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，将代表一种强大的策略来产生合适的细胞 供胰岛素依赖型糖尿病患者移植的肿块。 胰岛β细胞被认为是从存在于 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 导管上皮仍然难以捉摸。我们对人类的初步研究 胰腺显示细胞黏附分子DCC识别 导管细胞，可以在体外扩增，并诱导表达 重组为三维胰岛样细胞时的胰岛素 集群。更重要的是，重组的DCC+细胞移植 在免疫缺陷的裸鼠中导致功能性 体内的胰岛β细胞。我们进一步证明胎儿胰腺DCC+ 细胞产生细胞外基质(ECM)降解酶MMP2和 MMP9，而胎儿间充质细胞分泌化学诱导剂 Netrin-1，一种新发现的DCC配体。这些 观察表明，DCC+内分泌是一种分子途径 间充质趋化因子Netrin-1对祖细胞的刺激作用 从导管上皮迁移到周围的间质 通过ECM修饰酶打开的迁移线索。 这一提议的中心假设是胰腺导管细胞 以DCC的表达为特征的包括胰岛细胞的祖细胞 血统。基于最近发现的DCC是一种细胞黏附 调控上皮-间充质相互作用的分子，以及大多数 最近，作为化学吸引分子Netrin-1的受体，我们 还假设导管细胞与周围环境的相互作用 间充质和特异性MMPs的激活，调节生长 以及内分泌祖细胞在胰岛个体发育过程中的迁移。 最后，我们假设从其他细胞分离的DCC+细胞 胃肠道间隔具有共同的胚胎学起源 胰腺可能由胰岛祖细胞组成。 我们建议通过以下具体目标来检验这些假设：1) 胎儿DCC+细胞体外扩增能力的研究 和成人胰腺；2)研究直接上皮细胞的作用。 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测定；3)研究 基质金属蛋白酶MMP2和MMP9在肿瘤转移中的作用 DCC+细胞的增殖和分化；4)研究DCC+细胞 上皮细胞-细胞黏附分子对内分泌的作用 DCC+细胞的分化方案；5)评估DCC+细胞的能力 从胃肠道其他部分分离的细胞 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.