Optimized human cell products for research
and drug discovery

Human Lung Wild Type – Coming Soon

Human Lung Wild Type Cells

Def-Lung cells products are a population of  mature distal airway epithelium cells that express NKX2.1, GATA6, cystic fibrosis transmembrane conductance regulator (CFTR) and the cells secrete surfactant protein C (SFPTC) as well as the ABCA3 protein, a major component of the lamellar bodies that allow the secretion of surfactant proteins. Wild-type and cystic fibrosis (CF) disease modelled versions of the products are available. CF is one of the most common genetic disorders in the world, affecting as many as 1:2,000 births in Europe, with lung transplantation remaining the only effective treatment. CF is caused by mutations in the CFTR, which result in deregulation of chloride and water transport across the lung epithelium. The cell products have been used to predict the efficacy of small molecules to reverse the CFTR phenotype. For example our Def-Lung CF disease cell model has been incubated in the presence of the small molecule VX809. As expected  CF cells were unable to transport chloride, however cells treated with VX809 were able to transport chloride as efficiently as wild type Def-Lung cells using MQAE chloride influx assay. To further emphasise this result we performed immunocytochemistry using antibodies raised against human CFTR. Wild type cells show high levels of CFTR on the cell surface while delta-f-508 cells have an obvious deficiency of CFTR on the cell surface further emphasising the phenotype. After treatment with VX809 the delta-f-508 cells express levels of CFTR on the surface similar to that observed in wild type cells.

Product Specification

Product ID:
Def-LUNG WT
Format:
Fresh plate
Disease:
None (Healthy Donor)
Application:
Disease Modelling and Drug Discovery

Foregut Stem Cells Make Functional Airway Epithelium

Treating foregut stem cells with FGF10 and retinoic acid is essential to induce expression of the early lung genes NKX2.1 and FOXP2. In the absence of FGF signalling using and FGF inhibitor (SU) neither NKX2.1 or FOXP2 is expressed. Removal of retinoic acid resulted in the loss of FOXP2 expression.

Immunocytochemistry hFSCs

Figure 1. 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 = 100mM.

Lung endoderm was matured and expressed distal airway marker NKX2.1 and adult functionality marker pro-surfactant C.

Distal airway marker NKX2.1

Figure 2. Immunocytochemistry showing matured lung endoderm coexpressing disal airway markers (Pro-SFPTC, CK18, and NKX2.1).

More than 80% of the lung endoderm produced expressed CFTR and at least 80% expressed NKX2.1.

Flow cytometric analysis showing percentages of cells positive for distal airway markers

Figure 3. Flow cytometric analysis showing percentages of cells positive for distal airway markers (Pro-SFPTC, NKX2.1, and Pro-SFTPC). Red shading = positive stained Grey Shading = isotype/secondary control.

ELISA for mature surfactant protein showed that the lung epithelum is able to process and secrete surfactant. Using a chloride sensitive dye (MQAE) we showed that lung epithelium is able to transport chloride using the CFTR.

SFTPC enzyme-linked immunosorbent assay detection of SFTPC in tissue culture medium

Figure 4. SFTPC enzyme-linked immunosorbent assay detection of SFTPC in tissue culture medium from cultures of matured distal airway epithelium.

 

Chloride transport with CFTR

Figure 5. In the presence of chloride, the signal from the dye was quenched, in the absence of chloride the dye fluoresced indicating the movement of chloride in and out of the cell. When an inhibitor for CFTR was used (blue) no change in fluorescence was observed demonstrating the specificity of chloride transport with the CFTR. 

 

Cystic Fibrosis

Cystic Fibrosis (CF) is an inherited genetic disorder that affects many organs including the lungs. CF is caused by mutations in the cystic fibrosis transmemberane conductance regulator (CFTR) causing a disruption in chloride transport across the lung epithelium. This deficient chloride transport results in a thickening of lung mucous that obstructs the airways causing difficulty breathing, lung inflammation, chronic infection and ultimately sccarring and fibrosis. CF affects as many as 1:2000 births in Europe and the only effective treatment for the continuing effects of CF is a lung transplant. The worldwide average survival rate for CF is just 33 years making CF one of the most common and severe life shortening genetic diseases in the world.

 

Cystic Fibrosis

Figure 1. (A) When CFTR is functioning normally chloride is able to move down its electrochemical gradient out of the cell. Water naturally follows this osmotic gradient moving onto the airways of the lung helping to produce an aqueous layer of mucus. (B) In this case of cystic fibrosis the CFTR is rendered non-functional and as a consequence chloride is trapped within the cells of the airway epithelium. The results in reduced water movement to the lining of the airways and thickening of the mucus lining the airway. The thick mucus blocks airways, harbours bacteria that causes inflammation, difficulty breathing and fibrosis.

Foregut Stem Cells from Patients with the delta-F508 Mutation in CFTR Recapitulate the Disease in-vitro.

MQAE was used to measure chloride influx and efflux in the delta-f-508 cells compared to wild type.

Temperature dependent CFTR phenotype

Figure 2. Temperature dependent CFTR phenotype that was only present at 37C (red line) and not at 26C (blue line) when compare to wild-type cells (green line).

Platform to predict efficacy of small molecules to reverse the CFTR phenotype.

Platform to predict efficacy of small molecules to reverse the CFTR phenotype

Figure 3. Untreated delta f-508 lung epithelium were unable to transport chloride (Blue line), however cells treated with VX809 (green line) was able to transport chloride as efficiently as wild type cells (red line).

Immunocytochemistry using antibodies raised against human CFTR. Wild type cells show high levels of CFTR on the cell surface while delta-f-508 cells have an obvious deficiency of CFTR on the cell surface further emphasising the phenotype.

Immunocytochemistry using antibodies raised against human CFTR

Figure 4. After treatment with VX809 the delta-f-508 cells express levels of CFTR on the surface similar to that observed in wild type cells.