Pilot-Germ Cell Nuclear Factor & Embryonic Stem Cell Differentiation

说明（由申请人提供）：维持多元性是 胚胎干细胞（ES细胞），没有它ES细胞将自发分化。在小鼠ES（mES）细胞中多能性的维持方面已经取得了重大进展;然而，关于人ES（hES）细胞中多能性的维持知之甚少，这对于它们在组织工程定向分化中的实际应用至关重要。为了维持hES细胞在长期培养和维持plurification，可能与mES细胞，我们需要了解的因素，管理hES细胞中的plurification的维持。一个好的起点是确定在mES细胞中学习的信息是否适用于hES细胞。调节FGF 4表达的转录因子Oct 4和Sox 2在小鼠中已被证明对于维持ES细胞功能是必不可少的。在hES细胞中，正性和负性调节Oct 4和Sox 2表达的因子在调节多能性中也起重要作用。我的实验室专注于Oct 4基因的调控，该基因受核受体家族的几个成员的调控，包括类维生素A受体和SF-1，它们调节Oct 4的阳性表达，以及诸如孤儿受体GCNF的阴性因子。在小鼠中，我们已经证明GCNF是 在原肠胚形成过程中体细胞中抑制Oct 4表达所必需的， 与多元性相关。我们建议通过进行以下具体目标来确定在hES细胞中是否存在相同的Oct 4调节：首先，我们将确定Oct 4在hES细胞中表达的稳定性，并确定在任何细胞中是否存在Oct 4表达的丧失。然后确定Oct 4表达的丧失是否与SF-1的丧失或GCNF表达的获得相关。然后，我们将进行分化研究，以确定是否有Oct 4和SF-1在未分化细胞和GCNF在分化细胞的相互表达模式。最后，我们将进行RNAi以敲低GCNF和SF-1的表达，并观察对Oct 4表达的影响，以确定是否需要SF-1来维持Oct 4表达以及是否需要GCNF来抑制Oct 4。这些特定目标的实现将为调节hES细胞中的多能性的一些因子提供新的线索。

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.