The identification of human pancreatic endocrine stem cells which are capable of growing in vitro and differentiating into mature Beta-cells in vivo, would represent a powerful strategy to generate a suitable cell mass for transplantation in IDDM. Islet Beta-cells are thought to develop from precursor cells present in the pancreatic ductal epithelium. Yet, the phenotype of these endocrine progenitors, and the mechanisms regulating their emergence from the ductal epithelium remain elusive. Our preliminary studies in the human pancreas demonstrate that the cell adhesion molecule DCC identifies ductal cells, that can be expanded in vitro, and induced to express insulin when reaggregated into three-dimensional islet-like cell clusters. More importantly, transplantation of reaggregated DCC+ cells in immune deficient nude mice results in the development of functional islet Beta-cells in vivo. We further show that fetal pancreatic DCC+ cells produce the extracellular matrix (ECM)-degrading enzymes MMP2 and MMP9, whereas fetal mesenchymal cells secrete the chemo-attractant molecule Netrin-1, a newly identified ligand for DCC. These observations suggest a molecular pathway by which DCC+ endocrine progenitors are stimulated by the mesenchymal chemoattractant Netrin-1 to migrate out of the ductal epithelium into the surrounding mesenchyme through migration cues opened by ECM-modifying enzymes. The central hypothesis of this proposal is that pancreatic ductal cells marked by the expression of DCC comprise progenitors of the islet cell lineage. Based on the recent identification of DCC as a cell adhesion molecule regulating epithelial-mesenchymal interaction, and most recently as a receptor for the chemoattractant molecule Netrin-1, we also postulate that interactions of ductal cells with the surrounding mesenchyme and the activation of specific MMPs, regulate the outgrowth and migration of endocrine progenitors during pancreatic islet ontogeny. Finally, we hypothesize that DCC+ cells isolated from other gastrointestinal compartments sharing a common embryological origin with the pancreas, may comprise islet progenitors. We propose to test these hypothesis by the following Specific Aims: 1) To assess the proliferative potential of DCC+ cells isolated from fetal and adult human pancreas; 2) To study the role of direct epithelial- mesenchymal interactions and mesenchyme-derived factors on the growth, migration and endocrine determination of DCC+ cells; 3) To study the role of metalloproteinase MMP-2 and MMP-9 in the migration, proliferation, and differentiation of DCC+ cells; 4) To study the contribution of epithelial cell-cell adhesion molecules to the endocrine differentiation program of DCC+ cells; 5) To assess the ability of DCC+ cells isolated from other compartments of the gastrointestinal tract to enter an endocrine differentiation pathway.
