Proximal deposition of collagen IV by fibroblasts contributes to basement membrane formation by colon epithelial cells in vitro.

The basement membrane (BM) underlying epithelial tissue is a thin layer of extracellular matrix that governs tissue integrity and function. Epithelial BMs are generally assembled using BM components secreted from two origins: epithelium and stroma. Although de novo BM formation involves self-assembly processes of large proteins, it remains unclear how stroma-derived macromolecules are transported and assembled, specifically in the BM region. In this study, we established an in vitro co-culture model of BM formation in which DLD-1 human colon epithelial cells were cultured on top of collagen I gel containing human embryonic OUMS-36T-2 fibroblasts as stromal cells. A distinct feature of our system is represented by OUMS-36T-2 cells which are almost exclusively responsible for synthesis of collagen IV, a major BM component. Exploiting this advantage, we found that collagen IV incorporation was significantly impaired in culture conditions where OUMS-36T-2 cells were not allowed to directly contact DLD-1 cells. Soluble collagen IV, once diluted in the culture medium, did not accumulate in the BM region efficiently. Live imaging of fluorescently tagged collagen IV revealed that OUMS-36T-2 cells deposited collagen IV aggregates directly onto the basal surface of DLD-1 cells. Collectively, these results indicate a novel mode of collagen IV deposition in which fibroblasts proximal to epithelial cells exclusively contribute to collagen IV assembly during BM formation.