IDENTIFICATION & CHARACTERIZATION OF PANCREATIC ENDOCRINE PROGENITOR CELLS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。细胞粘附分子（CAM）调控的细胞-细胞相互作用最近已被证明是支持细胞功能如增殖、分化和存活的多种信号的调节者。我们的初步研究表明，细胞粘附分子Ep-CAM可以阻断人胰岛细胞的促有丝分裂信号。与此结果一致，我们发现Ep-CAM在转基因小鼠模型的胰腺导管细胞中的过表达导致胰腺显著增大，这是由于外分泌区室（5倍）和内分泌区室（12倍）的细胞数量增加。这些结果表明，在导管上皮中Ep-CAM的转基因过表达可能会导致外分泌和内分泌细胞谱系的推定祖细胞的扩增。重要的是，尽管胰岛显著增大，但这些小鼠没有显示出胰岛素产生和分泌的缺陷。因此，我们推测，与生长因子介导的细胞增殖不同，其可能与分化性状表达的降低有关，在这些转基因小鼠中，Ep-CAM介导的推定胰岛祖细胞（包含在导管上皮内）的细胞生长允许大胰岛质量的发展，对β细胞特异性基因表达没有明显的负面影响。 我们研究的中心假设是，通过腺病毒基因转移在人β细胞中瞬时过表达Ep-CAM可以诱导细胞生长，对内分泌细胞功能的影响最小。因此，基于由细胞粘附分子激活的独特分子途径，而不是由经典的可溶性生长因子诱导的细胞增殖，我们假设由人胰岛细胞中Ep-CAM的暂时有限的过表达提供的瞬时生长刺激将对胰岛素产生具有最小（如果有的话）的负面影响。 我们的目的是测试Ep-CAM是否可以用作分子开关，在体外瞬时激活人β细胞生长。此外，我们计划测试用可溶性重组Ep-CAM分子（我们实验室生产的）治疗胰岛β细胞是否也可以刺激细胞生长和/或改善胰岛β细胞的存活率。 1）研究腺病毒载体介导的Ep-CAM在人β细胞中的瞬时过表达对细胞生长、分化和存活的影响; 2）通过“细胞形态学”方法评估Ep-CAM过表达对胰岛内β细胞簇三维结构的形态调节作用。先进的显微技术”，如“双光子共聚焦显微镜”和“高压电子显微镜”。“由于胰岛细胞簇内β细胞的结构组织已被证明对胰岛素产生细胞的内分泌功能具有深远的功能影响，我们预测，拟议的研究将揭示粘附受体Ep-CAM介导的细胞-细胞相互作用的新机制，并最终可能有助于设计用于移植的胰岛细胞扩增的新方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell-cell interactions regulated by cell adhesion molecules (CAMs) have been recently shown to transduce a variety of signals supporting cellular functions as diverse as proliferation, differentiation, and survival. Our preliminary studies indicate that the cell adhesion molecule Ep-CAM can transduce mitogenic signals in human pancreatic islet cells. Consistently with this result, we find that over-expression of Ep-CAM in pancreatic ductal cells of a transgenic mouse model causes a significant enlargement of the pancreas, due to an increased cell number of both the exocrine (5-folds) and endocrine compartments (12-folds). These results indicate that transgenic over-expression of Ep-CAM in the ductal epithelium may cause the expansion of putative progenitors of both the exocrine and endocrine cell lineages. Importantly, despite the significant enlargement of islets, these mice show no defects of insulin production and secretion. Thus, we postulate that unlike growth factors-mediated cell proliferation, which can be associated with a decreased expression of differentiated traits, Ep-CAM-mediated cell growth of putative islet progenitors (comprised within the ductal epithelium) in these transgenic mice allows the development of a large islet mass with no apparent negative effects on beta-cell-specific genes¿ expression. The central hypothesis of our studies is that transient over-expression of Ep-CAM in human beta-cells by means of Adenoviral gene transfer may induce cell growth with minimal effects on the endocrine cell function. Thus, based on the unique molecular pathways activated by cell adhesion molecules as opposed to cell proliferation induced by classical soluble growth factors, we hypothesize that a transient growth stimulus provided by a temporally limited over-expression of Ep-CAM in human islet cells will have minimal (if any) negative effects on insulin production. Our objective is to test whether Ep-CAM can be used as a molecular switch to transiently activate human beta-cell growth in vitro. Furthermore, we plan to test whether treatment of islet beta-cells with a soluble recombinant Ep-CAM molecule (produced in our laboratory) may also stimulate cell growth, and/or improve islet beta-cells¿ survival. The proposed experimental plan focuses on the following Specific Aims: 1) To study the effects of transient over-expression of Ep-CAM in human adult beta-cells by means of Adenoviral vectors, and assess the effects on cell growth, differentiation, and survival; 2) To assess the mophoregulatory effects of Ep-CAM over-expression on the three-dimensional organization of beta-cell clusters within pancreatic islets by means of "advanced microscopic techniques" such as "two photon confocal microscopy," and "high voltage electron microscopy." Since the architectural organization of the beta-cells within the islet cell clusters has been shown to have a profound functional impact on the endocrine function of insulin-producing cells, we predict that the proposed studies will uncover novel mechanisms of cell-cell interactions mediated by the adhesion receptor Ep-CAM, and may ultimately help in the design of novel protocols of islet cell expansion for transplantation.