Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Airway disease decreases the therapeutic potential of epithelial stem cells.

BACKGORUND: Tissue-engineered tracheal grafts (TETG) can be recellularized by the host or pre-seeded with host-derived cells. However, the impact of airway disease on the recellularization process is unknown. METHODS: In this study, we determined if airway disease alters the regenerative potential of the human tracheobronchial epithelium (hTBE) obtained by brushing the tracheal mucosa during clinically-indicated bronchoscopy from 48 pediatric and six adult patients. RESULTS: Our findings revealed that basal cell recovery and frequency did not vary by age or region. At passage 1, all samples produced enough cells to cellularize a 3.5 by 0.5 cm2 graft scaffold at low cell density (~ 7000 cells/cm2), and 43.75% could cellularize a scaffold at high cell density (~ 100,000 cells/cm2). At passage 2, all samples produced the number of cells required for both recellularization models. Further evaluation revealed that six pediatric samples (11%) and three (50%) adult samples contained basal cells with a squamous basal phenotype. These cells did not form a polarized epithelium or produce differentiated secretory or ciliated cells. In the pediatric population, the squamous basal cell phenotype was associated with degree of prematurity (< 28 weeks, 64% vs. 13%, p = 0.02), significant pulmonary history (83% vs. 34%, p = 0.02), specifically with bronchopulmonary dysplasia (67% vs. 19%, p = 0.01), and patients who underwent previous tracheostomy (67% vs. 23%, p = 0.03). CONCLUSIONS: In summary, screening high-risk pediatric or adult population based on clinical risk factors and laboratory findings could define appropriate candidates for airway reconstruction with tracheal scaffolds. LEVEL OF EVIDENCE: Level III Cohort study.

A Review of Forensic Fellowship Training: Similar Challenges, Diverse Approaches.

OBJECTIVE: As the specialty of forensic psychiatry and the numbers of justice involved individuals with mental illness continue to grow, forensic education is receiving more attention. To add to this discussion, the authors reviewed current training practices in forensic psychiatry fellowship programs. METHODS: As part of a project funded by the state legislature looking at psychiatry and psychology training, with special focus on state hospital partnerships, the authors interviewed and surveyed forensic psychiatry fellowship program directors and conducted site visits to select programs. Given the mandate, special attention was given to programs with state hospital and psychology training affiliations. RESULTS: The literature and fellowship directors identified several key features and challenges for forensic training programs. Forensic fellowships focused primarily on evaluations and consultations but differed in their offerings related to training in treatment, scholarship, and particular types of evaluations. Common concerns included maintaining adequate funding, variety of training experiences, adequate faculty time, and adequate institutional support. Directors described cultivating institutional support by demonstrating mutual benefit as particularly important for securing resources. CONCLUSION: Forensic fellowships operational during the 2016-2017 academic year offered a range of forensic training activities, but experienced common challenges such as inadequate funding and faculty supervision. Empirical studies are needed to evaluate the relationship between programmatic offerings, the noted challenges, and acquired competencies.

Understanding the evolving phenotype of vascular complications in telomere biology disorders.

Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.

Regulation of Human Airway Epithelial Tissue Stem Cell Differentiation by ß-Catenin, P300, and CBP.

The wingless/integrase-1 (WNT)/ß-catenin signaling pathway is active in several chronic lung diseases including idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease. Although this WNT/ß-catenin pathway activity is associated with an increase in mucus cell frequency and a decrease in ciliated cell frequency, a cause and consequence relationship between signaling and cell frequency has not been established. We previously demonstrated that genetic stabilization of ß-catenin inhibited differentiation of mouse bronchiolar tissue stem cells (TSC). This study determined the effect of ß-catenin and its co-factors P300 (E1A-binding protein, 300 kDa) and cAMP response element binding (CREB)-binding protein (CBP) on human bronchial epithelial TSC differentiation to mucus and ciliated cells. We developed a modified air-liquid interface (ALI) culture system in which mucus and ciliated cell frequency is similar. These cultures were treated with the ß-catenin agonist CHIR99021 (CHIR) and antagonists to ß-catenin (XAV939), P300 (IQ1), and CBP (ICG001). We report that human TSC differentiation to mucus and ciliated cells can be divided into two stages, specification and commitment. CHIR treatment inhibited mucus and ciliated cell commitment while XAV939 treatment demonstrated that ß-catenin was necessary for mucus and ciliated cell specification. Additional studies demonstrate that a ß-catenin/P300 complex promotes mucus cell specification and that ß-catenin interacts with either P300 or CBP to inhibit ciliated cell commitment. These data indicate that activation of ß-catenin-dependent signaling in chronic lung disease leads to changes in mucus and ciliated cell frequency and that P300 and CBP tune the ß-catenin signal to favor mucus cell differentiation. Stem Cells 2018;36:1905-12.

