Reconstituted skin from murine embryonic stem cells.

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.

Immunohistochemistry of CFTR in native tissues and primary epithelial cell cultures.

Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties. However, such data are of the highest importance to understand the pathophysiology of CF. The purpose of this article is to outline several assays for the characterization of primary epithelial cultures and to review different CFTR immunostaining protocols.

Aquaporin-3 expression in human fetal airway epithelial progenitor cells.

Airway epithelium stem cells have not yet been prospectively identified, but it is generally assumed that both secretory and basal cells have the capacity to divide and differentiate. Previously, we developed a test for progenitor cells of the human airway epithelium, relying on the transplantation of fetal respiratory tissues into immunodeficient mice. In this study, we hypothesized that airway-repopulating epithelial progenitors can be marked with surface antigens, and we screened an array of such candidate markers, including lectin ligands, the CD44 and CD166 adhesion molecules, and the aquaporin-3 (AQP3) water channel. We observed that AQP3 is selectively expressed on the surface of basal cells, allowing the separation by flow cytometry of AQP3+ basal cells and AQP3- ciliated and secretory cells. Functional evaluation of sorted cells in vivo showed that AQP3+ cells can restore a normal pseudostratified, mucociliary epithelium as well as submucosal glands. AQP3- cells are also endowed with a similar potential, although faster engraftment suggests their inclusion of more committed progenitors. These results show that stem cell candidates in the human tracheo-bronchial mucosa can be positively selected with a novel marker but also, for the first time, that epithelial progenitors exist among both basal and suprabasal cell subsets within the human airway.

Embryonic stem cells generate airway epithelial tissue.

Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

Characterization of ion and fluid transport in human bronchioles.

The regulation of the volume and composition of airway surface liquid is achieved through epithelial ion transport processes. In humans, these processes have been characterized in proximal but not distal airways. Segments of human bronchioles were dissected from surgically removed lung pieces. The transmural potential difference of microperfused bronchioles was inhibited by luminal exposure to amiloride and increased when exposed to the Cl secretagogues forskolin and ATP in the presence of amiloride. Human bronchiolar epithelial cells were cultured on permeable supports and studied in Ussing chambers. They generated a short circuit current (Isc) that decreased in response to amiloride and increased in response to forskolin and to ATP in the presence of amiloride. In low-Cl Kreb's Ringer bicarbonate, the baseline Isc and amiloride-induced decrease in Isc were not different, whereas the forskolin- and ATP-induced increases in Isc were smaller. Fluid transport measurement in excised bronchioles revealed a basal absorptive flow that was reduced by amiloride, whereas forskolin and ATP combined induced a secretory flow in the presence of amiloride. We conclude that human bronchioles actively absorb Na and fluid in unstimulated conditions and are capable of active Cl and fluid secretion when exposed to forskolin and to ATP.

Stimulation of beta 2-adrenergic receptor increases cystic fibrosis transmembrane conductance regulator expression in human airway epithelial cells through a cAMP/protein kinase A-independent pathway.

PSD-95/Dlg-A/ZO-1 (PDZ) domains play an essential role in determining cell polarity. The Na(+)/H(+) exchanger regulatory factor (NHERF), also known as EBP50, contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. Moreover, it has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2)-adrenergic receptor (beta(2)AR) bind equally well to the PDZ1 domain of EBP50. We hypothesized that beta(2)AR activation may regulate CFTR protein expression. To verify this, we evaluated the effects of a pharmacologically relevant concentration of salmeterol (2.10(-7) m), a long acting beta(2)AR agonist, on CFTR expression in primary human airway epithelial cells (HAEC). beta(2)AR stimulation induced a time-dependent increase in apical CFTR protein expression, with a maximal response reached after treatment for 24 h. This effect was post-transcriptional, dependent upon the beta(2)AR agonist binding to beta(2)AR and independent of the known beta(2)AR agonist-mediated cAMP/PKA pathway. We demonstrated by immunohistochemistry that CFTR, beta(2)AR, and EBP50 localize to the apical membrane of HAEC. Analyses of anti-EBP50 protein immunoprecipitate showed that salmeterol induced an increase in the amount of CFTR that binds to EBP50. These data suggest that beta(2)AR activation regulates the association of CFTR with EBP50 in polarized HAEC.

Airway epithelial integrity is protected by a long-acting beta2-adrenergic receptor agonist.

