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Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

Hannan NR, Sampaziotis F, Segeritz CP, Hanley NA, Vallier L - Stem Cells Dev. (2015)

Bottom Line: 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.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 is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm.

View Article: PubMed Central - PubMed

Affiliation: 1 Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge , Cambridge, United Kingdom .

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: The Δf508 constitutes the most common mutation in CF. Skin fibroblasts obtained from a patient carrying the CFTR Δf508 mutation were reprogrammed into hIPSCs using non-integrative Sendai virus expressing the four factors Oct-4, Sox2, C-Myc, and KLF-4 [24]. The resulting CF-hIPSC lines were characterized for their pluripotent state, capacity of differentiation into the three germ layers, and absence of transgene integration as previously described (Supplementary Fig. S4A and data not shown) [19]. CF-hIPSCs were then differentiated into distal AECs as described earlier (Fig. 3A–D; Supplementary Fig. S4B, C), and the functionality of the CFTR protein in the resulting cells was tested using the MQAE assay. As expected, CF-derived lung cells did not show a change in fluorescence, indicating their CFTR was not functional (Fig. 3E). Interestingly, the Δf508 mutation causes a temperature-sensitive mis-folding of the CFTR whereby the activity of the Δf508-CFTR has been shown to be normal at ∼28°C with mis-folding at 37°C [25–27]. Accordingly, the MQAE assay showed a change in fluorescence in CF-hFSCs-derived AECs grown at 28°C for 6 h before the MQAE assay, demonstrating that the functionality of CFTR could be restored by a reduction in temperature (Fig. 3E). Taken together, these data demonstrate that our culture system enables the production of lung epithelial cells that recapitulate the basic biology of the Δf508 mutation in vitro.


Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

Hannan NR, Sampaziotis F, Segeritz CP, Hanley NA, Vallier L - Stem Cells Dev. (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: 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.
Mentions: The Δf508 constitutes the most common mutation in CF. Skin fibroblasts obtained from a patient carrying the CFTR Δf508 mutation were reprogrammed into hIPSCs using non-integrative Sendai virus expressing the four factors Oct-4, Sox2, C-Myc, and KLF-4 [24]. The resulting CF-hIPSC lines were characterized for their pluripotent state, capacity of differentiation into the three germ layers, and absence of transgene integration as previously described (Supplementary Fig. S4A and data not shown) [19]. CF-hIPSCs were then differentiated into distal AECs as described earlier (Fig. 3A–D; Supplementary Fig. S4B, C), and the functionality of the CFTR protein in the resulting cells was tested using the MQAE assay. As expected, CF-derived lung cells did not show a change in fluorescence, indicating their CFTR was not functional (Fig. 3E). Interestingly, the Δf508 mutation causes a temperature-sensitive mis-folding of the CFTR whereby the activity of the Δf508-CFTR has been shown to be normal at ∼28°C with mis-folding at 37°C [25–27]. Accordingly, the MQAE assay showed a change in fluorescence in CF-hFSCs-derived AECs grown at 28°C for 6 h before the MQAE assay, demonstrating that the functionality of CFTR could be restored by a reduction in temperature (Fig. 3E). Taken together, these data demonstrate that our culture system enables the production of lung epithelial cells that recapitulate the basic biology of the Δf508 mutation in vitro.

Bottom Line: 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.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 is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm.

View Article: PubMed Central - PubMed

Affiliation: 1 Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge , Cambridge, United Kingdom .

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.

No MeSH data available.


Related in: MedlinePlus