Career Enchancement Award for Stem Cell Research in Urology

描述(由申请人提供)： NIDDK的使命包括研究泌尿学和消化系统疾病，以改善美国人的健康。最近的研究表明，膀胱和胃等器官可能含有能够再生受损组织的干细胞。该奖项将培训Monica Liebert博士，她是尿路上皮(膀胱)生物学的高级研究员，掌握干细胞技术。利伯特将由乳腺癌干细胞生物学研究负责人Max Wicha和胃肠道器官发生专家Deborah Gumucio共同指导。Wicha将在基本干细胞分离、表征和培养技术方面对Liebert进行培训。然后，利伯特将把这种训练应用于胃干细胞的研究。Gumucio之前发现，由Villin启动子驱动的转基因在胃干细胞所在组织中的一组胃细胞中高水平表达。我们的假设是，这些细胞是更大的胃干细胞群体的子集。目的1分离并鉴定干细胞样胃干细胞。具有干细胞特征的细胞将通过磁性或流式细胞仪分离从组织中浓缩。这些细胞将被培养成不依赖于锚定的“胃球”，并对干细胞特性和基因表达进行评估。采用抗体染色、基因芯片和实时荧光定量聚合酶链式反应等方法对这些细胞进行检测，并将它们与由绒毛膜蛋白启动子驱动的绿色荧光蛋白表达鉴定的细胞进行比较。目的2将在体外或体内测试这些干细胞样细胞是否能分化为肠道或胃系。细胞将在分化条件下培养。来自ROSA-26小鼠和Villin-EGFP×ROSA 26杂交的丰富的胃干细胞群体，以及未分离的对照细胞群体，将被分离并移植到裸鼠身上。将评估异种移植的绿色荧光蛋白、β-半乳糖的表达以及肠或胃组织的形态发育。这些研究将证实不同潜在干细胞群体的繁殖和分化能力。这项研究将帮助我们了解胃对溃疡等疾病的反应，以及胃癌可能如何发展。在利伯特的培训结束后，她将把这些技术应用于膀胱发育、组织工程学和膀胱癌的研究。她未来在泌尿科的工作将开发治疗尿路疾病和膀胱癌的新方法。

DESCRIPTION (provided by applicant): NIDDK's mission includes research in urologic and digestive diseases to improve the health of Americans. Recent studies indicate that organs, such as the urinary bladder and stomach, may contain stem cells capable of regenerating damaged tissue. This award will train Dr. Monica Liebert, a senior investigator in urothelial (urinary bladder) biology, in stem cell techniques. Liebert will be co-mentored by Max Wicha, leader in breast cancer stem cell biology research, and Deborah Gumucio, a expert in gastrointestinal organogenesis. Wicha will train Liebert in basic stem cell isolation, characterization and culture techniques. Liebert will then apply that training to study gastric stem cells. Gumucio previously found that transgenes driven by the villin promoter are expressed at high levels in a subset of gastric cells located in tissue where gastric stem cells are thought to reside. Our hypothesis is that these cells are a subset of a larger gastric stem cell population. Aim 1 is to isolate and characterize stem-like gastric stem cells. Cells with stem cell characteristics will be enriched from tissues using magnetic or flow cytometric separation. These cells will be cultured to form anchorage-independent "gastrospheres," and evaluated for stem cell properties and gene expression. Antibody staining, microarray methods and real-time quantitative PCR will be used to examine the these cells and compare them to the cells identified by villin-promoter driven EGFP expression. Aim 2 will test whether these stem-like cells can give rise to intestinal or gastric lineages in vitro or in vivo. Cells will be cultured under differentiating conditions. Enriched populations of gastric stem cells from Rosa-26 mice and the Villin-EGFP x Rosa 26 cross, as well as a control population of unsorted cells, will be isolated and grafted on nude mice. Xenografts will be evaluated for EGFP, beta-gal expression and for morphologic development into intestinal or gastric tissues. These studies will confirm the ability of the different potential stem cell populations to reproduce and to differentiate. This research will help us understand how the stomach responds to diseases, such as ulcers, and how stomach cancer may develop. At the end of Liebert's training, she will apply these techniques to the study of urinary bladder development, tissue engineering, and bladder cancer. Her future work in urology will develop new treatments for urinary tract disorders and bladder cancer.