Specific Knockdown of OCT4 in Human Embryonic Stem Cells by Inducible Short Hairpin RNA Interference

Zafarana, Gaetano; Avery, Stuart; Avery, Katie; Moore, H. D. M.; Andrews, Peter W.

doi:10.1002/stem.5

Cited by 51 publications

(53 citation statements)

References 40 publications

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“…These sequences were annealed and cloned into the pSuperior-Neo plasmid, driven by the H1 promoter (OligoEngine). This shRNA system was previously shown to have a high knockdown efficiency in hESCs (Zafarana et al, 2009). The human NICD1 sequence was obtained from Addgene [plasmid 17623 (Yu et al, 2008)] and was subcloned into a vector containing the CAG promoter and a G418 resistance cassette.…”

Section: Methodsmentioning

confidence: 99%

Notch signaling regulates neural crest differentiation from human pluripotent stem cells

Noisa

Lund

Kanduri

et al. 2014

Journal of Cell Science

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Neural crest (NC) cells are specified at the border of neural plate and epiderm. They are capable of differentiating into various somatic cell types, including craniofacial and peripheral nerve tissues. Notch signaling plays significant roles during neurogenesis; however, its function during human NC development is poorly understood. Here, we generated self-renewing premigratory NC-like cells (pNCCs) from human pluripotent stem cells and investigated the roles of Notch signaling during the NC differentiation. pNCCs expressed various NC specifier genes, including SLUG, SOX10 and TWIST1, and were able to differentiate into most NC derivatives. Blocking Notch signaling during the pNCC differentiation suppressed the expression of NC specifier genes. In contrast, ectopic expression of activated Notch1 intracellular domain (NICD1) augmented the expression of NC specifier genes, and NICD1 was found to bind at their promoter regions. Notch activity was also required for the maintenance of premigratory NC state, and suppression of Notch led to generation of NC-derived neurons. Taken together, we provide a protocol for the generation of pNCCs, and show that Notch signaling regulates the formation, migration and differentiation of NC from hPSCs.

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Section: Methodsmentioning

confidence: 99%

Notch signaling regulates neural crest differentiation from human pluripotent stem cells

Noisa

Lund

Kanduri

et al. 2014

Journal of Cell Science

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“…To analyze SNON function, we first performed tetracycline (Tet)-inducible shRNA-mediated knockdown of SNON in S4TR5 cells, a derivative of the Shef4 hESC line that constitutively expresses the nuclear Tet repressor (TetRnls) (Zafarana et al 2009). Two independent and previously validated human SNON-shRNA target sequences-shSNON(954) and shSNON(1307)-were transfected into S4TR5 cells (Sarker et al 2005;Zhu et al 2007).…”

Section: Snon Knockdown Destabilizes Pluripotencymentioning

confidence: 99%

“…For feeder-free culture, HES3 and S4TR5 cells (Zafarana et al 2009) were maintained in mTeSR1 medium (Stem Cell Technologies) on 1:200 GFRMatrigel-coated (Becton Dickinson) dishes. DE differentiation was carried out as previously described (Teo et al 2012).…”

Section: Cell Culturementioning

confidence: 99%

The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells

et al. 2012

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Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.

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“…In vitro, Oct4 is also highly expressed in undifferentiated ESCs, and it is downregulated on differentiation. Knockdown of Oct4 in ESCs triggers primitive endoderm as well as trophectoderm differentiation (11)(12)(13). Of note, Niwa et al (13) found that up-regulation of Oct4 expression causes differentiation of ESCs into primitive endoderm and mesoderm.…”

mentioning

confidence: 99%

Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation

Sun

Gao

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Self-renewal and differentiation of embryonic stem cells (ESCs) are controlled by intracellular transcriptional factors and extracellular factor-activated signaling pathways. Transcription factor Oct4 is a key player maintaining ESCs in an undifferentiated state, whereas the Erk/MAPK pathway is known to be important for ESC differentiation. However, the manner in which intracellular pluripotency factors modulate extracellular factor-activated signaling pathways in ESCs is not well understood. Here, we report identification of a target gene of Oct4, serine/threonine kinase 40 (Stk40), which is able to activate the Erk/MAPK pathway and induce extraembryonicendoderm (ExEn) differentiation in mouse ESCs. Interestingly, cells overexpressing Stk40 exclusively contribute to the ExEn layer of chimeric embryos when injected into host blastocysts. In contrast, deletion of Stk40 in ESCs markedly reduces ExEn differentiation in vitro. Mechanistically, Stk40 interacts with Rcn2, which also activates Erk1/2 to induce ExEn specification in mouse ESCs. Moreover, Rcn2 proteins are specifically located in the cytoplasm of the ExEn layer of early mouse embryos. Importantly, knockdown of Rcn2 blocks Stk40-activated Erk1/2 and ESC differentiation. Therefore, our study establishes a link between the pluripotency factor Oct4 and the Erk/MAPK signaling pathway, and it uncovers cooperating signals in the Erk/ MAPK activation that control ExEn differentiation.embryonic stem cells | Rcn2 | Ras | Gata6

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Specific Knockdown of OCT4 in Human Embryonic Stem Cells by Inducible Short Hairpin RNA Interference

Cited by 51 publications

References 40 publications

Notch signaling regulates neural crest differentiation from human pluripotent stem cells

Notch signaling regulates neural crest differentiation from human pluripotent stem cells

The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells

Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation

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