IDENTIFICATION & CHARACTERIZATION OF PANCREATIC ENDOCRINE PROGENITOR CELLS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。近年来，细胞黏附分子(CAM)调控的细胞间相互作用被证明可以传递多种信号，支持细胞的增殖、分化和存活等多种功能。我们的初步研究表明，细胞黏附分子EP-CAM可以在人胰岛细胞中传递有丝分裂信号。与这一结果一致的是，我们发现在转基因小鼠模型的胰腺导管细胞中过表达EP-CAM导致胰腺显著增大，这是由于外分泌(5倍)和内分泌(12倍)的细胞数量增加。这些结果表明，转基因的EP-CAM在导管上皮细胞中的过度表达可能导致外分泌和内分泌细胞谱系的前体细胞的扩张。重要的是，尽管这些小鼠的胰岛显著增大，但它们并没有表现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，EP-CAM介导的细胞生长可能与分化特征的表达减少有关，在这些转基因小鼠中，EP-CAM介导的假定胰岛祖细胞(包括导管上皮内)的细胞生长允许发育成大的胰岛团块，而对β细胞特异性基因的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可能会诱导细胞生长，而对内分泌细胞功能的影响最小。因此，基于细胞黏附分子激活的独特的分子通路，而不是经典的可溶性生长因子诱导的细胞增殖，我们假设，在人胰岛细胞中，由时间有限的EP-CAM过度表达提供的瞬时生长刺激对胰岛素产生的负面影响将是最小的(如果有的话)。我们的目标是测试EP-CAM是否可以用作分子开关，在体外瞬时激活人β细胞的生长。此外，我们计划测试用我们实验室生产的可溶性重组EP-CAM分子处理胰岛β细胞是否也可以刺激细胞生长，和/或提高胰岛β细胞的存活率。该实验计划的重点在于以下具体目标：1)通过腺病毒载体研究EP-CAM在成人胰岛细胞中瞬时过表达的影响，并评估其对细胞生长、分化和存活的影响；2)通过“双光子共聚焦显微镜”和“高压电子显微镜”等“先进的显微技术”来评价EP-CAM过表达对胰岛内β细胞团三维结构的调控作用。由于胰岛细胞团中β细胞的构筑结构已被证明对胰岛素产生细胞的内分泌功能有深刻的功能影响，我们预测所提出的研究将揭示由黏附受体EP-CAM介导的细胞-细胞相互作用的新机制，并最终有助于设计新的胰岛细胞移植扩增方案。

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.