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Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

Mora-Castilla S, Tejedo JR, Tapia-Limonchi R, Díaz I, Hitos AB, Cahuana GM, Hmadcha A, Martín F, Soria B, Bedoya FJ - Stem Cells Int (2014)

Bottom Line: Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment.Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression.Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), University Pablo de Olavide, Biomedical Research Network (CIBER) of Diabetes and Related Metabolic Diseases, Red-Tercel, Avenida Américo Vespucio S/N, 41092 Seville, Spain ; Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.

ABSTRACT
The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

No MeSH data available.


Related in: MedlinePlus

Reexpression of pluripotency genes following exposure to DETA/NO. (a) Western blot analysis of Nanog and Oct4 proteins. D3 mESCs were grown in the presence (+LIF) or in the absence (−LIF) of LIF for 3 days. Cells were then cultured as indicated in Section 2 for additional periods of 1 and 2 days (day 4 and day 5). C: control cells. T: cells exposed to 1 mM DETA/NO for 19 h on day 4. Lanes 5 and 6 refer to cells exposed to DETA/NO and subsequently cultured for 1 and 2 days as in Section 2. (b) GFP expression in D3-Oct4 mESCs. Control cells were cultured in the presence of LIF for 6 days (+LIF/Control). Treated cells were cultured in the presence of 1 mM DETA/NO for 19 hours on day 4 and subsequently cultured as indicated in Section 2 for an additional period of 2 days in absence of LIF. PhC: phase contrast. Results are representative of 3 independent experiments. (c) Western blot analysis reveals Nanog and Oct4 reexpression after DETA/NO treatment. +/− LIF indicates that cells were cultured for 4 days in the presence of LIF, then LIF was removed and cells were subsequently exposed to 1 mM DETA/NO for 19 hours. Days 5 and 6 indicate that cells were subsequently cultured for 1 and 2 days in the absence of LIF. −/+LIF indicates cells that were cultured for 4 days in the absence of LIF; exposure to 1 mM DETA/NO for 19 hours was then performed in the presence of LIF. Days 5 and 6 indicate that cells were subsequently cultured for additional periods of 1 and 2 days, respectively, in the presence of LIF. Results are representative of 3 independent experiments.
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fig1: Reexpression of pluripotency genes following exposure to DETA/NO. (a) Western blot analysis of Nanog and Oct4 proteins. D3 mESCs were grown in the presence (+LIF) or in the absence (−LIF) of LIF for 3 days. Cells were then cultured as indicated in Section 2 for additional periods of 1 and 2 days (day 4 and day 5). C: control cells. T: cells exposed to 1 mM DETA/NO for 19 h on day 4. Lanes 5 and 6 refer to cells exposed to DETA/NO and subsequently cultured for 1 and 2 days as in Section 2. (b) GFP expression in D3-Oct4 mESCs. Control cells were cultured in the presence of LIF for 6 days (+LIF/Control). Treated cells were cultured in the presence of 1 mM DETA/NO for 19 hours on day 4 and subsequently cultured as indicated in Section 2 for an additional period of 2 days in absence of LIF. PhC: phase contrast. Results are representative of 3 independent experiments. (c) Western blot analysis reveals Nanog and Oct4 reexpression after DETA/NO treatment. +/− LIF indicates that cells were cultured for 4 days in the presence of LIF, then LIF was removed and cells were subsequently exposed to 1 mM DETA/NO for 19 hours. Days 5 and 6 indicate that cells were subsequently cultured for 1 and 2 days in the absence of LIF. −/+LIF indicates cells that were cultured for 4 days in the absence of LIF; exposure to 1 mM DETA/NO for 19 hours was then performed in the presence of LIF. Days 5 and 6 indicate that cells were subsequently cultured for additional periods of 1 and 2 days, respectively, in the presence of LIF. Results are representative of 3 independent experiments.

Mentions: D3 mESCs were cultured for 3 days with or without LIF and then exposed to 1 mM DETA/NO for 19 hours (day 4). A substantial reduction in Nanog and Oct4 protein level was observed in cells following treatment with DETA/NO (Figure 1(a)). To explore the stability of Nanog and Oct4 downregulation, recovery experiments were carried out. After DETA/NO treatment, cells were grown in complete medium until day 6 in the presence or absence of LIF. The results show that Nanog and Oct4 proteins were reexpressed following DETA/NO exposure. In the case of Nanog, the phenomenon was more apparent in the presence of LIF. A similar response was observed in D3 mESCs expressing the green fluorescence protein (GFP) under the control of the Oct4 promoter. Cell colonies with homogeneous fluorescence were formed when D3 cells were cultured in the presence of LIF (+LIF/control) (Figure 1(b), GFP Oct4). Exposure to DETA/NO led to loss of GFP fluorescence in cells cultured without LIF (Treatment). Similar results were observed in cells cultured in the presence of LIF (not shown). Loss of homogeneous colony morphology is also apparent upon DETA/NO treatment (Figure 1(b), Treatment Ph C). Heterogeneity in the recovery of GFP signal is apparent following 2 days of culture in complete medium even in the absence of LIF (day 6) and up to 4 days (day 8) (see Supplementary Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/379678) since clusters of GFP positive cells coexist with GFP negative cells (see PhC image). In addition, colony morphology is not recovered.


Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

Mora-Castilla S, Tejedo JR, Tapia-Limonchi R, Díaz I, Hitos AB, Cahuana GM, Hmadcha A, Martín F, Soria B, Bedoya FJ - Stem Cells Int (2014)

Reexpression of pluripotency genes following exposure to DETA/NO. (a) Western blot analysis of Nanog and Oct4 proteins. D3 mESCs were grown in the presence (+LIF) or in the absence (−LIF) of LIF for 3 days. Cells were then cultured as indicated in Section 2 for additional periods of 1 and 2 days (day 4 and day 5). C: control cells. T: cells exposed to 1 mM DETA/NO for 19 h on day 4. Lanes 5 and 6 refer to cells exposed to DETA/NO and subsequently cultured for 1 and 2 days as in Section 2. (b) GFP expression in D3-Oct4 mESCs. Control cells were cultured in the presence of LIF for 6 days (+LIF/Control). Treated cells were cultured in the presence of 1 mM DETA/NO for 19 hours on day 4 and subsequently cultured as indicated in Section 2 for an additional period of 2 days in absence of LIF. PhC: phase contrast. Results are representative of 3 independent experiments. (c) Western blot analysis reveals Nanog and Oct4 reexpression after DETA/NO treatment. +/− LIF indicates that cells were cultured for 4 days in the presence of LIF, then LIF was removed and cells were subsequently exposed to 1 mM DETA/NO for 19 hours. Days 5 and 6 indicate that cells were subsequently cultured for 1 and 2 days in the absence of LIF. −/+LIF indicates cells that were cultured for 4 days in the absence of LIF; exposure to 1 mM DETA/NO for 19 hours was then performed in the presence of LIF. Days 5 and 6 indicate that cells were subsequently cultured for additional periods of 1 and 2 days, respectively, in the presence of LIF. Results are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269087&req=5

fig1: Reexpression of pluripotency genes following exposure to DETA/NO. (a) Western blot analysis of Nanog and Oct4 proteins. D3 mESCs were grown in the presence (+LIF) or in the absence (−LIF) of LIF for 3 days. Cells were then cultured as indicated in Section 2 for additional periods of 1 and 2 days (day 4 and day 5). C: control cells. T: cells exposed to 1 mM DETA/NO for 19 h on day 4. Lanes 5 and 6 refer to cells exposed to DETA/NO and subsequently cultured for 1 and 2 days as in Section 2. (b) GFP expression in D3-Oct4 mESCs. Control cells were cultured in the presence of LIF for 6 days (+LIF/Control). Treated cells were cultured in the presence of 1 mM DETA/NO for 19 hours on day 4 and subsequently cultured as indicated in Section 2 for an additional period of 2 days in absence of LIF. PhC: phase contrast. Results are representative of 3 independent experiments. (c) Western blot analysis reveals Nanog and Oct4 reexpression after DETA/NO treatment. +/− LIF indicates that cells were cultured for 4 days in the presence of LIF, then LIF was removed and cells were subsequently exposed to 1 mM DETA/NO for 19 hours. Days 5 and 6 indicate that cells were subsequently cultured for 1 and 2 days in the absence of LIF. −/+LIF indicates cells that were cultured for 4 days in the absence of LIF; exposure to 1 mM DETA/NO for 19 hours was then performed in the presence of LIF. Days 5 and 6 indicate that cells were subsequently cultured for additional periods of 1 and 2 days, respectively, in the presence of LIF. Results are representative of 3 independent experiments.
Mentions: D3 mESCs were cultured for 3 days with or without LIF and then exposed to 1 mM DETA/NO for 19 hours (day 4). A substantial reduction in Nanog and Oct4 protein level was observed in cells following treatment with DETA/NO (Figure 1(a)). To explore the stability of Nanog and Oct4 downregulation, recovery experiments were carried out. After DETA/NO treatment, cells were grown in complete medium until day 6 in the presence or absence of LIF. The results show that Nanog and Oct4 proteins were reexpressed following DETA/NO exposure. In the case of Nanog, the phenomenon was more apparent in the presence of LIF. A similar response was observed in D3 mESCs expressing the green fluorescence protein (GFP) under the control of the Oct4 promoter. Cell colonies with homogeneous fluorescence were formed when D3 cells were cultured in the presence of LIF (+LIF/control) (Figure 1(b), GFP Oct4). Exposure to DETA/NO led to loss of GFP fluorescence in cells cultured without LIF (Treatment). Similar results were observed in cells cultured in the presence of LIF (not shown). Loss of homogeneous colony morphology is also apparent upon DETA/NO treatment (Figure 1(b), Treatment Ph C). Heterogeneity in the recovery of GFP signal is apparent following 2 days of culture in complete medium even in the absence of LIF (day 6) and up to 4 days (day 8) (see Supplementary Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/379678) since clusters of GFP positive cells coexist with GFP negative cells (see PhC image). In addition, colony morphology is not recovered.

Bottom Line: Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment.Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression.Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), University Pablo de Olavide, Biomedical Research Network (CIBER) of Diabetes and Related Metabolic Diseases, Red-Tercel, Avenida Américo Vespucio S/N, 41092 Seville, Spain ; Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.

ABSTRACT
The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

No MeSH data available.


Related in: MedlinePlus