A multicellular 3D stem cell model to define the role of stroma in epithelial differentiation

In aging men the disorders of prostate are a major medical problem. Benign prostatic hyperplasia and cancer are increasingly prevalent. To find cures for these diseases it is essential to understand how the prostate grows and functions normally. All organs have their own population of stem cells which grow and develop into a variety of cells which communicate to form correct organ architecture and function. This occurs as a result of signals from the stem cell's own genes but also from signals provided by neighbouring cells, known as stroma. In the prostate, how this occurs is unknown. We propose to develop a model to grow gland-like structures from adult stem cells in the laboratory. The model will be employed to understand how stromal cells influence prostate cellular architecture. We aim to identify proteins which act as signals from the stroma to change epithelial shape. The shape of a cell has important effects on cell function. These experiments will increase our knowledge of how tissues develop and function. Development of tissue-like models based on human cells will provide a valuable gap between results from animal models and human clinical studies, to help understand the basic mechanisms of human physiology and disease. Such model systems will reduce the need for animal experimentation, which is currently the best way to investigate complex cell interactions in tissues. We anticipate the model will aid university directed research into human differentiation and disease mechanisms, but also for the pharmaceutical industry to screen new drugs for efficacy and safety in humans before trial.

Recent advances in our lab have resulted in the isolation of human adult prostate stem cells and the development of 3D models of prostatic acini from basal cells. Results from 3D modelling indicate that stroma is important for epithelial morphogenesis and differentiation. Importantly, stromal cultures increase epithelial cell polarity and columnar cell shape. Using electron microscopy and RT-PCR our preliminary data has found that these morphological effects are accompanied by increased desmosomal expression. We now wish to develop our tissue engineering to produce a 3D model of prostatic acini using a homogeneous population of stem cells. A stem cell model will allow the study of full epithelial differentiation and the stem cell niche. It is important to model the prostate with human cells because the mouse prostate has a different anatomy, cell structure and protein function, and does not develop equivalent diseases to humans. The model will be used to investigate our hypothesis that 'stroma signals to control epithelial cell shape and polarity'. We will confirm which desmosomal isoforms are present in prostate epithelial acini and which are upregulated by stromal cultures, using Western Blotting and real time PCR. Upregulated desmosomal isoforms will be used as markers for epithelial cell polarity and shape. A differential gene expression profile will be generated from stroma grown with epithelial acini in 3D culture and stroma grown in 3D culture without acini, using microarray analysis. Candidate stromal genes will be identified that signal to upregulate epithelial polarity (desmosomal expression) and their function will be confirmed using siRNA knockdown studies. This is a novel pathway for epithelial cell differentiation which has not been studied before.