Effect of signal processing strategy and stimulation type on speech and auditory perception in adult cochlear implant users.

OBJECTIVE: The objective of this study was to investigate the effects of signal processing strategy on speech understanding and auditory function for adult cochlear implant (CI) users with a focus on the effects of sequential versus paired stimulation. DESIGN: Within-subjects, repeated measures design was utilised to compare performance between processing strategies and stimulation type on various measures of auditory function and subjective sound quality. Testing with subsequent strategies was completed after a total familiarisation time of two weeks. STUDY SAMPLE: Ten post-lingually deafened adult CI users were recruited from a clinical population. Participants had a minimum of 13 months CI experience. Ages ranged from 25-78 years. All participants had long-term experience with the optima strategy; eight with sequential stimulation and two with paired stimulation. RESULTS: We found no statistically significant effect of processing strategy. We observed an effect of stimulation type with sequential stimulation yielding significantly higher performance than paired stimulation for speech understanding in quiet and in noise, and subjective estimates of sound quality. No significant differences were noted across strategy or stimulation for music perception, spectral resolution or temporal resolution. CONCLUSIONS: Many patients utilise paired stimulation - the default stimulation type in the clinical software; however, sequential stimulation yielded significantly higher outcomes on multiple measures.

Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Impact of Donor Arterial Partial Pressure of Oxygen on Outcomes After Lung Transplantation in Adult Cystic Fibrosis Recipients.

INTRODUCTION: Donor PaO2 levels are used for assessing organs for lung transplantation (LTx), but survival implications of PaO2 levels in adult cystic fibrosis (CF) patients receiving LTx are unclear. METHODS: UNOS registry data spanning 2005-2013 were used to test for associations of donor PaO2 with patient survival and bronchiolitis obliterans syndrome (BOS) in adult (age ≥ 18 years) first-time LTx recipients diagnosed with CF. RESULTS: The analysis included 1587 patients, of whom 1420 had complete data for multivariable Cox models. No statistically significant differences among donor PaO2 categories of ≤200, 201-300, 301-400, or >400 mmHg were found in univariate survival analysis (log-rank test p = 0.290). BOS onset did not significantly differ across donor PaO2 categories (Chi-square p = 0.480). Multivariable Cox models of patient survival supported the lack of difference across donor PaO2 categories. Interaction analysis found a modest difference in survival between the two top categories of donor PaO2 when examining patients with body mass index (BMI) in the lowest decile (≤16.5 kg/m(2)). CONCLUSIONS: Donor PaO2 was not associated with survival or BOS onset in adult CF patients undergoing LTx. Notwithstanding statistically significant interactions between donor PaO2 and BMI, there was no evidence of post-LTx survival risk associated with donor PaO2 below conventional thresholds in any subgroup of adults with CF.

Epidermal growth factor receptor activity is necessary for mouse basal cell proliferation.

ERB family receptors (EGFR, ERB-B2, ERB-B3, and ERB-B4) regulate epithelial cell function in many tissue types. In the human airway epithelium, changes in ERB receptor expression are associated with epithelial repair defects. However, the specific role(s) played by ERB receptors in repair have not been determined. We aimed to determine whether ERB receptors regulate proliferation of the tracheobronchial progenitor, the basal cell. Receptor tyrosine kinase arrays were used to evaluate ERB activity in normal and naphthalene (NA)-injured mouse trachea and in air-liquid interface cultures. Roles for epidermal growth factor (EGF), EGFR, and ERB-B2 in basal cell proliferation were evaluated in vitro. NA injury and transgenic expression of an EGFR-dominant negative (DN) receptor were used to evaluate roles for EGFR signaling in vivo. EGFR and ERB-B2 were active in normal and NA-injured trachea and were the only active ERB receptors detected in proliferating basal cells in vitro. EGF was necessary for basal cell proliferation in vitro. The EGFR inhibitor, AG1478, decreased proliferation by 99, and the Erb-B2 inhibitor, AG825, decreased proliferation by ∼66%. In vivo, EGFR-DN expression in basal cells significantly decreased basal cell proliferation after NA injury. EGF and EGFR are necessary for basal cell proliferation. The EGFR/EGFR homo- and the EGFR/ERB-B2 heterodimer account for ∼34 and 66%, respectively, of basal cell proliferation in vitro. Active EGFR is necessary for basal cell proliferation after NA injury. We conclude that EGFR activation is necessary for mouse basal cell proliferation and normal epithelial repair.

