Endodermal progenitor cell characterization

描述(由申请人提供)：干细胞生物学为体外研究和疾病模型以及疾病的治疗提供了巨大的潜力。内胚层器官疾病，包括I型糖尿病和各种肝病，令人高度关注。目前没有足够的捐赠者提供治疗需要移植的患者所需的胰岛细胞或肝脏。使用干细胞群体来产生胰岛细胞和肝脏将为这个问题提供一个潜在的解决方案。目前，人类胚胎干细胞(ES)和诱导多能干细胞(IPS)的研究为这些疾病的治疗带来了希望。这两种细胞类型都可以在培养中扩增，并有可能分化为体内的任何细胞类型。不幸的是，在细胞替代疗法成为现实之前，有几个障碍需要克服。首先，ES和iPS细胞在直接移植时都会形成肿瘤。因此，移植纯分化的细胞类型至关重要。此外，从这些早期干细胞群体中产生成熟的功能细胞类型已被证明是困难的。为了解决这些问题，我们建议建立内皮祖细胞(EP)系。EP细胞和ES细胞和iPS细胞一样，可以在培养中扩增，但缺乏形成肿瘤的能力。EP细胞也可以分化为内皮细胞类型，如肝细胞和胰腺细胞。初步数据表明，我们已经找到了从人ES细胞产生EP细胞的培养条件。我们建议进一步鉴定EP细胞，从多个人ES和iPS细胞系中产生EP细胞，并将其分化为肝细胞和胰岛细胞。这些分化的后代将在各种体外和体内系统中进行功能测试，以确定ES细胞和iPS细胞来源的EP细胞系在功能上是否相同。最后，我们建议研究控制EP细胞产生和维持的机制。初步数据表明，Notch信号可能促进EP细胞的形成。我们将在这些发现的基础上，研究利用遗传系统和小分子来激活或抑制Notch通路的Notch信号，并分析其对EP细胞形成和维持的影响。此外，使用基因表达微阵列分析发现的其他可能的候选基因也将通过在EP细胞中的RNAi敲除来检测。然后，将在Notch信号的背景下研究EP细胞所需的基因，以建立EP细胞形成和维持的信号层次结构。这些信息可能会使产生EP细胞的效率更高，并可能导致更好地理解总体上控制干细胞群体的机制。 公共卫生相关性：该项目建议开发内皮干细胞株，这些干细胞株将有可能用于研究肝、胰腺、肺和胃肠道疾病。此外，生成患者特有的内皮线的技术可以用于生成用于细胞替代治疗的组织，而不会像包括胚胎干细胞在内的其他类型的干细胞那样具有肿瘤形成潜力。

DESCRIPTION (provided by applicant): Stem cell biology offers tremendous potential to both study and model disease in vitro as well as in the treatment of disease. Diseases of endodermal organs including type I diabetes and various liver diseases are of great concern. There are currently not sufficient donors for islet cells or livers necessary to treat the number of patients requiring transplants. The use of stem cell populations to generate pancreatic islet cells and liver would offer a potential solution to this problem. Currently, work with human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells offer hope in the treatment of these diseases. Both of these cell types can be expanded in culture and have the potential to differentiate into any cell type in the body. Unfortunately, there are several hurdles that need to be overcome before cell replacement therapy becomes a reality. First, both ES and iPS cells when transplanted directly can form tumors. Therefore, it is of critical importance to transplant pure differentiated cell types. In addition, the generation of mature function cell types from these early stem cell populations has proved difficult. To attempt to address these concerns we propose to generate endodermal progenitor cell (EP) lines. EP cells, like ES and iPS cells, can be expanded in culture but lack tumor forming potential. EP cells can also differentiate into endodermal cell types such as hepatocytes and pancreatic cells. Preliminary data suggests that we have found the culture conditions to generate EP cells from human ES cells. We propose to further characterize EP cells, generate EP cells from multiple human ES and iPS cell lines and to differentiate them into hepatocytes and pancreatic islet cells. These differentiated progeny will be functionally assayed in a variety of in vitro and in vivo systems to determine if both ES cell and iPS cell derived EP cell lines are functionally equivalent. Finally, we propose to investigate the mechanisms that control EP cell generation and maintenance. Preliminary data indicates that Notch signaling may promote EP cell formation. We will expand upon these findings, examining Notch signaling utilizing both genetic systems and small molecules to activate or repress the Notch pathway and assay the effects on EP cell formation and maintenance. In addition, other possible candidates discovered using gene expression microarray analysis will also be assayed by RNAi knockdown in EP cells. Genes found to be required in EP cells will then be studied in the context of Notch signaling to establish a signaling hierarchy for EP cell formation and maintenance. This information may make generating EP cells more efficient and may lead to a better understanding of the mechanisms controlling stem cell populations in general. PUBLIC HEALTH RELEVANCE: This project proposes to develop endodermal stem cell lines that will have the potential to be used to study diseases of liver, pancreas, lung and gastrointestinal track. In addition, the technology to generate patient specific endodermal lines could be used to generate tissue for cell replacement therapies without the tumor forming potential of other stem cells types including embryonic stem cells.