Corrigendum: Effect of elexacaftor-tezacaftor-ivacaftor on nasal potential difference and lung function in Phe508del rats.

[This corrects the article DOI: 10.3389/fphar.2024.1362325.].

Ultra-fastin vivodirectional dark-field x-ray imaging for visualising magnetic control of particles for airway gene delivery.

Objective.Magnetic nanoparticles can be used as a targeted delivery vehicle for genetic therapies. Understanding how they can be manipulated within the complex environment of live airways is key to their application to cystic fibrosis and other respiratory diseases.Approach.Dark-field x-ray imaging provides sensitivity to scattering information, and allows the presence of structures smaller than the detector pixel size to be detected. In this study, ultra-fast directional dark-field synchrotron x-ray imaging was utlilised to understand how magnetic nanoparticles move within a live, anaesthetised, rat airway under the influence of static and moving magnetic fields.Main results.Magnetic nanoparticles emerging from an indwelling tracheal cannula were detectable during delivery, with dark-field imaging increasing the signal-to-noise ratio of this event by 3.5 times compared to the x-ray transmission signal. Particle movement as well as particle retention was evident. Dynamic magnetic fields could manipulate the magnetic particlesin situ. Significance.This is the first evidence of the effectiveness ofin vivodark-field imaging operating at these spatial and temporal resolutions, used to detect magnetic nanoparticles. These findings provide the basis for further development toward the effective use of magnetic nanoparticles, and advance their potential as an effective delivery vehicle for genetic agents in the airways of live organisms.

Effect of elexacaftor-tezacaftor-ivacaftor on nasal potential difference and lung function in Phe508del rats.

Introduction: Phe508del is the most common cystic fibrosis transmembrane conductance regulator (CFTR) gene variant that results in the recessive genetic disorder cystic fibrosis (CF). The recent development of highly effective CFTR modulator therapies has led to significant health improvements in individuals with this mutation. While numerous animal models of CF exist, few have a CFTR mutation that is amenable to the triple combination therapy elexacaftor-tezacaftor-ivacaftor (ETI). Methods: To determine the responsiveness of Phe508del rats to ETI, a baseline nasal potential difference was measured. Subsequently, they received ETI daily for 14 days, after which post-treatment nasal potential difference, lung mechanics (via flexiVent) and lung ventilation (via X-ray Velocimetry) were assessed. Results: Chloride ion transport in nasal airways was restored in Phe508del rats treated with ETI, but neither lung mechanics nor ventilation were significantly altered. Discussion: These findings validate the usefulness of this rat model for future investigations of modulator therapy in CF.

Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine.

The Ca2+ activated Cl- channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

Noncontact Respiratory Motion Detection in Anesthetized Rodents.

Small animal physiology studies are often complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron radiation facilities. This is because these facilities are typically not designed specifically for biomedical research, and the animals and image detectors are located away from the researchers in a radiation enclosure. In respiratory X-ray imaging studies one challenge is the detection of respiration in free-breathing anaesthetised rodents, to enable images to be acquired at specific phases of the breath and for detecting changes in respiratory rate. We have previously used a Philtec RC60 sensor interfaced to a PowerLab data acquisition system and custom-designed timing hub to perform this task. Here we evaluated the Panasonic HL-G108 for respiratory sensing. The performance of the two sensors for accurate and reliable breath detection was directly compared using a single anesthetized rat. We also assessed how an infrared heat lamp used to maintain body temperature affected sensor performance. Based on positive results from these comparisons, the HL-G108 sensor was then used for respiratory motion detection in tracheal X-ray imaging studies of 21 rats at the SPring-8 Synchrotron, including its use for gated image acquisition. The results of that test were compared to a similar imaging study that used the RC60 for respiratory detection in 19 rats. Finally, the HL-G108 sensor was tested on 5 mice to determine its effectiveness on smaller species. The results showed that the HL-G108 is much more robust and easier to configure than the RC60 sensor and produces an analog signal that is amenable to stable peak detection. Furthermore, gated image acquisition produced sequences with substantially reduced motion artefacts, enabling the additional benefit of reduced radiation dose through the application of shuttering. Finally, the mouse experiments showed that the HL-G108 is equally capable of detecting respiration in this smaller species.

Repeat or single-dose lentiviral vector administration to mouse lungs? It's all about the timing.