Regulation of trachebronchial tissue-specific stem cell pool size.

Tissue-specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA) injury and then returned to normal. This study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:green fluorescent protein (GFP) model to purify basal cells that proliferated infrequently (GFP(bright) ) or frequently (GFP(dim) ) after NA injury. Both populations contained TSC but TSCs were 8.5-fold more abundant in the GFP(bright) population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally differentiated basal cells. The ratio of TSC to UPB was 5:1 in the GFP(bright) population and 1:5 in the GFP(dim) population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFP(bright) and GFP(dim) populations and passaged the clones seven times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFP(dim) lineage. Gene expression analysis showed that ß-catenin and Notch pathway genes were differentially expressed in freshly isolated TSC derived from GFP(bright) and GFP(dim) populations. We conclude that (a) TSC and UPB are members of a single lineage; (b) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and (c) an interaction between the ß-catenin and Notch pathways regulates the TSC-to-UPB differentiation process.

Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin signaling.

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated ß-catenin signaling, and this was verified by real-time PCR, nuclear translocation of ß-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of ß-catenin signaling in response to neutrophil transmigration. Knockdown of ß-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of ß-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of ß-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that ß-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Chemosensory brush cells of the trachea. A stable population in a dynamic epithelium.

Tracheal brush cells (BCs) are specialized epithelial chemosensors that use the canonical taste transduction cascade to detect irritants. To test whether BCs are replaced at the same rate as other cells in the surrounding epithelium of adult mice, we used 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells. Although scattered BrdU-labeled epithelial cells are present 5-20 days after BrdU, no BCs are labeled. These data indicate that BCs comprise a relatively static population. To determine how BCs are generated during development, we injected 5-day-old mice with BrdU and found labeled BCs and non-BC epithelial cells 5 days after BrdU. During the next 60 days, the percentage of labeled BCs increased, whereas the percentage of other labeled cell types decreased. These data suggest that BCs are generated from non-BC progenitor cells during postnatal tracheal growth. To test whether the adult epithelium retains the capacity to generate BCs, tracheal epithelial cells were recovered from adult mice and grown in an air-liquid interface (ALI) culture. After transition to differentiation conditions, BCs are detected, and comprise 1% of the total cell population by Day 14. BrdU added to cultures before the differentiation of BCs was chased into BCs, indicating that the increase in BC density is attributable to the proliferation of a non-BC progenitor. We conclude that: (1) BCs are normally a static population in adult mice; (2) BC progenitors proliferate and differentiate during neonatal development; and (3) BCs can be regenerated from a proliferative population resident in adult epithelium.

Tissue factor signals airway epithelial basal cell survival via coagulation and protease-activated receptor isoforms 1 and 2.

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

Human tracheobronchial basal cells. Normal versus remodeling/repairing phenotypes in vivo and in vitro.

Human tracheobronchial epithelial (TBE) basal cells (BCs) function as progenitors in normal tissue. However, mechanistic studies are typically performed in vitro and frequently use BCs recovered from patients who die of nonrespiratory disease. It is not known whether the cadaveric epithelium (1) is undergoing homeostatic remodeling and/or repair, or (2) yields BC clones that represent homeostatic processes identified in tissue. We sought to compare the phenotype of TBE-BCs with that of BCs cultured under optimal clone-forming conditions. TBE pathology was evaluated using quantitative histomorphometry. The cultured BC phenotype was determined by fluorescence-activated cell sorter analysis. Clone organization and cell phenotype were determined by immunostaining. The cadaveric TBE is 20% normal. In these regions, BCs are keratin (K)-5(+) and tetraspanin CD151(+), and demonstrate a low mitotic index. In contrast, 80% of the cadaveric TBE exhibits homeostatic remodeling/repair processes. In these regions, BCs are K5(+)/K14(+), and a subset expresses tissue factor (TF). Passage 1 TBE cells are BCs that are K5(+)/TF(+), and half coexpress CD151. Optimal clone formation conditions use an irradiated NIH3T3 fibroblast feeder layer (American Type Culture Collection, Frederick, MD) and serum-supplemented Epicult-B medium (Stemcell Technologies, La Jolla, CA). The TF(+)/CD151(-) BC subpopulation is the most clonogenic BC subtype, and is enriched with K14(+) cells. TF(+)/CD151(-) BCs generate clones containing BCs that are K5(+)/Trp63(+), but K14(-)/CD151(-). TF(+) cells are limited to the clone edge. In conclusion, clonogenic human TBE BCs (1) exhibit a molecular phenotype that is a composite of the normal and remodeling/reparative BC phenotypes observed in tissue, and (2) generate organoid clones that contain phenotypically distinct BC subpopulations.

