The authors have declared that no competing interests exist.
Conceived and designed the experiments: SVM RL PH GJ EMW. Performed the experiments: SVM MDdJ CM. Analyzed the data: SVM MC MLG DLH DP AA. Contributed reagents/materials/analysis tools: SVM MLG MDdJ DLH DP GJ EMW AA. Wrote the paper: SVM RL GJ EMW. Facilitated funding, travel and access to Advanced Light Source: PH MC MLG. Provided expertise, maintenance and data analysis for X-ray Fluorescence Microscopy experiments: MDdJ DLH DP.
Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the
The need for a cure of cystic fibrosis remains as urgent today as ever. Since the discovery of the
We have shown that placental-derived, human amnion epithelial cells (hAECs) offer promise for such a therapy. Cells isolated from the human placenta and fetal membranes have considerable advantages over other sources of stem cells such as embryonic or bone marrow-derived cells. They are plentiful, multipotent, immunosuppressive, have a low immunogenic profile, lack tumorigenicity, and present no ethical barriers to their collection
The collection and use of human tissues were undertaken with informed, written consent and with approval of Southern Health Human Research and Ethics Committee and the Monash University Human Ethics Committee (Approval No. 01067B: Establishment of a tissue bank and associated database for use by the Centre for Women’s Health Research). Human amnion epithelial cells (hAECs) were isolated as previously described
Total RNA was isolated using TRIZOL® reagent according to manufacturer’s instructions (Invitrogen, Mulgrave, VIC, Australia). RNA concentration was measured on Nanodrop (Thermo Fisher Scientific, North Ryde, NSW, Australia) and used immediately for polymerase chain reaction (PCR). Complementary DNA (cDNA) was synthesized from 500 ng total RNA in a final reaction volume of 20 µL using Thermoscript Reverse Transcription System (Invitrogen). After denaturing the template RNA, 10 mM dNTP Mix and Oligo (dT)20 primers at 65°C for 5 minutes, 15U of ThermoScript™ Reverse Transcriptase was added in the presence of 5X cDNA Synthesis Buffer (250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM magnesium acetate, stabilizer), 0.1 M Dithiothreitol (DTT), 40 U RNaseOUT™ and RNase-free water to complete the final volume. The reaction mixture (20 µL) was incubated at 60°C for 1 h and then stopped at 85°C for 5 minutes.
Quantitative RT-PCR analysis was performed with the TaqMan® Gene Expression Assay (Hs00357011_m1) targeted against CFTR, and (Hs03928990_g1) targeted against 18 S (Invitrogen), using the Applied Biosystems 7900HT fast real time PCR system (Applied Biosystems, Mulgrave, VIC, Australia). Human lung total RNA (Ambion, Invitrogen) was used as a positive control sample for this study. Quadruplicate 20 µL reactions were performed for both CFTR and 18 S. CFTR gene expression was normalized to the 18 S endogenous control to account for variability in the initial concentration and quality of the total RNA and in the conversion efficiency of the reverse transcription reaction. Results were analysed as per manufacturer’s instructions. Briefly, Sequence Detection System (SDS) software was used to automatically determine the baseline and threshold for the amplification curves. Relative quantitation was performed using the comparative CT method, comparing a CFTR gene expression against an internal calibrator sample from day 0. CFTR gene expression was expressed as a fold change from this internal calibrator sample. This was achieved by dividing the normalized test sample by the normalized calibrator sample.
Whole cell extracts were electrophoresed in polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with blocking buffer (5% w/v non-fat dry milk, 1X TBS, 0.1% Tween-20) followed by incubation with human CFTR antibody (#MAB25031) at 1 µg/mL (R&D Systems, Minneapolis, MN, USA) incubated overnight at 4°C. Membranes were then incubated with anti-mouse IgG-HRP secondary antibody (Cell Signaling #7076, Invitrogen) at 1∶10000 for 1 hr at room temperature. Bands were visualised using an Immobilon™ Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Nitrocellulose membranes were stripped using mild stripping buffer, blocked, and incubated with mouse anti-β-actin primary antibody (#A5441) at 1∶5000 (Sigma-Aldrich, St Louis, MO, USA) overnight at 4°C. Membranes were then incubated with anti-mouse IgG-HRP secondary antibody at 1∶10000 (Cell Signaling #7076, Invitrogen) for 1 hr at room temperature. Bands were visualised as described above.
Human amnion epithelial cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at 4°C and were permeabilized with 0.1% Triton-X-100 in PBS for 5 minutes at room temperature. DAKO Protein Block Serum-Free (Dako Australia Pty Ltd, Botany, NSW, Australia) was used to block non-specific binding for 10 minutes at room temperature and cells incubated in 8 µg/mL human CFTR antibody (#MAB25031, R&D Systems) diluted in DAKO antibody diluent for 1 hour at room temperature. Alexa Fluor 488 goat anti-mouse IgG (#A-11001, Life Technologies, Mulgrave, VIC, Australia) was used as a secondary antibody at 40 µg/mL for 1 hour at room temperature. Mouse IgG2A Isotype Control (#MAB0031, R&D Systems) was used for isotype control samples. Samples were analysed with the BD Biosciences FACSCalibur flow cytometer (BD Australia, North Ryde, NSW, Australia).
