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受到极大关注。目前没有足够的胰岛细胞或肝脏捐赠者来治疗需要移植的患者。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，人类胚胎干（ES）细胞和诱导多能干（iPS）细胞的研究为治疗这些疾病提供了希望。这两种细胞类型都可以在培养中扩增，并具有分化成体内任何细胞类型的潜力。不幸的是，在细胞替代疗法成为现实之前，还有几个障碍需要克服。首先，ES和iPS细胞直接移植时都可以形成肿瘤。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产生成熟的功能细胞类型已被证明是困难的。为了试图解决这些问题，我们提出产生内胚层祖细胞（EP）系。EP细胞，如ES和iPS细胞，可以在培养中扩增，但缺乏肿瘤形成潜力。EP细胞还可以分化成内胚层细胞类型，例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。我们建议进一步表征EP细胞，从多种人ES和iPS细胞系产生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多种体外和体内系统中进行功能测定，以确定ES细胞和iPS细胞衍生的EP细胞系是否在功能上等同。最后，我们建议研究控制EP细胞生成和维持的机制。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，研究Notch信号利用遗传系统和小分子激活或抑制Notch途径，并测定对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物也将通过在EP细胞中的RNAi敲低来测定。然后将在Notch信号传导的背景下研究EP细胞中所需的基因，以建立EP细胞形成和维持的信号传导层次。这些信息可能使产生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.