The first experiments were designed to assess the transducability of the two cell types (hABCs and hAECs) with a lentiviral (LV) vector containing the LacZ reporter gene. A separate group of cells were expanded, transduced with an LV vector containing the luciferase (Luc) transgene, and prepared for in vivo delivery. Transduced cells were delivered to normal mouse nasal airways after treatment with either a phosphate-buffered saline (PBS) sham control or PDOC by using two different intervals between epithelial disruption and cell delivery. Transplantation levels were assessed by bioluminescence imaging.
Cell culture production of hABCs and hAECs
Human primary airway cells (human bronchial epithelial cells, or HBECs, cc-2540 s, Lonza, Mount Waverley, VIC, Australia) were seeded onto 25-cm2 collagen-coated flasks to isolate the hABC population. Cells were expanded by using Bronchial Epithelial Cell Growth Medium (BEGM, cc-3170, Lonza), and passaged twice. Samples were taken during passaging to confirm the basal cell identity by cytokeratin 5 (Krt5) staining as previously published [8].
hAECs were obtained from the Amnion Cell Biology Group, the Ritchie Centre, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia. Cells were seeded onto 75-cm2 collagen-coated flasks, expanded and passaged once in Epi Growth Medium (215–500, Sigma-Aldrich, Sydney, NSW, Australia).
Lentiviral vector production
VSV-G–pseudotyped HIV-1–based LV vectors expressing either nuclear-localised LacZ under transcriptional control of the MPSV promoter (LV-LacZ) or Luc with the ef1α promoter (LV-Luc) were produced in accordance with previously published methods [9]. The titre of the LacZ vector was 1.3 × 109 Tu/mL for in vitro studies. The titre for the Luc vector used in cell transplantation studies was 6.8 × 108 Tu/mL, as assayed by quantitative polymerase chain reaction [10].
Assessment of in vitro LacZ transduction efficiency
In a previous short-term study, hABC cultures were transduced at multiplicities of infection (MOIs) of 0, 10 and 100, and the MOI of 10 produced close to 100% transduction [8]. To confirm that transgene expression was maintained in cells long-term, a subset of hABC-LacZ cultures (MOI 10) were grown for 4 weeks and processed for analysis of gene expression, and quantification was performed as previously described [8]. In the present study, the transduction efficiency of the same vector (LacZ) in hAECs at an MOI of 10 was assessed for comparison with transduction of hABCs. The hAEC cultures were seeded at a density of 2.5 × 105 cells per well onto Nunc™ six-well plates coated with type 1 rat tail collagen. Two hours later, when 75% confluent and cells had adhered, they were treated with the LV vector, diluted with PBS as appropriate, at an MOI of 0 (PBS only) or 10. Cell cultures were incubated at 37 °C and 5% CO2 in a humidified chamber. One day after LV vector treatment, the media was aspirated, cells were washed with PBS at room temperature, fresh pre-warmed (37 °C) BEGM was added, and the cultures were returned to the incubator and maintained for 3 days. Quantification of LacZ gene expression was performed in accordance with previously published methods [8].
Reporter gene transduction of hABCs and hAECs for in vivo delivery
hABCs and hAECs were grown on 75-cm2 collagen-coated flasks, expanded to 75% confluency, treated with the LV-Luc vector at the chosen MOI of 10, and incubated at 37 °C and 5% CO2 overnight in a humidified chamber. To confirm that cells expressed Luc prior to transplantation, a subset were examined by using bioluminescence imaging (IVIS Lumina XRMS, Xenogen Corporation, Alameda, CA, USA). Media was removed from the flasks, 2 mL of D-luciferin (30 mg/mL in PBS) was added, and imaging was performed. A circular region of interest (ROI) was used for quantification, and blank flasks containing no cells were imaged as controls.
On the following day, transduced cells (designated hABC-Luc and hAEC-Luc) were harvested by using HEPES-buffered saline, Trypsin/EDTA, and Trypsin neutralising solution (ReagentPack, cc-5034, Lonza) in accordance with the instructions of the manufacturer. Cells were re-suspended in PBS, cell counts were obtained, and 1.3 × 105 cells per aliquot were held on ice for immediate delivery into mouse airways.
Animal studies
All experiments were approved by the Women’s and Children’s Hospital and University of Adelaide animal ethics committees. Female C57BL/6 mice (6–8 weeks of age) were anaesthetised with an intraperitoneal (i.p.) injection containing a mixture of 10 μL/g body weight of medetomidine (Domitor, 0.1 mg/mL, Orion Corporation, Espoo, Finland) and ketamine (7.6 mg/mL, Parnell Laboratories, Alexandria, NSW, Australia). After each experimental procedure, anaesthesia was reversed with 1 μg/g body weight of atipamezole hydrochloride (Antisedan, Orion Corporation) also delivered as an i.p. injection, and mice were held in a humidified incubator until fully recovered. At termination of the animal studies, mice were humanely killed by 100% CO2 inhalation.