Antimicrobial activity of PLUNC protects against Pseudomonas aeruginosa infection.

Epithelial antimicrobial activity may protect the lung against inhaled pathogens. The bactericidal/permeability-increasing protein family has demonstrated antimicrobial activity in vitro. PLUNC (palate, lung, and nasal epithelium associated) is a 25-kDa secreted protein that shares homology with bactericidal/permeability-increasing proteins and is expressed in nasopharyngeal and respiratory epithelium. The objective of this study was to determine whether PLUNC can limit Pseudomonas aeruginosa infection in mice. Transgenic mice (Scgb1a1-hPLUNC) were generated in which human PLUNC (hPLUNC) was directed to the airway epithelium with the Scgb1a1 promoter. The hPLUNC protein (hPLUNC) was detected in the epithelium throughout the trachea and bronchial airways and in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid from transgenic mice exhibited higher antibacterial activity than that from wild type littermates in vitro. After in vivo P. aeruginosa challenge, Scgb1a1-hPLUNC transgenic mice displayed enhanced bacterial clearance. This was accompanied by a decrease in neutrophil infiltration and cytokine levels. More importantly, the overexpressed hPLUNC in Scgb1a1-hPLUNC transgenic mouse airway significantly enhanced mouse survival against P. aeruginosa-induced respiratory infection. These data indicate that PLUNC is a novel antibacterial protein that likely plays a critical role in airway epithelium-mediated innate immune response.

Regeneration of tracheal neotissue in partially decellularized scaffolds.

Extensive tracheal injury or disease can be life-threatening but there is currently no standard of care. Regenerative medicine offers a potential solution to long-segment tracheal defects through the creation of scaffolds that support the generation of healthy neotissue. We developed decellularized tracheal grafts (PDTG) by removing the cells of the epithelium and lamina propria while preserving donor cartilage. We previously demonstrated that PDTG support regeneration of host-derived neotissue. Here, we use a combination of microsurgical, immunofluorescent, and transcriptomic approaches to compare PDTG neotissue with the native airway and surgical controls. We report that PDTG neotissue is composed of native tracheal cell types and that the neoepithelium and microvasculature persisted for at least 6 months. Vascular perfusion of PDTG was established within 2 weeks and the graft recruited multipotential airway stem cells that exhibit normal proliferation and differentiation. Hence, PDTG neotissue recapitulates the structure and function of the host trachea and has the potential to regenerate.

Partial decellularization eliminates immunogenicity in tracheal allografts.

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long-segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T-cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

Roles for ß-catenin and doxycycline in the regulation of respiratory epithelial cell frequency and function.

The expression of ß-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant ß-catenin is termed DE3, and promotes the expression of ß-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent ß-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of ß-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element-Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the ß-catenin reporter transgene TOPGal demonstrated that ß-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+) mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.

Direct and indirect roles for ß-catenin in facultative basal progenitor cell differentiation.

The conducting airway epithelium is maintained and repaired by endogenous progenitor cells. Dysregulated progenitor cell proliferation and differentiation is thought to contribute to epithelial dysplasia in chronic lung disease. Thus modification of progenitor cell function is an attractive therapeutic goal and one that would be facilitated by knowledge of the molecular pathways that regulate their behavior. We modeled the human tracheobronchial epithelium using primary mouse tracheal epithelial cell cultures that were differentiated by exposure to the air-liquid-interface (ALI). A basal cell subset, termed facultative basal cell progenitors (FBP), initiate these cultures and are the progenitor for tracheal-specific secretory cells, the Clara-like cell, and ciliated cells. To test the hypothesis that ß-catenin is necessary for FBP function, ALI cultures were generated from mice homozygous for the Ctnb(flox(E2-6)) allele. In this model, exons 2-6 of the ß-catenin gene are flanked by LoxP sites, allowing conditional knockout of ß-catenin. The ß-catenin locus was modified through transduction with Adenovirus-5-encoding Cre recombinase. This approach generated a mosaic epithelium, comprised of ß-catenin wild-type and ß-catenin knockout cells. Dual immunostaining and quantitative histomorphometric analyses demonstrated that ß-catenin played a direct role in FBP-to-ciliated cell differentiation and that it regulated cell-cell interactions that were necessary for FBP-to-Clara-like cell differentiation. ß-catenin was also necessary for FBP proliferation and long-term FBP viability. We conclude that ß-catenin is a critical determinant of FBP function and suggest that dysregulation of the ß-catenin signaling pathway may contribute to disease pathology.