Children's Hospital, Vanderbilt University, Nashville, Tennessee, USA.
Division of Pulmonary Biology, Cincinnati Children's Hospital, Cincinnati, Ohio, USA.


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.

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