Differentiation Of Nasal Epithelial Cells Research Articles

Characterizing well-differentiated culture of primary human nasal epithelial cells for use in wound healing assays

The nasal epithelium is the initial contact between the external environment and the respiratory tract and how it responds to noxious stimuli and repairs epithelial damage is important. Growing airway epithelial cells in culture at air-liquid interface allows for a physiologically relevant model of the human upper airways. The aim of the present study was to characterize human primary nasal epithelial cells grown at the air-liquid interface and establish a model for use in wound healing assays. This study determined the time required for full differentiation of nasal epithelial cells in an air-liquid interface culture to be at least 7 weeks using the standardized B-ALI media. Also, a model was established that studied the response to wounding and the effect of EGFR inhibition on this process. Nasal epithelial cultures from healthy subjects were differentiated at air-liquid interface and manually wounded. Wounds were monitored over time to complete closure using a time lapse imaging microscope with cultures identified to have a rate of wound healing above 2.5%/h independent of initial wound size. EGFR inhibition caused the rate of wound healing to drop a significant 4.6%/h with there being no closure of the wound after 48 h. The robust model established in this study will be essential for studying factors influencing wound healing, including host disease status and environmental exposures in the future.Damage to the airway epithelium can be caused by numerous environmental factors. This study describes the development and characterization of a wound healing model from nasal epithelial cells grown at the air-liquid interface. It also identifies the rate of wound closure from healthy donors and the effect epidermal growth factor receptor inhibition has on wound healing.

Hyaluronan antagonizes the differentiation effect of TGF-β1 on nasal epithelial cells through down-regulation of TGF-β type I receptor

Although hyaluronan (HA)-based biomaterials have been proposed to promote mucociliary differentiation of nasal epithelial cells (NECs), the mechanism by which HA affects the growth and differentiation of NECs has not been thoroughly explored. This study investigates the effect and mechanism of HA on the differentiation of NECs. The experiment cultures human NECs in four conditions, namely controls, transforming growth factor (TGF)-β1, TGF-β1 + HA and HA groups. In the TGF group, the NECs become irregular shape without formation of tight junction and mucociliary differentiation of NECs is inhibited. Epithelial–mesenchymal transition (EMT) of NECs also occurs in the TGF group. However, with addition of HA in TGF groups, NECs reveal the mucociliary phenotypes of epithelial cells with tight junction expression. Incubation of TGF-β1 in an NEC culture leads to an increase in phosphorylated type 1 TGF-β receptors (p-TβRI). This increase is attenuated when NECs are cultured in the presence of HA. Similar expressions are observed in phosphorylated smad2/smad3. Additionally, HA-dependent inhibition of TGF-β1 signalling is inhibited by co-incubation with a blocking antibody to CD44. Experimental results indicate that HA can antagonize TGF-β1 effect on EMT and mucociliary differentiation of NECs by down-regulation of TβR I, which is via CD44.

Chitosan promotes aquaporin formation and inhibits mucociliary differentiation of nasal epithelial cells through increased TGF-β1 production.

Although endoscopic sinus surgery is the mainstay surgical treatment for chronic rhinosinusitis, over 15% of patients require a repeat operation wherein postoperative adhesion formation is one of the main causes of failure. Several recently proposed chitosan-based biomaterials promote mucosal healing, reduce postoperative adhesion formation and restore mucociliary function of sinonasal mucosa. However, the effects of chitosan on cellular morphology, re-epithelization, and mucociliary differentiation of nasal epithelial cells (NECs) during the wound healing process have not been thoroughly investigated. The present study investigates the direct effects of chitosan on cellular growth, cellular migration, mucociliary differentiation and aquaporin (AQP) formation of NECs to elucidate the role of chitosan in sinonasal applications. Wound healing assay reveals that proliferation and migration of NECs are inhibited by incubation of chitosan. The NECs become irregular in shape without formation of tight junction and mucociliary differentiation of NECs is inhibited during a culture period with incubation of chitosan. However, AQP3 and AQP5 formation in NECs is significantly higher in chitosan groups than in control groups. Further, expressions of transforming growth factor (TGF)-β1, Smad2, and Smad3 are significantly higher in the chitosan groups compared with controls. The results of the comparison indicate that chitosan inhibits proliferation, migration and mucociliary differentiation of NECs through increasing production of TGF-β1. Copyright © 2016 John Wiley & Sons, Ltd.

