Defining the role of REST in regulating human embryonic stem cell fate

Includes bibliographical references (pages 58-62)Human embryonic stem cells (hESCs) have the unique ability to undergo self-renewal\ud and are pluripotent, which enables them to differentiate into all three germ layers. Harnessing\ud these properties could result in significant therapeutic outcomes for repair or cell\ud replacement in many disease settings. However, many hurdles remain prior to use of\ud these cells in cell-based therapies including lack of understanding of what signals regulate\ud pluripotency and how to maintain stability of hESCs. OCT4, NANOG and SOX2\ud are transcription factors that regulate multi-gene networks involved in control of cell fate\ud decisions important for maintaining self-renewal and stability of hESCs. RE1-Silencing\ud Transcription Factor (REST) is one of the direct targets of this network which is a well\ud known transcriptional repressor. REST acts by binding to RE1 sites in greater than 1900\ud genes in the genome. The main role of REST is to act as a transcriptional repressor in nonneuronal\ud cell types to repress neuronal differentiation. This repression has been shown in\ud some but not all mouse embryonic stem cell studies, however, its role in hESCs is not clear. To examine the role of REST in hESCs, we have performed lentiviral mediated knockdown of REST (REST KD) in three different cell lines and we have evaluated the effects\ud of the loss of REST on hESC self-renewal, differentiation, genetic stability. REST KD\ud hESCs seem to exhibit enhanced self-renewal and the ablation of REST does not have an\ud effect on the pluripotency of hESCs. We also found that three out of four hESC lines with\ud REST KD were genetically unstable, whereas control hESC lines were karyotypically normal.\ud To determine the gene networks involved in this instability, we have examined several\ud candidate genes differentially expressed upon REST KD by performing qPCR of individual\ud REST targets. Some of the genes that are consistently and significantly up-regulated\ud include ATM, RTEL1, CDK4 and E2F1. These genes have been instigated as direct REST\ud targets and most are up-regulated upon DNA damage which is consistent with our previous\ud result of chromosomal instability upon ablation of REST. We were also interested in changes to the expression of lineage markers in embryoid\ud bodies (EBs) from REST KD and control cells in order to examine the effects of the loss of REST on differentiation. Upon REST KD, hESC EBs exhibited a bias towards endoderm and mesendoderm lineage. Contrary to our expectations, there was a reduction in ectodermal\ud cells upon REST KD. Since our results confirmed a endoderm/mesoderm bias in vitro\ud we next examined the signaling pathways that may be involved in producing the aforementioned\ud lineage commitment. Using protein and RNA analysis we have shown a slight\ud reduction in pMEK and pSMAD upon REST KD indicating that REST may be involved in\ud the MAPK pathway as well as the TGF pathway. In summary, our work has shown that REST is a regulator of hESC genetic stability.\ud Elucidating the targets mis-regulated upon REST KD could shed light on the gene\ud networks regulating instability and differentiation in pluripotent stem cell cultures. This\ud could improve our ability to stably expand and differentiate hESCs for use in regenerative\ud medicine