Lentiviral vectors are attractive delivery vehicles for cystic fibrosis gene therapy owing to their low immunogenicity and ability to integrate into the host cell genome, thereby producing long-term, stable gene expression. Nonetheless, repeat dosing may be required to increase initial expression levels, and/or boost levels when they wane. The primary aim of this study was to determine if repeat dosing of a VSV-G pseudotyped LV vector delivered into mouse lungs is more effective than a single dose. C57Bl/6 mouse lungs were conditioned with lysophosphatidylcholine, followed one-hour later by a LV vector carrying the luciferase reporter gene, using six different short-term (≤1 wk) and long-term (>1 wk) dosing schedules. Luciferase expression was quantified using bioluminescence imaging over 12 months. Most dosing schedules produced detectable bioluminescence over the 12-month period, but the shorter intervals (≤1 wk) produced higher levels of flux than the longest interval (five doses at least 1-month apart). Ex vivo lung analysis at 12 months showed that the estimated mean flux for the group that received two doses 1-week apart was significantly greater than the single dose group and the two groups that received doses over a period greater than 1-week. These results suggest that early consecutive multiple doses are more effective at improving gene expression in mouse lungs at 12 months, than longer repeat dosing intervals.

To bead or not to bead: A review of Pseudomonas aeruginosa lung infection models for cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterised by recurring bacterial infections resulting in inflammation, lung damage and ultimately respiratory failure. Pseudomonas aeruginosa is considered one of the most important lung pathogens in those with cystic fibrosis. While multiple cystic fibrosis animal models have been developed, many fail to mirror the cystic fibrosis lung disease of humans, including the colonisation by opportunistic environmental pathogens. Delivering bacteria to the lungs of animals in different forms is a way to model cystic fibrosis bacterial lung infections and disease. This review presents an overview of previous models, and factors to consider when generating a new P. aeruginosa lung infection model. The future development and application of lung infection models that more accurately reflect human cystic fibrosis lung disease has the potential to assist in understanding the pathophysiology of cystic fibrosis lung disease and for developing treatments.

Effective viral-mediated lung gene therapy: is airway surface preparation necessary?

Gene-based therapeutics are actively being pursued for the treatment of lung diseases. While promising advances have been made over the last decades, the absence of clinically available lung-directed genetic therapies highlights the difficulties associated with this effort. Largely, progress has been hindered by the presence of inherent physical and physiological airway barriers that significantly reduce the efficacy of gene transfer. These barriers include surface mucus, mucociliary action, cell-to-cell tight junctions, and the basolateral cell membrane location of viral receptors for many commonly used gene vectors. Accordingly, airway surface preparation methods have been developed to disrupt these barriers, creating a more conducive environment for gene uptake into the target airway cells. The two major approaches have been chemical and physical methods. Both have proven effective for increasing viral-mediated gene transfer pre-clinically, although with variable effect depending on the specific strategy employed. While such methods have been explored extensively in experimental settings, they have not been used clinically. This review covers the airway surface preparation strategies reported in the literature, the advantages and disadvantages of each method, as well as a discussion about applying this concept in the clinic.

Comparison of Physical Perturbation Devices for Enhancing Lentiviral Vector-Mediated Gene Transfer to the Airway Epithelium.

Natural airway defenses currently impede the efficacy of viral vector-mediated airway gene therapy. Conditioning airways before vector delivery can disrupt these barriers, improving viral vector access to target receptors and airway stem cells. This study aimed to assess and quantify the in vivo histological and gene transfer effects of physical perturbation devices to identify effective conditioning approaches. A range of flexible wire baskets with varying configurations, a Brush, biopsy forceps, and a balloon catheter were examined. We first evaluated the histological effects of physical perturbation devices in rat tracheas that were excised 10 min after conditioning. Based on the histological findings, a selection of devices was used to condition rat tracheas in vivo before delivering a lentiviral vector containing the LacZ reporter gene. After 7 days, excised tracheas were X-gal processed and examined en face to quantify the area of LacZ staining. Histological observations 10 min after conditioning found that physical perturbation dislodged cells from the basement membrane to varying degrees, with some producing significant levels of epithelial cell removal. When a subset of devices was assessed for their ability to enhance gene transfer, only the NGage® wire basket (Cook Medical) produced a significant increase in the proportion of X-gal-stained area when compared with unconditioned tracheas (eightfold, p = 0.00025). These results suggest that a range of factors contribute to perturbation-enhanced gene transfer. Overall, this study supports existing evidence that physical perturbation can assist airway gene transfer and will help to identify the characteristics of an effective device for airway gene therapy.

