Human Bronchial Epithelial Cells Differentiate to 3D Glandular Acini on Basement Membrane Matrix

Abstract

To create a model system that investigates mechanisms resulting in hyperplasia and hypertrophy of respiratory tract submucosal glands, we developed an in vitro three-dimensional (3D) system wherein normal human bronchial epithelial (HBE) cells differentiated into glandular acini when grown on a basement membrane matrix. The differentiation of primary HBE cells into glandular acini was monitored temporally by light microscopy. Apoptosis-induced lumen formation was observed by immunofluorescence analysis. The acinar cells expressed and secreted MUC5B mucin (marker for glandular mucous cells) and lysozyme, lactoferrin, and zinc-a2-glycoprotein (markers for glandular serous cells) at Day 22. b-Tubulin IV, a marker for ciliated cells, was not detected. Expression of mucous and serous cell markers in HBE glandular acini demonstrated that HBE cells grown on a basement membrane matrix differentiated into acini that exhibit molecular characteristics of respiratory tract glandular acinar cells. Inhibition studies with neutralizing antibodies resulted in a marked decrease in size of the spheroids at Day 7, demonstrating that laminin (a major component of the basement membrane matrix), the cell surface receptor integrin a6, and the cell junction marker E-cadherin have functional roles in HBE acinar morphogenesis. No significant variability was detected in the average size of glandular acini formed by HBE cells from two normal individuals. These results demonstrated that this in vitro model system is reproducible, stable, and potentially useful for studies of glandular differentiation and hyperplasia.

Three-dimensional culture model to distinguish normal from malignant human bronchial epithelial cells

“…3) [23], AZGP1 (a glandular serous cell marker) and b-tubulin IV (a ciliated cell marker) [24]. As described by WU et al [19], in HBECs, MUC5B and AZGP1 were distributed generally in luminal cells suspended within the lumen of the spheres and some were localised at the apical membrane ( fig. 3a and b, upper panels).…”

“…HBECs on two-dimensional monolayer cultures grew exponentially ( fig. 1a), as previously described [19]. Cells were then embedded in Matrigel matrix as single cells ( fig.…”

“…Lung epithelial cells from distal regions of rodent airways have also been shown to grow in Matrigel [17,18] and more recently in normal HBECs [19]. In their study, WU et al [19] have shown that normal human bronchial epithelial cells could differentiate into three-dimensional spheroids when grown on Matrigel. FRANZDÓ TTIR et al [20] have also shown that human bronchial epithelial cell line could be induced by endothelial cells to form branching bronchioalveolar-like structure in three-dimensional culture.…”

“…Different studies have used a reconstituted Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) derived from the Englebreth-Holm-Swarm tumour, composed of laminin, collagen IV and entactin, to study the morphology of human epithelial cell in three-dimensional culture from breast [11], intestine [12,13], pancreas [14,15] and metastatic tumour cells [16]. Lung epithelial cells from distal regions of rodent airways have also been shown to grow in Matrigel [17,18] and more recently in normal HBECs [19]. In their study, WU et al [19] have shown that normal human bronchial epithelial cells could differentiate into three-dimensional spheroids when grown on Matrigel.…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…While the focus of this section of the current review is on the surface epithelium, it is also worth noting that primary human bronchial epithelial cells can differentiate into three-dimensional spheroids with a lumen and cells that secrete MUC5B mucin (a marker for glandular mucous cells) together with lysozyme, lactoferrin and zinc-a2-glycoprotein (markers for glandular serous cells) when grown on Matrigel™ (Wu et al, 2011). This in vitro model system of respiratory tract glandular acini is relevant for investigations into mechanisms that lead to the submucosal glandular hyperplasia seen in COPD, CF and chronic rhinosinusitis.…”