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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

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.
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f1: 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.

Mentions: To generate early lung progenitors in vitro from hFSCs, we decided to study the effect of multiple growth factors known to direct lung specification, including FGF10 and RA [14,15,17]. We first treated hFSCs with a combination of FGF2, FGF4, and FGF10 (Fig. 1A, B) and found that all three FGFs are sufficient to induce expression of lung bud markers NKX2.1, GATA6, IRX1, and FOXA2 at levels similar to those of human FL (Fig. 1B and data not shown). However, in contrast to FGF10, FGF2 induced HHEX and PAX8 mRNA expression along with NKX2.1, suggesting that this pathway could promote hFSCs differentiation into a thyroid-like cell population (Fig. 1B, C; Supplementary Fig. S1A). Analyses of other signaling pathways such as transforming growth factor (TGF)β revealed that they did not play a significant role in inducing the pulmonary endoderm phenotype (Supplementary Fig. S1A, B). Considered together, these results demonstrate that FGF signaling is sufficient and necessary for specification of hFSCs into lung and thyroid progenitors with FGF2 inducing thyroid markers and FGF10 more specific for lung markers.


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)

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

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getmorefigures.php?uid=PMC4499787&req=5

f1: 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.
Mentions: To generate early lung progenitors in vitro from hFSCs, we decided to study the effect of multiple growth factors known to direct lung specification, including FGF10 and RA [14,15,17]. We first treated hFSCs with a combination of FGF2, FGF4, and FGF10 (Fig. 1A, B) and found that all three FGFs are sufficient to induce expression of lung bud markers NKX2.1, GATA6, IRX1, and FOXA2 at levels similar to those of human FL (Fig. 1B and data not shown). However, in contrast to FGF10, FGF2 induced HHEX and PAX8 mRNA expression along with NKX2.1, suggesting that this pathway could promote hFSCs differentiation into a thyroid-like cell population (Fig. 1B, C; Supplementary Fig. S1A). Analyses of other signaling pathways such as transforming growth factor (TGF)β revealed that they did not play a significant role in inducing the pulmonary endoderm phenotype (Supplementary Fig. S1A, B). Considered together, these results demonstrate that FGF signaling is sufficient and necessary for specification of hFSCs into lung and thyroid progenitors with FGF2 inducing thyroid markers and FGF10 more specific for lung markers.

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