Improved in-vivo airway gene transfer via magnetic-guidance, with protocol development informed by synchrotron imaging.

Gene vectors to treat cystic fibrosis lung disease should be targeted to the conducting airways, as peripheral lung transduction does not offer therapeutic benefit. Viral transduction efficiency is directly related to the vector residence time. However, delivered fluids such as gene vectors naturally spread to the alveoli during inspiration, and therapeutic particles of any form are rapidly cleared via mucociliary transit. Extending gene vector residence time within the conducting airways is important, but hard to achieve. Gene vector conjugated magnetic particles that can be guided to the conducting airway surfaces could improve regional targeting. Due to the challenges of in-vivo visualisation, the behaviour of such small magnetic particles on the airway surface in the presence of an applied magnetic field is poorly understood. The aim of this study was to use synchrotron imaging to visualise the in-vivo motion of a range of magnetic particles in the trachea of anaesthetised rats to examine the dynamics and patterns of individual and bulk particle behaviour in-vivo. We also then assessed whether lentiviral-magnetic particle delivery in the presence of a magnetic field increases transduction efficiency in the rat trachea. Synchrotron X-ray imaging revealed the behaviour of magnetic particles in stationary and moving magnetic fields, both in-vitro and in-vivo. Particles could not easily be dragged along the live airway surface with the magnet, but during delivery deposition was focussed within the field of view where the magnetic field was the strongest. Transduction efficiency was also improved six-fold when the lentiviral-magnetic particles were delivered in the presence of a magnetic field. Together these results show that lentiviral-magnetic particles and magnetic fields may be a valuable approach for improving gene vector targeting and increasing transduction levels in the conducting airways in-vivo.

Single-Dose Lentiviral Mediated Gene Therapy Recovers CFTR Function in Cystic Fibrosis Knockout Rats.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective ion transport in the airways. Addition of a functioning CFTR gene into affected airway cells has the potential to be an effective treatment for lung disease. The therapeutic efficacy of airway gene transfer can be quantified in animal models by assessing ion transport in the treated nasal epithelium using the nasal potential difference (PD) measurement technique. The nasal PD technique is routinely used in CF mice, however when applied to a recently developed CF rat model those animals did not tolerate the initial nasal PD assessment, therefore the procedure was firstly optimised in rats. This study evaluated the effect of lentiviral (LV)-mediated CFTR airway gene delivery on nasal PD in a CFTR knockout rat model. LV gene vector containing the CFTR gene tagged with a V5 epitope tag (LV-V5-CFTR) was delivered to the nasal epithelium of CF rats, and one week later nasal PD was analysed. This study demonstrated for the first time that LV-V5-CFTR treatment produced a mean correction of 46% towards wild-type chloride response in treated CF rats. Transduced cells were subsequently identifiable using V5 immunohistochemical staining. These findings in the nose validate the use of airway gene therapy for future lung based experiments.

The Effects of Conditioning and Lentiviral Vector Pseudotype on Short- and Long-Term Airway Reporter Gene Expression in Mice.

A gene addition therapy into the conducting airway epithelium is a potential cure for cystic fibrosis lung disease. Achieving sustained lung gene expression has proven difficult due to the natural barriers of the lung. The development of lentiviral (LV) vectors pseudotyped with viral envelopes that have a natural tropism to the airway has enabled persistent gene expression to be achieved in vivo. The aims of this study were to compare the yields of hemagglutinin (HA) and vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped HIV-1 vectors produced under the same conditions by our standard LV vector production method. We then sought to measure gene expression in mouse airways and to determine whether lysophosphatidylcholine (LPC) conditioning enhances short- and long-term gene expression. C57Bl/6 mouse airways were conditioned with 10 µL of 0.1% LPC or saline control, followed 1 h later by a 30 µL dose of an HA or VSV-G pseudotyped vector carrying either the LacZ or luciferase reporter genes. LacZ expression was assessed by X-gal staining after 7 days, while lung luminescence was quantified regularly for up to 18 months by bioluminescent imaging. The HA pseudotyped vectors had functional titers 25 to 60 times lower than the VSV-G pseudotyped vectors. Conditioning the lung with LPC significantly increased the total number of LacZ-transduced cells for both pseudotypes compared to saline control. Regardless of LPC conditioning, the VSV-G pseudotype produced higher initial levels of gene expression compared to HA. LPC conditioning did not increase the number of transduced basal cells for either pseudotype compared to saline, and was not required for long-term gene expression. Both pseudotyped vectors effectively transduced the upper conducting airways of wild-type mice. The use of LPC conditioning before vector delivery was not required in mouse lungs to produce long-term gene expression, but did improve short-term gene expression.

