Abstract
Pluripotent Human Embryonic Stem Cells (hESCs) have the unconstrained capacity for long-term stable undifferentiated growth in culture and unrestricted developmental capacity. Packaging of the eukaryotic genome into chromatin confers higher order structural control over maintaining stem cell plasticity and directing differentiation. We recently reported the establishment of a defined culture system for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards functional lineages. To unveil the epigenetic mechanism in maintaining the epiblast pluripotence of hESCs, in this study, the global chromatin dynamics in the pluripotent hESCs maintained under the defined culture were examined. This study shows that the genomic plasticity of pluripotent hESCs is enabled by an acetylated globally active chromatin maintained by Oct-4. The pluripotency of hESCs that display normal stable expansion is associated with high levels of expression and nuclear localization of active chromatin remodeling factors that include acetylated histone H3 and H4, Brg-1, hSNF2H, HAT p300, and HDAC1; weak expression or cytoplasmic localization of repressive chromatin remodeling factors that are implicated in transcriptional silencing; and residual H3 K9 methylation. A dynamic progression from acetylated to transient hyperacetylated to hypoacetylated chromatin states correlates with loss-of-Oct4-associated hESC differentiation. RNA interference directed against Oct-4 and HDAC inhibitor analysis support this pivotal link between chromatin dynamics and hESC differentiation. These findings reveal an epigenetic mechanism for placing global chromatin dynamics as central to tracking the normal pluripotency and lineage progression of hESCs.
Highlights
Undifferentiated human embryonic stem cells, as essentially the in vitro equivalent of the inner cell mass (ICM) of the blastocyst, have the potential for differentiation into any somatic cell type
The undifferentiated Human Embryonic Stem Cell (hESC) maintained under the defined culture form the typical tightly packed colonies of small compact cells that are Oct-4 positive, while large differentiated cells migrate outside the colonies and become Oct-4 negative (Figure 1A, 1B)
We observed high levels of expression and nuclear localization of acetylated histone H3 and H4 in Oct-4-positive undifferentiated hESCs inside the colony, suggesting an acetylated active chromatin state in the pluripotent hESCs (Figure 1A, 1B), consistent with previous observation for hESCs maintained on feeders [12,13]
Summary
Undifferentiated human embryonic stem cells (hESCs), as essentially the in vitro equivalent of the inner cell mass (ICM) of the blastocyst, have the potential for differentiation into any somatic cell type This pluripotent state of hESCs is associated with the expression of a unique group of genes, including Oct-4, alkaline phosphatase, SSEA-4, Tra-1-60, Tra-1-80, though none of these markers, in isolation, is exclusively expressed by undifferentiated hESCs [1]. A dynamic progression from acetylated to transient hyperacetylated to hypoacetylated chromatin states correlates with loss-of-Oct4associated hESC differentiation, as assessed by Oct-4 RNA interference (RNAi) and histone deacetylase (HDAC) inhibitor analysis These findings reveal an epigenetic mechanism for placing global chromatin dynamics as central to tracking pluripotency and lineage progression of hESCs
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