Pilot-Germ Cell Nuclear Factor & ES Cell Differentiation

Pilot-生殖细胞核因子

• 批准号: 7004367
• 负责人: Austin J Cooney
• 金额: $ 5万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7004367
• 关键词: RNA interference; cell differentiation; cytogenetics; gene expression; germ cells; human embryonic stem cell line; human tissue; immunofluorescence technique; nuclear proteins; pluripotent stem cells; tissue /cell culture; transcription factor

Description (provided by applicant): The maintenance of pluripotence is central to the function of embryonic stem (ES) cells, without which ES cells will spontaneously differentiate. Significant headway has been made in understanding the maintenance of pluripotence in mouse ES (mES) cells; however, little is known about the maintenance of pluripotence in human ES (hES) cells, which is crucial for their practical application in directed differentiation for tissue engineering. In order to maintain hES cells in long-term culture and maintain pluripotence, as is possible with mES cells, we need to understand the factors that govern the maintenance of pluripotence in hES cells. A good starting point is to determine whether the information that has been learned in mES cells is applicable to hES cells. The transcription factors Oct4 and Sox 2, which regulate FGF4 expression, have been shown in the mouse to be essential for the maintenance of ES cell function. The factors that positively and negatively regulate the expression of Oct4 and Sox2 in hES cells also play essential roles in regulating pluripotence. My laboratory has focused on the regulation of the Oct 4 gene, which is regulated by several members of the nuclear receptor family including retinoid receptors and SF-1, which regulate positive expression of Oct4, and negative factors such as the orphan receptor GCNF. In mice we have shown that GCNF is essential for repressing Oct4 expression in somatic cells during gastrulation and thus inversely correlates with pluripotence. We propose to determine if the same regulation of Oct4 exists in hES cells by carrying out the following specific aims: First we will determine the stability of expression of Oct4 in hES cells and determine if there is loss of Oct4 expression in any of the cells. Then determine whether the loss of Oct4 expression correlates with loss of SF-1 or gain of GCNF expression. Then we will perform differentiation studies to determine if there is a reciprocal expression pattern for Oct4 and SF-1 in undifferentiated cells and GCNF in differentiated cells. Lastly we will perform RNAi to knock down the expression of GCNF, and SF-1 and look at the effect on Oct4 expression to determine if SF-1 is required to maintain Oct4 expression and whether GCNF is required for Oct4 repression. The culmination of these specific aims will shed new light on some of the factors that regulate pluripotence in hES cells.

描述（由申请人提供）：多能的维护对于功能至关重要 胚胎干细胞（ES）细胞，没有ES细胞会自发区分。在了解小鼠ES（MES）细胞中多能性的维持方面取得了显着的进展。但是，对于人ES（HES）细胞中多能的维持知之甚少，这对于它们在组织工程的定向分化中的实际应用至关重要。为了在长期培养中维持HES细胞并保持多功能，MES细胞可能是可能的，我们需要了解控制HES细胞中多能维持的因素。一个好的起点是确定MES细胞中学习的信息是否适用于HES细胞。调节FGF4表达的转录因子Oct4和Sox 2在小鼠中已显示对于维持ES细胞功能至关重要。在HES细胞中积极和负调节Oct4和Sox2表达的因素在调节多能性方面也起着至关重要的作用。我的实验室专注于对10月4日基因的调节，该基因由核受体家族的几个成员调节，包括类视黄素受体和SF-1，这些成员调节了OCT4的阳性表达，以及诸如孤儿受体GCNF之类的负因子。在小鼠中，我们已经表明GCNF是 在胃结构过程中抑制体细胞中的OCT4表达至关重要 与多能相关。我们建议通过执行以下特定目的来确定HES细胞中OCT4的相同调节：首先，我们将确定HES细胞中OCT4表达的稳定性，并确定任何细胞中OCT4表达是否丧失。然后确定OCT4表达的丢失是与SF-1的丧失或GCNF表达的增益相关。然后，我们将进行分化研究，以确定在分化细胞中未分化的细胞和GCNF中OCT4和SF-1是否存在相互表达模式。最后，我们将执行RNAi来击倒GCNF和SF-1的表达，并查看对OCT4表达的影响，以确定是否需要SF-1维持OCT4表达以及是否需要GCNF进行OCT4抑制。这些特定目的的高潮将使一些调节HES细胞中多能性的因素开发新的启示。