Abstract
BackgroundREST 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.MethodsEmploying biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.ResultsWe 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.ConclusionsREST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.
Highlights
Pluripotent mouse embryonic stem cells derived from the inner cell mass of the mammalian blastocyst are capable of forming all the tissues of the organism and possess the ability to self-renew in an undifferentiated manner
Rest+/2 and Rest2/2 AB-1 embryonic stem cells (ESCs) cultured in the absence of feeder cells show impaired self-renewal that is restored upon prolonged culture Previously, using two Rest+/2 mutant and their parental
E14Tg2a.4 mouse embryonic stem cells (mESCs) cell lines cultured in the absence of mouse embryonic fibroblast feeder cells, we found that REST maintains the self-renewal and pluripotency of ESCs [25]
Summary
Pluripotent mouse embryonic stem cells (mESCs) derived from the inner cell mass of the mammalian blastocyst are capable of forming all the tissues of the organism and possess the ability to self-renew in an undifferentiated manner. The recent successes with reprogramming somatic cells into induced pluripotent stem-like cells have brought intense enthusiasm to this area of research [4,5,6,7,8,9,10,11] These findings suggest that factors such as Oct, Nanog, and Sox are core components of a large, interconnected network that regulates self-renewal and pluripotency in both mouse and human ESCs [2,12,13,14]. REST has been reported to maintain mESC self-renewal and pluripotency by directly suppressing miR21 [25] In these studies, loss of REST in ESCs accompanied expression of many differentiation markers, including Gata. These reports highlight gaps in our knowledge of the ESC network
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