Role of IFN-γ, IL-13, and IL-17 on mucociliary differentiation of nasal epithelial cells in chronic rhinosinusitis with nasal polyps.

Mucociliary dysfunction is a prominent pathophysiological feature of chronic rhinosinusitis with nasal polyps (CRSwNP); however, the precise mechanisms underlying mucociliary dysfunction are still unclear. The aim of this study was therefore to evaluate the effects of IFN-γ, IL-13, and IL-17 on human nasal mucociliary differentiation and ciliary beat frequency (CBF) in patients with CRSwNP. Human nasal epithelial cells from tissue of patients with CRSwNP and control subjects were established as air-liquid interface (ALI) primary cultures. Confluent cultures were incubated with10 ng/mL each of IFN-γ, IL-13, or IL-17 for 14 days and assessed for expression of specific morphological markers and factors associated with mucociliary differentiation, the percentage of ciliated and goblet cells, and CBF. In comparison with control subjects, percentage of ciliated cells and CBF were decreased; while percentage of goblet cells, FOXJ1, and MUC5AC mRNA expression were increased in nasal polyp-derived epithelial cultures. Treatment with IFN-γ and IL-13 significantly decreased the expression of β-tubulin IV (specific cilia marker), ciliated cell number, and expression of FOXJ1 and DNAI2, in epithelial cultures derived from both CRSwNP patients and control subjects. Furthermore, while both IFN-γ and IL-13 treatment significantly decreased the CBF of cells from both CRSwNP patients and control subjects, IL-13 additionally significantly increased goblet cell number and the expression of MUC5AC and CLCA1, in these cultures. IL-17 treatment did not significantly affect ciliated or goblet cell differentiation, CBF, nor MUC5AC and CLCA1 expression, but increased both MUC5B mRNA and protein expression in these cultures. The demonstration that IFN-γ and IL-13 both significantly reduce ciliated cell differentiation and CBF in CRSwNP patients, and IL-13 additionally induces significant goblet cell hyperplasia and MUC5AC mucin expression, as well as IL-17 significantly increases MUC5B mucin expression, suggests that these inflammatory cytokines may be potential therapeutic targets in the management of CRSwNP.

Muco‐ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro

Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which transforming growth factor-beta1 (TGF-beta1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-beta1. Basal, mucus, and ciliated cells were characterized by cytokeratin-14, MUC5AC, and betaIV tubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-beta1 supplementation. Using RT-PCR, the effects of TGF-beta1 on MUC5AC and DNAI1 genes, specifically transcribed in mucus and ciliated cells, were evaluated. In situ, high TGF-beta1 expression was associated with low MUC5AC and betaIV tubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while betaIV tubulin expression increased. Transforming growth factor-beta1 supplementation downregulated MUC5AC and betaIV tubulin expression as well as MUC5AC and DNAI1 transcripts. After a wound, differentiation into mucus and ciliated cells was possible and partially inhibited in vitro by TGF-beta1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.

Culture of nasal epithelial cells using chitosan‐based membranes

The aim of this study was to evaluate whether chitosan-based membranes can be used as scaffolds for growth and differentiation of nasal epithelial cells (NECs). Our final goal was to establish a novel methodology for enhancing the regeneration of the respiratory system. Prospective study. Human NECs were cultured on three various substrates, e.g., chitosan membranes, collagen, and chitosan-collagen membranes. Morphology of NECs was examined via light and electron microscopy, the area of ciliated cells was measured by confocal microscopy, and ciliary beat frequency was also evaluated. Expression of mucin genes was investigated with reverse-transcription polymerase chain reaction. NECs were found to be successfully adhesive with collagen and chitosan-collagen membranes at day 3 after seeding, but not with chitosan membranes. The cilia area on collagen were 6.1% +/- 1.2%, 8.4% +/- 1.4%, and 12.5% +/- 1.9% at days 7, 14, and 21 after confluence, respectively, compared with 5.1% +/- 0.9%, 8.6% +/- 1.6%, and 12.3% +/- 2.1% in chitosan-collagen membranes, exhibited nonsignificant difference (P > .05). There were no significant differences in ciliary beat frequency between each group. The expression levels of mucin genes, namely, MUC5AC, MUC5B, and MUC2, in NECs on both collagen and chitosan-collagen membranes did not differ significantly (P > .05). A small amount collagen mixed with chitosan substrate may improve the biocompatibility and promote the mucociliary differentiation in NECs. It appears that chitosan-collagen membrane is a promising scaffold for culture of the nasal epithelium, which sets the stage for studying tissue regeneration in the respiratory system.

