Abstract
Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.
Highlights
Pluripotent embryonic stem (ES) cells derived from the inner cell mass of mouse preimplantation-stage embryos retain the capacity to self-renewal in vitro indefinitely[1,2] in the presence of external stimuli such as leukemia inhibitory factor (LIF) and BMP4 or serum[3]
We further reveal that LIF-independent inducible expression of OCT4 (iOCT4) Embryonic stem (ES) cells are transcriptionally more similar to wild-type ES cells than epiblast stem cells (EpiSCs), differentiated embryoid bodies (EBs), or mouse embryonic fibroblasts (MEFs)
These results suggest that external activation of LIF-STAT3 signaling by addition of LIF in the culture media may drive a transcriptional network that cooperates with OCT4 to promote self-renewal
Summary
Pluripotent embryonic stem (ES) cells derived from the inner cell mass of mouse preimplantation-stage embryos retain the capacity to self-renewal in vitro indefinitely[1,2] in the presence of external stimuli such as leukemia inhibitory factor (LIF) and BMP4 or serum[3]. LIF-independent iOCT4 ES cells and wild-type ES cells exhibit overall similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells, global expression analysis demonstrated that a subset of STAT3 targets are downregulated in LIF-independent ES cells, while a subset of OCT4/STAT3 co-bound targets are upregulated. These results suggest that OCT4 may promote self-renewal in the absence of LIF/STAT3 signaling by driving expression of genes essential for maintaining pluripotency. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal
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