Department of Genetics, The University of Texas M D Anderson Cancer Center, Houston, Texas, United States of America.
PLoS One. 2012;7(8):e43659. doi: 10.1371/journal.pone.0043659. Epub 2012 Aug 28.
REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network.
Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.
We show that Rest+/- and Rest-/- AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/- and Rest-/- AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest-/- AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency.
REST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.
REST 在小鼠胚胎干细胞 (ESC) 中大量表达。许多全基因组分析发现 REST 是 ESC 多能性网络的一个组成部分。然而，实验系统得出了相互矛盾的发现：(1) REST 是维持 ESC 多能性所必需的，REST 的缺失会导致分化标记物的表达增加，(2) REST 不是维持 ESC 多能性所必需的，REST 的缺失不会改变分化标记物的表达，以及 (3) REST 不是维持 ESC 多能性所必需的，但 REST 的缺失会导致分化标记物的表达减少。这些报告突出了我们对 ESC 网络知识的差距。
我们采用生化和全基因组分析各种培养条件和 ESC 系，试图解决文献中的一些差异。
我们表明，在饲养细胞存在的情况下，Rest+/- 和 Rest-/- AB-1 突变 ESC 没有表现出 REST 在 ESC 多能性中的作用，但与亲本细胞相比，在无饲养细胞培养条件下自我更新能力受损，但仅在早期传代细胞中。在晚期传代细胞中，Rest+/- 和 Rest-/- AB-1 ESC 均恢复了多能性，表明存在与传代和培养条件相关的反应。全基因组分析随后的生化验证支持了这一反应，并进一步表明多能性的恢复与 ESC 多能性因子表达的增加有关。E14Tg2a.4 ESC 中 REST 的敲低，以前在无饲养细胞条件下培养时表现出 REST 依赖性多能性，以及 Rest-/- AB-1 ESC，在饲养细胞或层粘连蛋白存在的情况下培养时均未表现出 REST 依赖性多能性，表明细胞外基质成分可以挽救 REST 在 ESC 多能性中的作用。
REST 以培养条件和 ESC 系依赖的方式调节 ESC 多能性，需要在上下文相关的方式下评估 ESC 多能性。
