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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

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.
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f2: 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.

Mentions: We next decided to characterize the capacity of the NKX2.1+/FOXP2+ lung progenitors to mature into a more adult-like airway epithelium. In the mouse, the lung bud branches and invades the surrounding mesenchyme in a process largely driven by FGF10 from the surrounding mesenchyme. The production of distal airway epithelial cells that occurs gradually over the canalicular and saccular stages of FL development is also dependant on a continuous FGF signal. We therefore decided to test the effect of prolonged FGF10 signaling on the NKX2.1/FOXP2-positive population. Cells cultured in medium containing FGF10 and HGF for 10 days and then FGF10 alone for a further 15 days continued to express markers indicative of the distal tip airway epithelium (NKX2.1 and GATA6) and downregulated the lung bud markers (FOXP2) (Fig. 2A), suggesting that cells were adopting a more distal REC fate. Indeed, FGF10 exposure induced expression of several mature lung epithelium markers, predominantly characteristic of AECTII (ABCA3, CFTR, MUC1, SFTPB, and SFTPC) and AECTI (AQP5, PDPN and P2X7) (Fig. 2A and data not shown) in the absence of markers of other lineages such as the forebrain (PAX6) or the thyroid (HHEX, PAX8, PAX9, and TG; Supplementary Fig. S2A). Coexpression of NKX2.1 and the AECTII marker Pro-SFTPC; Pro-SFTPC and the epithelial marker CK18; as well as a cell surface marker CD26 and CFTR was further confirmed by immunostaining (Fig. 2B; Supplementary Fig. S2B). Of note, CFTR is expressed in a variety of endodermal cell types, including cells of the intestines and pancreas; however, co-immunocytochemistry of the CFTR with PDX1 (Pancreas) or CDX2 (intestine) revealed cells that were negative for these lineage markers and instead expressed high levels of the distal airway marker NKX2.1 (Supplementary Fig. S2D). Flow cytometric analyses revealed that 80% of cells expressed NKX2.1 and CFTR, and 70% expressed Pro-SFTPC (Fig. 2C). Importantly, the resulting cells did not exhibit significant expression of secretory cell markers (MUC5AC) or markers of ciliated cells (FOXJ1, SOX17), suggesting that FGF10 supports development of more distal lineages rather than proximal airway cell types (Supplementary Fig. S2A).


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)

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.
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Related In: Results  -  Collection

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f2: 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.
Mentions: We next decided to characterize the capacity of the NKX2.1+/FOXP2+ lung progenitors to mature into a more adult-like airway epithelium. In the mouse, the lung bud branches and invades the surrounding mesenchyme in a process largely driven by FGF10 from the surrounding mesenchyme. The production of distal airway epithelial cells that occurs gradually over the canalicular and saccular stages of FL development is also dependant on a continuous FGF signal. We therefore decided to test the effect of prolonged FGF10 signaling on the NKX2.1/FOXP2-positive population. Cells cultured in medium containing FGF10 and HGF for 10 days and then FGF10 alone for a further 15 days continued to express markers indicative of the distal tip airway epithelium (NKX2.1 and GATA6) and downregulated the lung bud markers (FOXP2) (Fig. 2A), suggesting that cells were adopting a more distal REC fate. Indeed, FGF10 exposure induced expression of several mature lung epithelium markers, predominantly characteristic of AECTII (ABCA3, CFTR, MUC1, SFTPB, and SFTPC) and AECTI (AQP5, PDPN and P2X7) (Fig. 2A and data not shown) in the absence of markers of other lineages such as the forebrain (PAX6) or the thyroid (HHEX, PAX8, PAX9, and TG; Supplementary Fig. S2A). Coexpression of NKX2.1 and the AECTII marker Pro-SFTPC; Pro-SFTPC and the epithelial marker CK18; as well as a cell surface marker CD26 and CFTR was further confirmed by immunostaining (Fig. 2B; Supplementary Fig. S2B). Of note, CFTR is expressed in a variety of endodermal cell types, including cells of the intestines and pancreas; however, co-immunocytochemistry of the CFTR with PDX1 (Pancreas) or CDX2 (intestine) revealed cells that were negative for these lineage markers and instead expressed high levels of the distal airway marker NKX2.1 (Supplementary Fig. S2D). Flow cytometric analyses revealed that 80% of cells expressed NKX2.1 and CFTR, and 70% expressed Pro-SFTPC (Fig. 2C). Importantly, the resulting cells did not exhibit significant expression of secretory cell markers (MUC5AC) or markers of ciliated cells (FOXJ1, SOX17), suggesting that FGF10 supports development of more distal lineages rather than proximal airway cell types (Supplementary Fig. S2A).

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