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

描述(申请人提供)：人类胚胎干细胞(HuE)有可能产生无限来源的β/胰岛细胞用于移植，但这一过程所需的生物因素一直难以确定，部分原因是缺乏特定阶段的体外细胞分析。最近，我们建立了一种小鼠体外胰腺干细胞/祖细胞集落试验，用于确定β细胞/胰岛分化和扩增所需的因素。在此小鼠实验的基础上，我们在这项研究中建议开发一种基于色调的分析方法，以加速发现以前未确定但需要人类因素来产生β细胞/胰岛。第一阶段的目标是：1)将我们明确定义的小鼠胰腺祖细胞集落分析转化为基于HuES细胞的人类分析，以及2)创建一种插入增强型绿色荧光蛋白(EGFP)的报告Hues细胞系，该细胞系由胰腺内分泌前体细胞标记物神经原蛋白(NGN)3的启动子驱动。一旦基于色调的祖细胞分析到位，我们将过渡到第二阶段研究，以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已确定的生物介体商业化，用于细胞替代治疗或刺激内源性胰腺细胞前体细胞再生以治疗1型糖尿病。摘要：人类胚胎干细胞具有为移植提供无限来源的β/胰岛细胞的潜力，但这一过程所需的生物学因素一直难以确定，部分原因是缺乏阶段特异性的体外细胞分析。这个第一阶段的提案将建立一种基于HuES细胞的胰腺前体细胞检测方法，以便在未来的第二阶段研究中筛选生物介质。我们的最终目的是将已确定的生物介体商业化，用于细胞替代治疗或刺激内源性胰腺细胞前体细胞再生以治疗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.