Increased Communication Among Nasal Epithelial Cells in Air‐Liquid Interface Culture

The retinoid acid (RA) sufficient air-liquid interface (ALI) cell culture model, but not the classical submerged single layer (SSL) cell culture model, can achieve ciliary differentiation of nasal epithelial cells. Because gap junction mediated intercellular communication (GJIC) may contribute to differentiation in numerous cell types, this study compared the extent of GJIC and the expression of Connexin 43 (Cx43) in nasal epithelial cells in both SSL and ALI cultures. Cell morphology was examined via optical and scanning electron microscope, and the number of cells with ciliary beating were counted. Lucifer Yellow dye transfer test using the scrape loading method was performed to assess the GJIC. Cx43 expression was measured with reverse-transcription polymerase chain reaction (RT-PCR) and quantitative (Q)-PCR. Nasal epithelial cells in ALI culture exhibited increased numbers of ciliated cells compared with SSL culture during the 3-week culture period. On day 20, GJIC was increased in ALI culture (ALI % - SSL % = 9.6 +/- 1.2%, n = 5). Accordingly, Cx43 expression was increased via RT-PCR (4.22-fold) and Q-PCR (5.3 +/- 1.1-fold, n = 5) examination. RA sufficient ALI culture manifested more differentiated nasal epithelial cell status with ciliogenesis. Cx43, being the responsible molecule for GJIC, increased in parallel. Consequently, as in primary cultured limbal epithelial cells, Cx43 expression and extent of GJIC may serve as markers for the differentiation status of nasal epithelial cells.

Growth and differentiation of human nasal epithelial cells in culture. Serum-free, hormone-supplemented medium and proteoglycan synthesis.

Ham's F12 medium supplemented with insulin (Ins), transferrin (Tf), epidermal growth factor (EGF), hydrocortisone (HC), T3, cholera toxin (CT), and bovine hypothalamus extract (BHE) was developed for in vitro growth of human nasal epithelial (HNE) cells. The HNE cells were dissociated from freshly excised nasal polyps or turbinates with protease. Colony-forming efficiency of primary HNE cells was approximately 5%. Growth studies showed Ins, BHE, and CT were essential for growth; HC, EGF, Tf, and T3 were also stimulatory for growth. The growth rate in this serum-free, hormone-supplemented medium was 24 h per population doubling. Up to 20 population doublings and 3 passages of dissociated HNE cells could be achieved. Addition of serum to this culture medium inhibited epithelial cell growth. Vitamin A had no apparent effect on cell growth but induced an alteration in the morphologic characteristics of the cell. The epithelial nature of cultured cells was confirmed by positive staining with antihuman keratin antibody, ultrastructural studies, and by formation of a columnar, ciliated epithelium in denuded tracheal grafts repopulated by these cultured HNE cells. Biochemical analyses of glycoproteins (labeled with 3H-glucosamine and/or 35S-sulfate) secreted by cultured HNE cells were unable to demonstrate the secretion of mucinlike glycoproteins in culture. Instead, major secretory products of cultured cells were hyaluronate and heparan sulfate. These results were in agreement with morphologic observations that showed no mucus-secreting granules in cultured cells. Dome formation was observed in high cell density cultures. We conclude that HNE cells can be cultured in well-defined culture media. As indicated by formation of domes, these cells may be useful for in vitro ion transport studies. Further differentiation, however, may be required for studies of mucin synthesis.