To image 3D structures formed by hAECs cultured in SAGM, cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and 4′,6-diamidino-2-phenylindole (DAPI). Briefly, cell cultures were incubated in 5 µM CFSE in PBS for 15 minutes at 37°C. CFSE solution was removed and cultures washed three times in PBS. Cells were counterstained with DAPI for 10 minutes at room temperature. Alternatively, for CFTR immunostaining human amnion epithelial cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at 4°C and permeabilized with 0.1% Triton-X-100 in PBS for 5 minutes at room temperature. DAKO Protein Block Serum-Free was used to block non-specific binding (10 minutes at room temperature) and cells were incubated overnight at 4°C in 8 µg/mL human CFTR C-Terminus antibody (#MAB25031, R&D Systems) diluted in DAKO antibody diluent. Alexa Fluor 488 goat anti-mouse IgG (#A-11001, Life Technologies) was used as a secondary antibody at 40 µg/mL for 1 hour at room temperature. Cells were counterstained with DAPI for 10 minutes at room temperature. Imaging was performed with the Leica DMI 6000 Advanced Fluorescence Imaging System (Leica Microsystems Pty Ltd, North Ryde, NSW, Australia).
Human amnion epithelial cells were fixed on plastic coverslips in 4% paraformaldehyde in PBS for 15 minutes at 4°C, frozen at −80°C overnight and freeze dried using the Thermo Scientific SuperModulyo Freeze Dryer system (Thermo Fisher Scientific). Samples were gold sputter coated using the Hummer sputtering system (Anatech Ltd, Battle Creek, MI, USA). Scanning Electron Microscopy (SEM) was performed using the Hitachi S-2600N Scanning Electron Microscope (Hitachi High Techologies, Tokyo, Japan) under high vacuum (HV) between 100x and 500x magnification.
A single cell solution of hAECs were obtained by disassociation with 0.05% trypsin for 5 minutes. Cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at 4°C and permeabilized with 0.1% Triton-X-100 in PBS for 5 minutes at room temperature. Cells were incubated with rabbit anti-CFTR primary antibody at 1∶1000 (Cell Signaling #2269, Invitrogen) and then with 1∶1000 Alexa Fluor 488 FluoroNanogold-conjugated secondary antibody (Molecular Probes #A-24922, Invitrogen), each for 1 hr at room temperature. Visualisation of gold particles was enhanced using the LI Silver enhancement kit (Invitrogen). X-ray datasets were acquired using the XM-2 soft X-ray microscope operated by the National Center for X-ray Tomography on beamline 2.1 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, Berkeley, CA, USA. The XM-2 soft X-ray microscope is designed to combine visualization of detailed cellular structure with the location of molecules tagged with gold nanoparticles in cryogenically frozen biological samples with a spatial resolution of approximately 50 nm
Plastic cultured hAECs were maintained in either SAGM or DMEM/F12 with 10% FBS for up to 28 days without passage. Primary fetal rat lung epithelial (FRLE) cells were provided as a gift from Dr Annie McDougall, Monash Institute of Medical Research (Clayton, VIC, Australia) and were used as a positive control. Functional analysis of CFTR was performed using the colorimetric technique of Tang and Wildey
hAECs were maintained on silicon nitride membrane windows (Silson, Blisworth, Northampton, England) in SAGM for 28 days without passage. Primary fetal rat lung epithelial (FRLE) cells were utilized as a positive control. Cells were incubated in iodine-loading buffer (150 mM NaI, 2 mM CaCl2, 0.8 mM NaH2PO4, 1 mM of MgCl2, and 5.4 mM of KI; adjusted to pH 7.4 with 1 N sodium hydroxide) and CFTR was activated through addition of 100 µM forskolin in DPBS as described above. After the cells had been treated windows were washed in a 0.2 M ammonium acetate solution in DPBS, and excess liquid removed by gently touching to a paper towel. Silson windows were then flash frozen in liquid nitrogen-cooled isopentane and immediately transferred to a 0.5 mL Eppendorf tube on dry ice. Windows were freeze-dried overnight and stored in a desiccator at 4°C.
