Julia T. Arnold, Ph.D.

Dissertation research performed under the guidence of Dr. David G. Kaufman

ABSTRACT
    Regulation of epithelial cell function and morphogenesis by the mesenchyme or stroma has been well-established in animal studies. Control of epithelial cell growth and differentiation is very complex. In steroid target tissues such as the endometrium, breast, or prostate, this stromal-mediated regulation involves the hormonal state of the stroma, the presence of appropriate ECM and the expression of autocrine and/or paracrine signals. Aspects of this regulation, especially the ECM contribution, have previously not been reproduced in cell culture due to lack of appropriate coculture models. The coculture model described below provides a valuable tool towards understanding these interactions in vitro. This model demonstrates that endometrial stromal cells can regulate the growth and differentiation of both primary endometrial epithelial cells and endometrial cancer (Ishikawa) cells and that normal paracrine relationships can be reconstituted in vitro. The methods of cell coculture that have been developed maintain more normal patterns of epithelial and stromal cell growth and differentiation. Culture media ingredients including concentrations of fetal bovine serum were optimized. The growth and morphology of stromal and epithelial cultures were compared on growth substrates including tissue culture plastic and the basement membrane extract (BME), Matrigel. The interactions between the two cell types were compared when cells were in contact within the BME to when they were separated by a filter. The regulation of epithelial cell proliferation by stromal cells in BME was quantitated and compared to proliferation of epithelial cells in coculture with stromal cells grown on plastic substrate. Profound growth inhibitory effects on the epithelial cells were observed when the stromal cells were cultured in basement membrane extract (BME). Regulation of epithelial cell gene expression by stroma also depended on BME. Stromal cells cultured in BME were able to control epithelial cell differentiation as measured by glycodelin expression. Normal human stromal cells also may restore a more normal phenotype in cells from a well-differentiated endometrial cancer cell line as measured by regulation of growth and by induction of glycodelin production. Following the establishment of this coculture model, this new technology was applied to begin to identify the mechanisms and paracrine mediators involved in stromal regulation of epithelial cells. By comparing gene expression in stromal cells cultured in BME with stromal cells grown on plastic, stromal steroid receptor status, selected growth factor production and integrin expression was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). The coculture studies described herein show great promise to begin to investigate interactions between endometrial stromal and epithelial cells and to understand the molecular basis for these interactions that regulate normal growth and differentiation of cells.