Breaching the Delivery Barrier: Chemical and Physical Airway Epithelium Disruption Strategies for Enhancing Lentiviral-Mediated Gene Therapy.

The lungs have evolved complex physical, biological and immunological defences to prevent foreign material from entering the airway epithelial cells. These mechanisms can also affect both viral and non-viral gene transfer agents, and significantly diminish the effectiveness of airway gene-addition therapies. One strategy to overcome the physical barrier properties of the airway is to transiently disturb the integrity of the epithelium prior to delivery of the gene transfer vector. In this study, chemical (lysophosphatidylcholine, LPC) and physical epithelium disruption using wire abrasion were compared for their ability to improve airway-based lentiviral (LV) vector mediated transduction and reporter gene expression in rats. When luciferase expression was assessed at 1-week post LV delivery, LPC airway conditioning significantly enhanced gene expression levels in rat lungs, while a long-term assessment in a separate cohort of rats at 12 months revealed that LPC conditioning did not improve gene expression longevity. In rats receiving physical perturbation to the trachea prior to gene delivery, significantly higher LacZ gene expression levels were found when compared to LPC-conditioned or LV-only control rats when evaluated 1-week post gene transfer. This proof-of-principle study has shown that airway epithelial disruption strategies based on physical perturbation substantially enhanced LV-mediated airway gene transfer in the trachea.

Towards Human Translation of Lentiviral Airway Gene Delivery for Cystic Fibrosis: A One-Month CFTR and Reporter Gene Study in Marmosets.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the luciferase (Luc), LacZ, and hCFTR transgenes was delivered into the trachea through a miniature bronchoscope. We examined whether a single-dose delivery of LV vector after LPC conditioning could increase levels of transgene expression in the trachea and lungs compared with control (phosphate-buffered saline [PBS]) conditioning. At 1 month, bioluminescence was detected in vivo in the trachea of three of the six animals within the PBS control group, compared with five of the six LPC-treated animals. When examined ex vivo there was weak evidence that LPC improves tracheal Luc expression levels. In the lungs, bioluminescence was detected in vivo in four of the six PBS-treated animals, compared with five of the six LPC-treated animals; however, bioluminescence was present in all lungs when imaged ex vivo. LacZ expression was predominantly observed in the alveolar regions of the lung. hCFTR was detected by qPCR in the lungs of five animals. Basal cells were successfully isolated and expanded from marmoset tracheas, but no LacZ-positive colonies were detected. There was no evidence of an inflammatory response toward the LV vector at 1 month postdelivery, with cytokines remaining at baseline levels. In conclusion, we found weak evidence that LPC conditioning improved gene transduction in the trachea, but not in the marmoset lungs. We also highlight some of the challenges associated with translational lung gene therapy studies in NHPs.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Towards automated in vivo tracheal mucociliary transport measurement: Detecting and tracking particle movement in synchrotron phase-contrast x-ray images.

Accurate in vivo quantification of airway mucociliary transport (MCT) in animal models is important for understanding diseases such as cystic fibrosis, as well as for developing therapies. A non-invasive method of measuring MCT behaviour, based on tracking the position of micron sized particles using synchrotron x-ray imaging, has previously been described. In previous studies, the location (and path) of each particle was tracked manually, which is a time consuming and subjective process. Here we describe particle tracking methods that were developed to reduce the need for manual particle tracking. The MCT marker particles were detected in the synchrotron x-ray images using cascade classifiers. The particle trajectories along the airway surface were generated by linking the detected locations between frames using a modified particle linking algorithm. The developed methods were compared with the manual tracking method on simulated x-ray images, as well as on in vivo images of rat airways acquired at the SPring-8 Synchrotron. The results for the simulated and in vivo images showed that the semi-automatic algorithm reduced the time required for particle tracking when compared with the manual tracking method, and was able to detect MCT marker particle locations and measure particle speeds more accurately than the manual tracking method. Future work will examine the modification of methods to improve particle detection and particle linking algorithms to allow for more accurate fully-automatic particle tracking.

Methods for dynamic synchrotron X-ray respiratory imaging in live animals.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Dynamics of Lead Bioavailability and Speciation in Indoor Dust and X-ray Spectroscopic Investigation of the Link between Ingestion and Inhalation Pathways.

