PANCREATIC BETA CELL DEVELOPMENT FROM HUMAN DCC+ STEM C

人胰腺内分泌干细胞的鉴定 能够在体外生长并分化为成熟的β细胞 在体内，将代表产生合适细胞的有力策略 用于IDDM患者的移植。 胰岛β细胞被认为是从存在于胰岛中的前体细胞发育而来的。 胰腺导管上皮 然而，这些内分泌的表型 祖细胞，以及调节它们从细胞中出现的机制。 导管上皮仍然难以捉摸。 我们对人类的初步研究 胰腺表明，细胞粘附分子DCC识别 导管细胞，可以在体外扩增，并诱导表达 当胰岛素重新聚集成三维胰岛样细胞时 集群更重要的是，移植重组的DCC+细胞 在免疫缺陷的裸鼠中， 体内胰岛β细胞。 我们进一步表明，胎儿胰腺DCC+ 细胞产生细胞外基质（ECM）降解酶MMP 2， MMP 9，而胎儿间充质细胞分泌化学引诱物 分子Netrin-1，一种新鉴定的DCC配体。 这些 观察表明DCC+内分泌通过一种分子途径 祖细胞被间充质化学引诱物Netrin-1刺激 从导管上皮迁移到周围的间质中 通过ECM修饰酶打开的迁移线索。 这个提议的中心假设是胰腺导管细胞 以DCC表达为标志的胰岛细胞包括胰岛细胞的祖细胞 脉 基于最近将DCC鉴定为细胞粘附 调节上皮-间质相互作用的分子， 最近，作为化学引诱物分子Netrin-1的受体，我们 还假设导管细胞与周围环境的相互作用 间充质和特定MMPs的激活，调节生长 以及胰岛个体发育过程中内分泌祖细胞的迁移。 最后，我们假设从其他组织中分离的DCC+细胞， 胃肠道隔室共享一个共同的胚胎起源， 胰腺可包含胰岛祖细胞。 我们建议通过以下具体目标来检验这些假设：1） 评估从胎儿分离的DCC+细胞的增殖潜力 2）研究直接上皮细胞在胰腺癌中的作用， 间充质相互作用和间充质衍生因子对生长的影响， DCC+细胞的迁移和内分泌决定; 金属蛋白酶MMP-2和MMP-9在迁移中的作用， DCC+细胞的增殖和分化; 上皮细胞-细胞粘附分子对内分泌的作用 DCC+细胞的分化程序; 5）评估DCC+细胞的分化能力， 从胃肠道的其他隔室分离的细胞， 进入内分泌分化途径。

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.