Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells

Velkey, J. Matthew; O’Shea, K. Sue

doi:10.1002/gene.10218

Cited by 94 publications

(77 citation statements)

References 27 publications

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“…As previously described for expression of shRNAs, annealed DNA oligonucleotides corresponding to nucleotides 670-688 of Oct-4 were cloned behind the human H1 RNA promoter in the pRetro-H1G vector (Velkey and O'Shea 2003). Upon electroporation of Hif-2␣ KI/KI ES cells, stable cell lines were selected by treatment at 2 µg/mL puromycin.…”

Section: Oct-4 Rnaimentioning

confidence: 99%

HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

Covello¹,

Kehler²,

Yu³

et al. 2006

Genes Dev.

747

590

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The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1␣ with HIF-2␣ results in expanded expression of HIF-2␣-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2␣, but not HIF-1␣, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2␣ KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2␣ severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2␣-specific target gene and indicate that HIF-2␣ can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2␣'s tumor promoting activity.[Keywords: HIF; hypoxia; HIF-2␣; Oct-4; VEGF; TGF-␣; stem cells; cancer] Supplemental material is available at http://www.genesdev.org.

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Section: Oct-4 Rnaimentioning

confidence: 99%

HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

Covello¹,

Kehler²,

Yu³

et al. 2006

Genes Dev.

747

590

View full text Add to dashboard Cite

show abstract

“…2 Briefly, fluorescence images of the cells were acquired at 3 days after induction of cell differentiation using a Zeiss Observer.Z1 inverted fluorescence microscope. The integral total fluorescence intensity of cells in each well was determined using Cellprofiler (http://www.cellprofiler.org/).…”

Section: Image Acquisition and Analysismentioning

confidence: 99%

“…For example, gene silencing was used to identify tumorsuppressor genes, 1 genes essential for maintaining stemness 2 or controlling cell differentiation. 3,4 In addition to understanding biological processes, RNAi has been proposed as a novel strategy for cancer therapy, antiviral therapy, treatment of genetic or metabolic disorders as well as cell reprogramming for personalized regenerative medicine.…”

Section: Introductionmentioning

confidence: 99%

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

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Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (aSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize aSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-b1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased aSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

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“…The ability to drive differentiation of hESC towards trophoblast by silencing genes essential for maintaining pluripotency was first demonstrated in murine cells after knock down of OCT4 (POU5F1) [31][32][33]. Silencing of NANOG or SOX2 also induces markers of trophoblast differentiation [34][35][36].…”

Section: Hesc As Models For Trophoblast Developmentmentioning

confidence: 99%

Human Embryonic Stem Cells as Models for Trophoblast Differentiation

et al. 2008

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Trophectoderm is specified from pluripotent blastomeres at some time prior to blastocyst formation. Proliferating cytotrophoblast derived from trophectoderm is the forerunner of the entire trophoblast component of the mature human placenta, including extra-villous cytotrophoblast and syncytiotrophoblast. Recently human embryonic stem cells (hESC) have been employed to study these events in an in vitro situation. Here we review some of the work in this emerging area of trophoblast biology. We concentrate primarily on a model in which colonies of hESC are exposed to BMP4 in stem cell growth medium lacking FGF2. Under both low (4 %) and high (20 %) O 2 conditions, differentiation proceeds unidirectionally towards trophoblast from the outside of the colonies inwards, with progression fastest under high O 2 . Immunohistochemical observations performed on whole colonies combined with microarray analysis of mRNA can be employed to track developmental transitions as they occur over time and in two-dimensional space as the cells respond to BMP4.

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Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells

Cited by 94 publications

References 27 publications

HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

Human Embryonic Stem Cells as Models for Trophoblast Differentiation

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