Airway epithelial integrity may be impaired by bacterial exoproducts, which are able to degrade tight junction-associated proteins such as zonula occludens 1 (ZO-1). We have investigated the protective effect of salmeterol, a long-acting beta(2)-adrenergic agonist, on Pseudomonas aeruginosa-induced alteration of the epithelial junctional barrier. We demonstrate in human airway epithelial cells (HAEC) that salmeterol induces a time-dependent increase in ZO-1 protein, although no significant change in ZO-1 transcripts was observed. When HAEC cultures were exposed to P. aeruginosa (PAO1) supernatants, apical expression of ZO-1 protein was maintained in salmeterol-pretreated HAEC cultures, whereas it disappeared after PAO1 exposure in cultures not pretreated with salmeterol. Western blot experiments showed that the 220-kD ZO-1 protein was decreased after PAO1 incubation but was still present in salmeterol-pretreated HAEC extracts. The functional activity of ZO-1 protein was monitored by measuring transepithelial resistance and analyzing the diffusion of a low molecular weight tracer through the intercellular spaces. After PAO1 incubation, the epithelial integrity of HAEC was impaired, as shown by a decrease in transepithelial resistance and increased paracellular permeability, but was not significantly altered after salmeterol preincubation. These results demonstrate that salmeterol may contribute to the protection of the airway epithelium barrier against bacterial virulence factors.

Polarized expression of cystic fibrosis transmembrane conductance regulator and associated epithelial proteins during the regeneration of human airway surface epithelium in three-dimensional culture.

We have previously shown that, in normal human airway tissue, localization of the cystic fibrosis transmembrane conductance regulator (CFTR) can be affected by epithelial maturation, polarity, and differentiation and that CFTR trafficking and apical localization depend on the integrity of the airway epithelium. In this study, we addressed the question of whether the three-dimensional (3-D) organization of adult human airway epithelial cells in suspension culture under rotation, leading to spheroid-like structures, could mimic the in vivo phenomenon of differentiation and polarization. The kinetics of the differentiation, polarity, and formation of the CFTR-ZO-1-ezrin complex was analyzed by transmission, scanning, and immunofluorescence microscopy. Functional activity of the airway surface epithelium was assessed by monitoring the degree of cAMP-stimulated chloride efflux from cultured cells. Our results show that after the initial step of dedifferentiation, characterized by a loss of ciliated cells and disappearance of epithelial subapical CFTR-ezrin-ZO-1 complex, the isolated cells formed 3-D spheroid structures within 24 hours. After 15 days, progressive ciliogenesis was observed and secretory cells could be identified. After 35 days of 3-D culture, ZO-1, CFTR, ezrin, and CD59 were apically or subapically located, and well-differentiated secretory and ciliated cells were identified. CFTR functionality was assessed by analyzing the Cl(-) secretion after amiloride and forskolin perfusion. After 35 days of culture of spheroids in suspension, a significant increase in Cl(-) efflux was observed in well-differentiated ciliated cells.

Dynamic interaction between airway epithelial cells and Staphylococcus aureus.

Staphylococcus aureus is a major cause of pulmonary infection, particularly in cystic fibrosis (CF) patients. However, few aspects of the interplay between S. aureus and host airway epithelial cells have been investigated thus far. We investigated by videomicroscopy the time- and bacterial concentration-dependent (10(4), 10(6), and 10(8) CFU/ml) effect of S. aureus on adherence, internalization, and the associated damage of the airway epithelial cells. The balance between the secretion by S. aureus of the alpha-toxin virulence factor and by the airway cells of the antibacterial secretory leukoproteinase inhibitor (SLPI) was also analyzed. After 1 h of interaction, whatever the initial bacterial concentration, a low percentage of S. aureus (<8%) adhered to airway cells, and no airway epithelial cell damage was observed. In contrast, after 24 h of incubation, more bacteria adhered to airway epithelial cells, internalized bacteria were observed, and a bacterial concentration-dependent effect on airway cell damage was observed. At 24 h, most airway cells incubated with bacteria at 10(8) CFU/ml exhibited a necrotic phenotype. The necrosis was preceded by a transient apoptotic process. In parallel, we observed a time- and bacterial concentration-dependent decrease in SLPI and increase in alpha-toxin expression. These results suggest that airway cells can defend against S. aureus in the early stages of infection. However, in later phases, there is a marked imbalance between the bactericidal capacity of host cells and bacterial virulence. These findings reinforce the potential importance of S. aureus in the pathogenicity of airway infections, including those observed early in CF patients.