The distribution of intracellular elements was mapped at the X-ray Fluorescence Microscopy beamline of the Australian Synchrotron
To determine
CFTR expression in hAECs cultured in DMEM:F12 (white bars) and hAECs cultured in Small Airway Epithelial Growth Medium (SAGM) (black bars) for 7, 14, 21 and 28 days (A) (n = 3). hAECs cultured in SAGM for 28 days had a 389.4±70.0 fold increase in CFTR gene expression (
We visualized cultured hAECs using carboxyfluorescein succinimidyl ester (CFSE) and 4′,6-diamidino-2-phenylindole (DAPI). We observed that, while hAECs cultured in DMEM:F12 remained as a confluent monolayer (n = 3,
hAECs cultured in DMEM/F12 visualized with fluorescent dyes (CFSE (green) and DAPI (blue)) remained as a confluent, plastic-adherent monolayer (A) (n = 3). hAECs cultured in SAGM for 28 days formed layers of cells clustered into a honeycomb-like morphology growing above a plastic-adherent hAEC monolayer (B) (n = 3). Scanning Electron Microscopy (SEM) analysis of the honeycomb-like 3D structures reveal close interactions between the plastic adherent hAECs and hAECs within the 3D honeycomb-like structures (C (100x) and D (500x)) (n = 3). CFTR expressing cells were found within all 3D structures observed in culture (E–G) and appeared to localize to the edges of the 3D cell clusters (H–J) (n = 3). X-ray tomographic image of the subcellular distribution of CFTR protein using X-ray tomography (K) (n = 4). Shown is a single hAEC from a culture of hAECs after 28 days in SAGM. We were able to visualize cell nuclei (blue), and localize the gold-labeled, silver-enhanced CFTR antibodies (red). CFTR was abundant on the cell membrane, and localized to an area covering approximately half of the plasma membrane.
Next, to localize the cells that expressed CFTR within the 3D structures we labelled cells with an anti-CFTR primary antibody and an Alexa Fluor® 488-conjugated secondary antibody. We found CFTR expressing cells within the 3D structures (
To assess whether the increased CFTR protein production induced in the hAECs cultured in SAGM was functional we used a colorimetric technique
Supernatant iodine concentration was measured and CFTR channel function was calculated and plotted as supernatant iodine concentration vs forskolin concentration (A) (error bars represent SD, n = 9). hAECs cultured in SAGM demonstrated forskolin-activated CFTR function. Treatment of cultures with CFTR-inhibitor CFTR-172 reversed forskolin activation of CFTR function. Fetal rat lung epithelial (FRLE) cells were utilized as a positive control in this study and displayed similar CFTR stimulation and inhibition as observed in SAGM-cultured hAECs. We did not detect any significant forskolin-activated CFTR function in undifferentiated hAECs. 2D elemental mapping of the forskolin-activated flux of iodine across the cell membrane as well as 2D elemental maps for sulfur, potassium and calcium (B) supported the observed forskolin-activated ion flux across the membrane (n = 3).
CFTR expression and function is essential for normal respiratory airway function and is defective in individuals with cystic fibrosis. With a view to developing a cell-based therapy for cystic fibrosis we have shown that human amnion epithelial cells (hAECs) can be induced to express functional cystic fibrosis transmembrane conductance regulator (CFTR)
We have previously reported that hAECs can be induced to produce surfactant proteins A, B, C and D and contain lamellar body–like organelles both
The observation that hAECs formed 3 dimensional cell aggregates following culture on plastic surfaces in SAGM was new. In our previous work when we cultured hAECs in SAGM to induce surfactant protein expression we did not observe the formation of such structures. However, in those experiments hAECs were grown on glass coverslips or on transwells coated with collagen type IV. hAECs have previously been utilized as feeder cells for limbal epithelial progenitor cells
In addition to localization of the CFTR expressing cells, we were interested in localisation of CFTR within individual cells. In the airway, CFTR is localized to the apical plasma membrane of ciliated cells, a localization that is disrupted by CFTR mutations
An important function of CFTR in airway epithelial cells is the regulation of ion and water flows across airway surfaces. It is a lack of this regulation that leads to the tenacious mucus and respiratory consequences of cystic fibrosis. Forskolin activates CFTR via protein kinase A (PKA) dependent phosphorylation of the regulatory domain of CFTR
In summary, we have shown that by culturing hAECs in SAGM we could induce hAECs to form 3D “alveoli-like” structures and to express functional CFTR ion channels on the luminal aspects of those structures. We have previously demonstrated that hAECs are anti-inflammatory and reduce fibrosis in animal models of acute lung injury
X-ray tomographic image of the subcellular distribution of CFTR protein using synchrotron soft X-ray microscopy. Shown is a single hAEC from a culture of hAECs after 28 days in SAGM. We were able to visualize cell nuclei (blue), and localize the gold-labeled, silver-enhanced CFTR antibodies (red). CFTR was abundant on the cell membrane, and localized to an area covering approximately half of the plasma membrane (n = 4).
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This research was partly undertaken on the XM-2 beamline at the National Center for X-ray Tomography (NCXT) at the Advanced Light Source (ALS) Berkeley, CA and at the X-ray Fluorescence Microscopy (XFM) beamline at the Australian Synchrotron, Victoria, Australia. Thanks to Christian Knoechel for data collection and Dula Parkinson for image reconstruction at the Advanced Light Source, Lawrence Berkeley National Laboratory. Also thanks to Annie McDougall at Monash Institute of Medical Research for providing fetal rat lung epithelial (FRLE) cells and to the Leica Research application specialists for assistance with fluorescence microscopy.