Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells - PubMed (original) (raw)

Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells

Nicholas R F Hannan et al. Stem Cells Dev. 2015.

Abstract

Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

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Figures

**FIG. 1.

RA and FGF10 pattern foregut endoderm into NKX2.1/FOXP2-positive lung endoderm. (A) Protocol to produce lung endoderm progenitors and distal lung epithelium. (B) Quantitative polymerase chain reaction (QPCR) analysis showing hFSCs cultured in medium containing FGF10 with or without additional growth factors express early lung progenitor genes (NKX2.1, FOXP2, and GATA6) and do not express more mature lung genes (SOX17, FOXJ1) or genes associated with thyroid progenitors (HHEX). (C) Immunocytochemistry showing hFSCs cultured in medium containing FGF10 and RA coexpress early lung progenitor genes (NKX2.1, FOXP2), while hFSCs cultured in the presence of FGF inhibitor SU5402, FGF10 without RA, or FGF2 do not express these genes. _White bars_=100 μM. *_P_≤0.05, **_P_≤0.01, ***_P_≤0.001, ****_P_≤0.0001. RA, retinoic acid; FGF, fibroblast growth factor; hFSCs, human foregut stem cells; FL, human fetal lung control; AL, adult lung control; C, undifferentiated hESC control; hESC, human embryonic stem cell; ns, not significant.

**FIG. 2.

NKX2.1/FOXP2-positive lung progenitors mature into distal AEC. (A) QPCR analysis showing NKX2.1+/FOXP2+-positive lung progenitor cells cultured for a further 25 days downregulate lung bud markers (FOXP2) and express genes of the distal airway epithelium (NKX2.1, GATA6), AECTII (SFTPC, SFTPB, ABCA3, and P2X7), and AECTI (AQP5). (B) Immunocytochemistry showing matured lung endoderm coexpressing distal airway markers (Pro-SFPTC, CK18, and NKX2.1). (C) Flow cytometric analysis showing percentages of cells positive for distal airways markers (CFTR, NKX2.1, and Pro-SFTPC). Red shading=positive stained Gray shading=isotype/secondary control. (D) SFTPC enzyme-linked immunosorbent assay detection of SFTPC in tissue culture medium from cultures of matured distal airway epithelium. (E) Fluorescent trace of Cl− indicator dye (MQAE) showing CFTR activity in matured airway epithelium. No fluorescent signal was detected within lung epithelium using Cl− containing medium (_t_=0 s), CFTR activity was shown by an increase in cell fluorescence in medium containing NO3− (_t_=15 s) and then a loss of fluorescence when Cl− medium was added back to cells (_t_=115 s). Cells treated with NO3− medium and a CFTR inhibitor showed no increase in fluorescence inside lung epithelial cells (_t_=230 s). _White bars_=100 μM. *_P_≤0.05, ***_P_≤0.001, ****_P_≤0.0001. AEC, alveolar epithelial cell; CFTR, cystic fibrosis transmembrane conductance regulator; FL, human foetal lung control; AL, adult lung control; C, undifferentiated hESC control; D25, airway epithelium cultured for 25 days; MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromine.

**FIG. 3.

CFTR-Δf508-derived hFSCs can differentiate into lung epithelium. (A) QPCR analysis showing that hFSCs derived from patients with the CFTR mutation Δf508 can differentiate into early lung endoderm and express early markers (NKX2.1, FOXP2, GATA6, and FOXA2) while they are negative for more mature markers (FOXJ1, SOX2, and SOX17) and thyroid markers (HHEX). (B) QPCR analysis showing that CFTR mutant lung progenitors can develop into mature airway epithelium and continue to express distal airway markers (NKX2.1, GATA6) as well as AECTII markers (SFTPB, SFTPC, and ABCA3) and AECTI markers (AQP5, P2X7, and PDPN). (C) Immunocytochemistry showing distal airway epithelium expresses NKX2.1, SFTPC, and CK18. (D) Confocal microscopy showing localization of Pro-SFTPC in the cytoplasm of the distal airway epithelium. (E) Trace of chloride influx and efflux in mature airway epithelium from CFTR-human-induced pluripotent stem cell showing the temperature sensitivity of CFTR. Chloride influx and efflux was measured in mature airway epithelium expressing the wild-type CFTR (green trace) cultured at 37°C, the Δf508 mutation (red trace) cultured at 37°C, and the Δf508 mutation (blue trace) cultured at 28°C. Addition of Cl− or NO3− indicated with solid arrowheads. _White bars_=100 μM. *_P_≤0.05, **_P_≤0.01, ***_P_≤0.001, ****_P_≤0.0001. FL, human fetal lung control; AL, adult lung control; C, undifferentiated hESC control; Δf508, airway epithelium from cystic fibrosis patients cultured for 25 days.

**FIG. 4.

The CFTR-Δf508 mis-folded receptor can be rescued to a functional state using the small molecule VX809. (A) Chloride influx and efflux trace in mature airway epithelial cells expressing the wild-type CFTR (red trace), the ΔF508 mutation in the CFTR (blue trace), or the ΔF508 mutation in the CFTR but treated with the small molecule VX809 (green trace). (B) Confocal microscopy showing expression of CFTR in matured airway epithelium from wild-type cells, cells with the ΔF508 mutation in the CFTR, and cells with the ΔF508 mutation in the CFTR but treated with the small molecule VX809. _White bars_=100 μM. Addition of Cl− or NO3− indicated with solid arrowheads.

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Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells - PubMed (original) (raw)