Progenitor assay to screen proteins/molecules for treatment of type1 diabetes

描述（由申请人提供）：人类胚胎干细胞（HuES）具有产生用于移植的β /胰岛细胞的无限来源的潜力，然而，由于缺乏阶段特异性的体外细胞测定，这一过程所需的生物学因素难以确定。最近，我们开发了一种小鼠体外胰腺干细胞/祖细胞集落试验，我们正在使用该试验来确定β细胞/胰岛分化和扩增所需的因子。在此小鼠实验的基础上，我们建议在本研究中开发一种基于HuES的实验，以加速发现以前未识别但需要的β细胞/胰岛生成的人为因素。第一阶段的目标是：1)将我们定义明确的小鼠胰腺祖细胞集落试验转化为基于HuES细胞的人类试验，2)创建一个报告HuES细胞系，插入增强型绿色荧光蛋白（EGFP），由神经原素（Ngn） 3启动子驱动，这是胰腺内分泌祖细胞的标记物。一旦基于HuES的祖细胞检测到位，我们将进入II期研究，筛选可用于扩大祖细胞数量的蛋白质或分子。我们的最终目的是将鉴定的生物介质商业化，用于细胞替代疗法或刺激内源性胰腺细胞祖细胞再生，以治疗1型糖尿病。摘要：人胚胎干细胞（HuES）具有产生用于移植的β /胰岛细胞的无限来源的潜力，然而，由于缺乏阶段特异性的体外细胞测定，这一过程所需的生物学因素难以确定。该I期提案将建立一个基于HuES细胞的胰腺祖细胞测定方法，以便在未来的II期研究中筛选生物介质。我们的最终目的是将鉴定的生物介质商业化，用于细胞替代疗法或刺激内源性胰腺细胞祖细胞再生，以治疗1型糖尿病。

DESCRIPTION (provided by applicant): Human embryonic stem cells (HuES) have the potential to generate an unlimited source of beta/islet cells for transplantation, however the biologic factors required for this process have been difficult to ascertain partly due to the lack of stage specific in vitro cell assays. Recently, we have developed a murine in vitro pancreatic stem/progenitor colony assay that we are using to identify factors required for beta cell /islet differentiation and expansion. Based on this murine assay, we propose in this study to develop a HuES based assay that would accelerate the discovery of previously unidentified but required human factors for the generation of beta cell/islets. The phase I goals are: 1) to translate our well-defined murine pancreatic progenitor colony assay to a human assay based on HuES cells, and 2) to create a reporter HuES cell line with insertion of enhanced green fluorescent protein (EGFP) that is driven by promoter of neurogenin (Ngn) 3, a marker for pancreatic endocrine progenitors. Once a HuES based progenitor assay is in place, we will then transition into phase II study to screen for proteins or molecules that could be used to expand the number of progenitors. Our ultimate intention is to commercialize the identified biological mediators for the purpose of cell replacement therapy or stimulation of regeneration of endogenous pancreatic ¿ cell progenitors to treat type 1 diabetes. Lay Abstract: Human embryonic stem cells (HuES) have the potential to generate an unlimited source of beta/islet cells for transplantation, however the biologic factors required for this process have been difficult to ascertain partly due to the lack of stage specific in vitro cell assays. This phase I proposal will establish an HuES cell based pancreatic progenitor assay for the purpose of screening for biological mediators in the future phase II study. Our ultimate intention is to commercialize the identified biological mediators for the purpose of cell replacement therapy or stimulation of regeneration of endogenous pancreatic ¿ cell progenitors to treat type 1 diabetes.