In Vitro Reconstitution of the Tracheal Epithelium

Abstract

We have developed a unique in vitro reconstitution system for tracheal epithelia of guinea pigs. In the system, a human amnion membrane was used as a basement membrane and the tracheal epithelial cells were cultured on the epithelial side of the membrane. Three weeks later, the tracheal fibroblasts were co-cultured on the serosal side of the amnion membrane and the culturing was continued for an additional 10 d. The morphology of the cultured epithelial cells consisted of a pseudostratified columnar ciliated epithelium from cuboidal ciliated epithelium during the last 10 d of the culture period. Epithelial cells included both goblet-like and basal cells. In addition, the frequency of each type of differentiated cells was almost identical to that of in vivo tracheas. Interestingly, the same results were obtained when the conditioned medium of the tracheal fibroblasts was used instead of the fibroblasts themselves. These results suggest that epithelial-mesenchymal interaction is likely involved in growth and differentiation of epithelial cells in vivo in a soluble factor(s)-mediated manner. As well as the epithelial cells, the fibroblasts also formed a multilayer during the last 10 d of co-culturing. This indicates that in vitro reconstitution of tracheal epithelia is achieved without addition of any exogenous growth or differentiation factors. The reconstitution system is shown to be useful for investigating the cellular and molecular interaction of epithelial and mesenchymal cells. Possible applications of the culture system and possible factors involved in growth and differentiation of epithelial cells are discussed.

Autologous Implantation of Bilayered Tissue-Engineered Respiratory Epithelium for Tracheal Mucosal Regenesis in a Sheep Model

“…Respiratory epithelial cells and fibroblasts were cultured using the coculture system described in our previous studies [Ziegelaar et al, 2002;Nur Adelina et al, 2007]. Stimulatory effects of fibroblasts facilitated epithelial cell differentiation into mainly basal, ciliated and goblet cells with increased mucin secretion [Goto et al, 1999;Kobayashi et al, 2006Kobayashi et al, , 2007. Respiratory epithelium cells obtained using a coculture system produce significantly more basal cells and stem cell gene expression, suggesting a high potential to regenerate a normal tracheal epithelium in vivo [Hong et al, 2004;Nur Adelina et al, 2007].…”

“…The basement membrane was reported to influence differentiation, migration and proliferation of epithelial cells and is im- portant in maintaining structural tissue integrity [Kobayashi et al, 2006[Kobayashi et al, , 2007Nomoto et al, 2008]. Another previous study suggested that epithelial-fibroblast interaction plays a crucial role in the organogenesis, morphogenesis and differentiation of the tracheal epithelial cells [Goto et al, 1999]. Although the implantation of the cell and fibrin construct demonstrated the regeneration of pseudostratified columnar epithelium lining, the cells found within this epithelium 4 weeks after surgery actually comprised cells in heterogenous morphologies.…”

Respiratory Tissue Engineering: Current Status and Opportunities for the Future

“…3). Recognising the effects of mesenchymal cells on epithelial proliferation and differentiation from earlier work (197) (198). The presence of hMSCs altered the temporal pattern of mucin secretion by the epithelial monolayer, but the authors noted a prominent limitation in their model that when the MSCs were induced towards a chondrogenic lineage, they detached from the insert before any discernible effects on the bronchial epithelium could be observed, negating the coculture system.…”

Tracheal Tissue Engineering

“…However, this biomimetic seeding strategy has had some success in humans, when seeding chondrocytes differentiated from MSCs on the exterior surface and epithelial cells on the interior surface [146]. Other combinations of cells utilized for trachea repair are epithelial cells and fibroblasts [172], endothelial cells and epithelial cells [173], chondrocytes and fibroblasts [136], fibroblasts and muscle cells [148], and bone marrow stromal cell-derived chondrocytes and epithelial cells [152,146]. Walles et al evaluated an even greater number of cell types; seeding chondrocytes, bone marrow-derived smooth muscle cells, bone marrowderived endothelial cells, or respiratory epithelium on a decellularized matrix [150].…”