Endodermal progenitor cell characterization

描述（由申请人提供）：干细胞生物学为体外疾病研究和模型以及疾病治疗提供了巨大的潜力。包括I型糖尿病和各种肝脏疾病在内的内胚层器官疾病备受关注。目前没有足够的胰岛细胞或肝脏供体来治疗需要移植的患者数量。使用干细胞群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，对人类胚胎干细胞（ES）和诱导多能干细胞（iPS）的研究为治疗这些疾病带来了希望。这两种细胞类型都可以在培养中扩增，并有可能分化成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，胚胎干细胞和iPS细胞在直接移植时都能形成肿瘤。因此，移植纯分化细胞类型具有重要意义。此外，从这些早期干细胞群体中产生成熟功能细胞类型已被证明是困难的。为了尝试解决这些问题，我们建议产生内胚层祖细胞（EP）系。EP细胞与ES和iPS细胞一样，在培养中可以扩增，但缺乏形成肿瘤的潜力。EP细胞也可以分化为内胚层细胞类型，如肝细胞和胰腺细胞。初步数据表明，我们已经找到了从人类胚胎干细胞中产生EP细胞的培养条件。我们打算进一步表征EP细胞，从多种人类胚胎干细胞和iPS细胞系中生成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分析，以确定胚胎干细胞和iPS细胞衍生的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。我们将扩展这些发现，利用遗传系统和小分子来激活或抑制Notch信号通路，并分析对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在EP细胞中通过RNAi敲除进行检测。发现EP细胞所需的基因将在Notch信号的背景下进行研究，以建立EP细胞形成和维持的信号传导层次。这一信息可能使生成EP细胞更有效，并可能导致更好地理解控制干细胞群体的机制。

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.