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DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

Czysz K, Minger S, Thomas N - PLoS ONE (2015)

Bottom Line: Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO.Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4.Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

View Article: PubMed Central - PubMed

Affiliation: GE Healthcare Life Sciences, The Maynard Centre, Cardiff, Wales, United Kingdom.

ABSTRACT

Background: Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells.

Materials and methods: Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG), definitive endoderm (SOX17, CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures.

Results: Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

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Transcriptional analysis of regulation of OCT4, SOX17 and ALB expression by addition of different concentrations of DMSO to DE medium.(A). Addition of DMSO to the Activin A-based medium during DE formation dose-dependently regulated the correlation between gene expression of the pluripotency marker OCT4 and the DE marker SOX17 during definitive endoderm formation. (B) Expression of the hepatic marker ALB at the end of differentiation linked to the prior expression of SOX17 at day 4 of DE formation. (C) Correlation was also observed between the down regulation of the pluripotency marker OCT4 at day 4 of DE formation and an increase in the expression of ALB at the end of hepatic differentiation. The housekeeping gene GAPDH was used for the initial normalization of the qRT-PCR results. Further standardization to the Activin A culture condition was performed to show relations between all of the investigated conditions. qRT-PCR data is mean ± SD of 3 replicates, Student’s t test: n = 3, (**) p ≤ 0.01, (***) p ≤ 0.001.
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pone.0117689.g006: Transcriptional analysis of regulation of OCT4, SOX17 and ALB expression by addition of different concentrations of DMSO to DE medium.(A). Addition of DMSO to the Activin A-based medium during DE formation dose-dependently regulated the correlation between gene expression of the pluripotency marker OCT4 and the DE marker SOX17 during definitive endoderm formation. (B) Expression of the hepatic marker ALB at the end of differentiation linked to the prior expression of SOX17 at day 4 of DE formation. (C) Correlation was also observed between the down regulation of the pluripotency marker OCT4 at day 4 of DE formation and an increase in the expression of ALB at the end of hepatic differentiation. The housekeeping gene GAPDH was used for the initial normalization of the qRT-PCR results. Further standardization to the Activin A culture condition was performed to show relations between all of the investigated conditions. qRT-PCR data is mean ± SD of 3 replicates, Student’s t test: n = 3, (**) p ≤ 0.01, (***) p ≤ 0.001.

Mentions: To further investigate the optimal concentration of DMSO at specific time points during Activin A-based DE formation and to assess its potency to increase the overall efficiency of hepatocyte differentiation, DE cells were primed through intermediate hepatoblasts to hepatic-like cells. Hepatic-like cells derived from all DE-culture conditions were harvested after 28 days and analysed for expression of the hepatocyte marker albumin (ALB). At day 4 of DE differentiation a dose-dependent correlation was observed between DMSO-induced up regulation of SOX17 and down regulation of OCT4 (Fig. 6A) confirming the beneficial effects of DMSO in the Activin A-driven process of definitive endoderm formation. Moreover, in cultures where 0.5% of DMSO was present during the first 2 days during DE formation followed by 0.25% DMSO during the remaining 2 days, cells showed lower expression of OCT4 than in cultures where the concentrations of DMSO were transposed (0.25% then 0.5%D) or maintained continuously at 0.25%. Down regulated expression of OCT4 in the presence of 0.5% then 0.25% DMSO was paralleled by up regulation of the DE marker SOX17, suggesting that a rapid and efficient decrease of the pluripotency status of cells at the very beginning of commitment to differentiation potentiates their ability to respond to DE-priming factors.


DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

Czysz K, Minger S, Thomas N - PLoS ONE (2015)

Transcriptional analysis of regulation of OCT4, SOX17 and ALB expression by addition of different concentrations of DMSO to DE medium.(A). Addition of DMSO to the Activin A-based medium during DE formation dose-dependently regulated the correlation between gene expression of the pluripotency marker OCT4 and the DE marker SOX17 during definitive endoderm formation. (B) Expression of the hepatic marker ALB at the end of differentiation linked to the prior expression of SOX17 at day 4 of DE formation. (C) Correlation was also observed between the down regulation of the pluripotency marker OCT4 at day 4 of DE formation and an increase in the expression of ALB at the end of hepatic differentiation. The housekeeping gene GAPDH was used for the initial normalization of the qRT-PCR results. Further standardization to the Activin A culture condition was performed to show relations between all of the investigated conditions. qRT-PCR data is mean ± SD of 3 replicates, Student’s t test: n = 3, (**) p ≤ 0.01, (***) p ≤ 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4320104&req=5

pone.0117689.g006: Transcriptional analysis of regulation of OCT4, SOX17 and ALB expression by addition of different concentrations of DMSO to DE medium.(A). Addition of DMSO to the Activin A-based medium during DE formation dose-dependently regulated the correlation between gene expression of the pluripotency marker OCT4 and the DE marker SOX17 during definitive endoderm formation. (B) Expression of the hepatic marker ALB at the end of differentiation linked to the prior expression of SOX17 at day 4 of DE formation. (C) Correlation was also observed between the down regulation of the pluripotency marker OCT4 at day 4 of DE formation and an increase in the expression of ALB at the end of hepatic differentiation. The housekeeping gene GAPDH was used for the initial normalization of the qRT-PCR results. Further standardization to the Activin A culture condition was performed to show relations between all of the investigated conditions. qRT-PCR data is mean ± SD of 3 replicates, Student’s t test: n = 3, (**) p ≤ 0.01, (***) p ≤ 0.001.
Mentions: To further investigate the optimal concentration of DMSO at specific time points during Activin A-based DE formation and to assess its potency to increase the overall efficiency of hepatocyte differentiation, DE cells were primed through intermediate hepatoblasts to hepatic-like cells. Hepatic-like cells derived from all DE-culture conditions were harvested after 28 days and analysed for expression of the hepatocyte marker albumin (ALB). At day 4 of DE differentiation a dose-dependent correlation was observed between DMSO-induced up regulation of SOX17 and down regulation of OCT4 (Fig. 6A) confirming the beneficial effects of DMSO in the Activin A-driven process of definitive endoderm formation. Moreover, in cultures where 0.5% of DMSO was present during the first 2 days during DE formation followed by 0.25% DMSO during the remaining 2 days, cells showed lower expression of OCT4 than in cultures where the concentrations of DMSO were transposed (0.25% then 0.5%D) or maintained continuously at 0.25%. Down regulated expression of OCT4 in the presence of 0.5% then 0.25% DMSO was paralleled by up regulation of the DE marker SOX17, suggesting that a rapid and efficient decrease of the pluripotency status of cells at the very beginning of commitment to differentiation potentiates their ability to respond to DE-priming factors.

Bottom Line: Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO.Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4.Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

View Article: PubMed Central - PubMed

Affiliation: GE Healthcare Life Sciences, The Maynard Centre, Cardiff, Wales, United Kingdom.

ABSTRACT

Background: Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells.

Materials and methods: Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG), definitive endoderm (SOX17, CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures.

Results: Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

Show MeSH
Related in: MedlinePlus