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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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Analysis of transcriptional changes in cells primed to the stage of hepatoblasts with or without prior DE specification.hESCs; human ES cells. DE; definitive endoderm. DE D4; cells transitioned through DE stage and harvested at day 4. Prog D4; cells cultured directly in hepatic progenitor medium and harvested at day4. DE D12: cells transitioned through DE and harvested at day 12. Prog D12; cells cultured directly in hepatic progenitor medium and harvested at day 12. The housekeeping gene GAPDH was used for normalization of the qRT-PCR results. uHepG2; internal control for efficiency of hepatoblast formation. qRT-PCR data are mean ± 1SD of 3 biological replicates.
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pone.0117689.g005: Analysis of transcriptional changes in cells primed to the stage of hepatoblasts with or without prior DE specification.hESCs; human ES cells. DE; definitive endoderm. DE D4; cells transitioned through DE stage and harvested at day 4. Prog D4; cells cultured directly in hepatic progenitor medium and harvested at day4. DE D12: cells transitioned through DE and harvested at day 12. Prog D12; cells cultured directly in hepatic progenitor medium and harvested at day 12. The housekeeping gene GAPDH was used for normalization of the qRT-PCR results. uHepG2; internal control for efficiency of hepatoblast formation. qRT-PCR data are mean ± 1SD of 3 biological replicates.

Mentions: To confirm that formation of DE via the NODAL signalling pathway is a prerequisite for subsequent hepatic specification, hESC were exposed to either Activin A + 0.5% DMSO DE-inducing signals or to conditions that directly stimulate hepatoblast formation. After four days of culture cells from both conditions were differentiated further to hepatoblasts (Fig. 4A). At each of the indicated time-points cells were harvested and analysed by qRT-PCR and immunocytochemistry. The HepG2 cell line was used as a positive control for hepatoblast marker expression analysis. Real-time quantitative reverse transcription PCR analysis confirmed distinct gene expression profiles between the two culture conditions (Fig. 5). Widespread immunoreactivity for OCT4 was detected in cells that did not transition through the definitive endoderm stage (Fig. 4B; DE-) contrasting with relatively low expression of this transcription marker revealed by qRT-PCR, therefore suggesting its decreased level of expression per single OCT4-positive cell. Importantly, cells which were not exposed to the definitive endoderm culture condition were also negative for expression of the DE-specific genes SOX17 and HHEX at day 4 of specification which contrasted with high levels of expression of these markers in cells exposed to the DE-priming condition (Fig. 5C,D, S4 Fig). Undetectable AFP expression and low SOX7 expression in this culture condition at day 4 suggests that the SOX17- and HHEX-positive cells committed to definitive and not to the visceral endoderm cell lineage [1]. Moreover, fetal hepatic identity was acquired by cells by the end of stage two of differentiation only when pluripotent stem cells were transitioned first through the stage of definitive endoderm, highlighting the necessity of activating the Nodal signalling pathway before pursuing further cellular differentiation of cell types which are derived from this germ layer (Fig. 4B, Fig. 5E).


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)

Analysis of transcriptional changes in cells primed to the stage of hepatoblasts with or without prior DE specification.hESCs; human ES cells. DE; definitive endoderm. DE D4; cells transitioned through DE stage and harvested at day 4. Prog D4; cells cultured directly in hepatic progenitor medium and harvested at day4. DE D12: cells transitioned through DE and harvested at day 12. Prog D12; cells cultured directly in hepatic progenitor medium and harvested at day 12. The housekeeping gene GAPDH was used for normalization of the qRT-PCR results. uHepG2; internal control for efficiency of hepatoblast formation. qRT-PCR data are mean ± 1SD of 3 biological replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320104&req=5

pone.0117689.g005: Analysis of transcriptional changes in cells primed to the stage of hepatoblasts with or without prior DE specification.hESCs; human ES cells. DE; definitive endoderm. DE D4; cells transitioned through DE stage and harvested at day 4. Prog D4; cells cultured directly in hepatic progenitor medium and harvested at day4. DE D12: cells transitioned through DE and harvested at day 12. Prog D12; cells cultured directly in hepatic progenitor medium and harvested at day 12. The housekeeping gene GAPDH was used for normalization of the qRT-PCR results. uHepG2; internal control for efficiency of hepatoblast formation. qRT-PCR data are mean ± 1SD of 3 biological replicates.
Mentions: To confirm that formation of DE via the NODAL signalling pathway is a prerequisite for subsequent hepatic specification, hESC were exposed to either Activin A + 0.5% DMSO DE-inducing signals or to conditions that directly stimulate hepatoblast formation. After four days of culture cells from both conditions were differentiated further to hepatoblasts (Fig. 4A). At each of the indicated time-points cells were harvested and analysed by qRT-PCR and immunocytochemistry. The HepG2 cell line was used as a positive control for hepatoblast marker expression analysis. Real-time quantitative reverse transcription PCR analysis confirmed distinct gene expression profiles between the two culture conditions (Fig. 5). Widespread immunoreactivity for OCT4 was detected in cells that did not transition through the definitive endoderm stage (Fig. 4B; DE-) contrasting with relatively low expression of this transcription marker revealed by qRT-PCR, therefore suggesting its decreased level of expression per single OCT4-positive cell. Importantly, cells which were not exposed to the definitive endoderm culture condition were also negative for expression of the DE-specific genes SOX17 and HHEX at day 4 of specification which contrasted with high levels of expression of these markers in cells exposed to the DE-priming condition (Fig. 5C,D, S4 Fig). Undetectable AFP expression and low SOX7 expression in this culture condition at day 4 suggests that the SOX17- and HHEX-positive cells committed to definitive and not to the visceral endoderm cell lineage [1]. Moreover, fetal hepatic identity was acquired by cells by the end of stage two of differentiation only when pluripotent stem cells were transitioned first through the stage of definitive endoderm, highlighting the necessity of activating the Nodal signalling pathway before pursuing further cellular differentiation of cell types which are derived from this germ layer (Fig. 4B, Fig. 5E).

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