Lead (Pb) exposure from household dust is a major childhood health concern because of its adverse impact on cognitive development. This study investigated the absorption kinetics of Pb from indoor dust following a single dose instillation into C57BL/6 mice. Blood Pb concentration (PbB) was assessed over 24 h, and the dynamics of particles in the lung and gastro-intestinal (GI) tract were visualized using X-ray fluorescence (XRF) microscopy. The influence of mineralogy on Pb absorption and particle retention was investigated using X-ray absorption near-edge structure spectroscopy. A rapid rise in PbB was observed between 0.25 and 4 h after instillation, peaking at 8 h and slowly declining during a period of 24 h. Following clearance from the lungs, Pb particles were detected in the stomach and small intestine at 4 and 8 h, respectively. Analysis of Pb mineralogy in the residual particles in tissues at 8 h showed that mineral-sorbed Pb and Pb-phosphates dominated the lung, while organic-bound Pb and galena were the main phases in the small intestines. This is the first study to visualize Pb dynamics in the lung and GI tract using XRF microscopy and link the inhalation and ingestion pathways for metal exposure assessment from dust.

In Vitro, in Vivo, and Spectroscopic Assessment of Lead Exposure Reduction via Ingestion and Inhalation Pathways Using Phosphate and Iron Amendments.

This study compared lead (Pb) immobilization efficacies in mining/smelting impacted soil using phosphate and iron amendments via ingestion and inhalation pathways using in vitro and in vivo assays, in conjunction with investigating the dynamics of dust particles in the lungs and gastro-intestinal tract via X-ray fluorescence (XRF) microscopy. Phosphate amendments [phosphoric acid (PA), hydroxyapatite, monoammonium phosphate (MAP), triple super phosphate (TSP), and bone meal biochar] and hematite were applied at a molar ratio of Pb:Fe/P = 1:5. Pb phosphate formation was investigated in the soil/post-in vitro bioaccessibility (IVBA) residuals and in mouse lung via extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structures (XANES) spectroscopy, respectively. EXAFS analysis revealed that anglesite was the dominant phase in the ingestible (<250 µm) and inhalable (<10 µm) particle fractions. Pb IVBA was significantly reduced (p < 0.05) by phosphate amendments in the <250 µm fraction (solubility bioaccessibility research consortium assay) and by PA, MAP, and TSP in the <10 µm fraction (inhalation-ingestion bioaccessibility assay). A 21.1% reduction in Pb RBA (<250 µm fraction) and 56.4% reduction in blood Pb concentration (<10 µm fraction) were observed via the ingestion and inhalation pathways, respectively. XRF microscopy detected Pb in the stomach within 4 h, presumably via mucociliary clearance.

Epithelial disruption: a new paradigm enabling human airway stem cell transplantation.

BACKGROUND: Airway disease is a primary cause of morbidity and early mortality for patients with cystic fibrosis (CF). Cell transplantation therapy has proven successful for treating immune disorders and may have the potential to correct the airway disease phenotype associated with CF. Since in vivo cell delivery into unconditioned mouse airways leads to inefficient engraftment, we hypothesised that disrupting the epithelial cell layer using the agent polidocanol (PDOC) would facilitate effective transplantation of cultured stem cells in mouse nasal airways. METHODS: In this study, 4 µL of 2% PDOC in phosphate-buffered saline was administered to the nasal airway of mice to disrupt the epithelium. At 2 or 24 h after PDOC treatment, two types of reporter gene-expressing cells were transplanted into the animals: luciferase-transduced human airway basal cells (hABC-Luc) or luciferase-transduced human amnion epithelial cells (hAEC-Luc). Bioluminescence imaging was used to assess the presence of transplanted luciferase-expressing cells over time. Data were evaluated by using two-way analysis of variance with Sidak's multiple comparison. RESULTS: Successful transplantation was observed when hABCs were delivered 2 h after PDOC but was absent when transplantation was performed 24 h after PDOC, suggesting that a greater competitive advantage for the donor cells is present at the earlier time point. The lack of transplantation of hAECs 24 h after PDOC supports the importance of choosing the correct timing and cell type to facilitate transplantation. CONCLUSIONS: These studies into factors that may enable successful airway transplantation of human stem cells showed that extended functioning cell presence is feasible and further supports the development of methods that alter normal epithelial layer integrity. With improvements in efficacy, manipulating the airway epithelium to make it permissive towards cell transplantation may provide another option for safe and effective correction of CF transmembrane conductance regulator function in CF airways.