Effect of PDOC treatment on nasal airway epithelium prior to cell transplantation
To assess the effect of PDOC on the epithelium at 2 and 24 h [11] post-delivery, the nasal airways of mice (n = 3) were exposed to 4 μL of 2% PDOC (3055–99-0, Sigma-Aldrich, St. Louis, MO, USA) in PBS. At either 2 or 24 h later, animals were humanely killed, relevant nasal tissue samples were collected and processed, and serial sections were examined histologically. Haematoxylin and eosin (H&E) staining was performed on 5-μm sections mounted on glass slides. Antibody staining for Krt5 was performed on adjacent 5-μm sections. After sodium citrate antigen retrieval, sections were stained with rabbit anti-Krt5 antibody (ab52635, Abcam, Melbourne, VIC, Australia; 1:350). The slides were placed in a humid incubation chamber and stored overnight at 4 °C. Goat anti-rabbit IgG H&L HRP (ab97051, Abcam; 1:500) was used as a secondary antibody for 1–2 h. An Abcam DAB Substrate Kit (ab64238) was applied for 10 min, and the colour development was stopped by rinsing tissue sections with PBS/0.05% Tween 20. Sections were counterstained with Meyer’s haematoxylin.
In vivo cell transplantation
In the transplantation trials, the nasal airways were exposed to 4 μL of PBS or 2% PDOC in PBS delivered into the right nostril to disrupt the epithelium prior to cell delivery. At either 2 or 24 h after PDOC or PBS delivery, 3 × 10 μL aliquots of cells (hABC-Luc or hAEC-Luc) were delivered to the previously treated nasal passage at 10-min intervals between bolus aliquots.
Bioluminescence imaging
One week after hABC-Luc or hAEC-Luc transplantation, mice were anaesthetised and Luc expression was assessed 10 min after a 50-μL intranasal bolus of D-luciferin (15 mg/mL in PBS). The nasal airways of treated mice were imaged by using the IVIS Lumina XRMS and auto-exposure with mice in a supine position. The resultant bioluminescent flux (photons/s) was measured after background image subtraction in a 1.5-cm2 square ROI in accordance with the instructions of the manufacturer (Igor Pro 4.09A Living Image Software, Xenogen Corporation). Mice were re-imaged at 3, 5 and 8 weeks after cell transplantation.
Assessment of circulating luciferase antibodies
Blood was removed by submandibular puncture directly after each bioluminescence imaging event and via cardiac puncture after study termination. Blood was processed and circulating antibodies to the Luc protein were assessed as previously published [9].
Detection of human cells in hABC-Luc– and hAEC-Luc–treated nasal airways
Mouse paraffin embedded nasal airway tissue sections were de-paraffinised and re-hydrated through sequential incubation twice in xylene for 3 min, twice in 100% ethanol for 2 min and 90% ethanol, 70% ethanol and milli-Q water for 2 min each. Antigen retrieval was carried out by placing sections in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at 90–100 °C for 20 min and allowed to cool to room temperature for 20 min. Sections were blocked with hydrogen peroxidase (ab64218, Abcam) for 10 min at room temperature, washed briefly twice in wash buffer (PBS/0.05% Tween-20) and blocked in blocking buffer (2% sheep serum, 0.1% bovine serum albumin, 0.1% gelatin, 0.1% Triton X-100 and 0.05% Tween-20) for 1 h at room temperature. Sections were washed twice in wash buffer and incubated with 1:1000 primary antibody to human mitochondria (Anti-Mitochondria, ab92824, Abcam) diluted in blocking buffer at room temperature for 1 h. Sections were washed with wash buffer and incubated with 1:400 secondary anti-body, Anti-Mouse IgG, Horseradish Peroxidase-Linked Species-Specific Whole Antibody (from sheep) (NA931, GE Life Sciences, ECL™, Parramatta, NSW, Australia) in blocking buffer for 1 h at room temperature. Sections were washed with wash buffer and secondary antibody was stained for 10 min with DAB substrate kit (ab64238, Abcam) (30 μL DAB Chromogen/1.5 mL DAB substrate) and washed with wash buffer. Sections were counterstained by rinsing in milli-Q water followed by Meyer’s haematoxylin for 90 s. Sections were washed in milli-Q water and sections were dipped into Scott’s tap water substitute for 5 s. Sections were de-hydrated by sequentially incubating the sections in milli-Q water, 70% ethanol, 90% ethanol, twice in 100% ethanol for 10 s each and xylene twice for 1 min, and a cover slip was added with mounting media. Slides were examined for the presence of human cells under 20× magnification.
Statistics
Results are represented as a mean and standard error. Data were tested for normality assumptions and analysed by using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at P = 0.05. Analysis of two treatment groups was performed by using a t test, and multiple treatment groups were analysed by two-way analysis of variance (ANOVA) with Sidak’s multiple comparison.
