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A novel chemically directed route for the generation of definitive endoderm from human embryonic stem cells based on inhibition of GSK-3.

Bone HK, Nelson AS, Goldring CE, Tosh D, Welham MJ - J. Cell. Sci. (2011)

Bottom Line: Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics, that is expressing α-fetoprotein and HNF4α.Furthermore, 1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin.These findings describe, for the first time, the utility of GSK-3 inhibition, in a chemically directed approach, to a method of DE generation that is robust, potentially scalable and applicable to different hESC lines.

View Article: PubMed Central - PubMed

Affiliation: Centre for Regenerative Medicine and Department of Pharmacy and Pharmacology, University of Bath, Bath BA27AY, UK.

ABSTRACT
The use of small molecules to 'chemically direct' differentiation represents a powerful approach to promote specification of embryonic stem cells (ESCs) towards particular functional cell types for use in regenerative medicine and pharmaceutical applications. Here, we demonstrate a novel route for chemically directed differentiation of human ESCs (hESCs) into definitive endoderm (DE) exploiting a selective small-molecule inhibitor of glycogen synthase kinase 3 (GSK-3). This GSK-3 inhibitor, termed 1m, when used as the only supplement to a chemically defined feeder-free culture system, effectively promoted differentiation of ESC lines towards primitive streak (PS), mesoderm and DE. This contrasts with the role of GSK-3 in murine ESCs, where GSK-3 inhibition promotes pluripotency. Interestingly, 1m-mediated induction of differentiation involved transient NODAL expression and Nodal signalling. Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics, that is expressing α-fetoprotein and HNF4α. Furthermore, 1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin. These findings describe, for the first time, the utility of GSK-3 inhibition, in a chemically directed approach, to a method of DE generation that is robust, potentially scalable and applicable to different hESC lines.

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Treatment of hESCs with GSK-3 inhibitors induces differentiation. Shef-3 hESCs were treated with BIO, 1m or vehicle (DMSO) or left untreated (UT) and cultured for 7 days on either MEFs or Matrigel in mTeSR1 medium. (A) Images show the typical colonies that are formed. Scale bar: 1 mm. (B) hESCs were analysed by flow cytometry following immunostaining with antibodies against the pluripotency markers Tra-1-60 and SSEA4. Data show the mean percentage of positive cells (±s.e.m.) from at least three independent experiments. Statistical analysis was conducted using ANOVA and Dunnett's post hoc test to compare each treatment with the untreated control cells. *P<0.05; **P<0.01. An example of a histogram plot from a representative experiment is shown in supplementary material Fig. S2A. (C) RNA was extracted from the cells and RT-PCR analyses were performed using primers specific to the pluripotency genes OCT4 and NANOG and to the house-keeping β-actin-encoding gene. (D) Cell lysates (20 μg) were separated by SDS-PAGE and immunoblotting was performed using an antibody against OCT4. Blots were stripped and re-probed with anti-GAPDH antibodies to assess equal loading.
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Figure 1: Treatment of hESCs with GSK-3 inhibitors induces differentiation. Shef-3 hESCs were treated with BIO, 1m or vehicle (DMSO) or left untreated (UT) and cultured for 7 days on either MEFs or Matrigel in mTeSR1 medium. (A) Images show the typical colonies that are formed. Scale bar: 1 mm. (B) hESCs were analysed by flow cytometry following immunostaining with antibodies against the pluripotency markers Tra-1-60 and SSEA4. Data show the mean percentage of positive cells (±s.e.m.) from at least three independent experiments. Statistical analysis was conducted using ANOVA and Dunnett's post hoc test to compare each treatment with the untreated control cells. *P<0.05; **P<0.01. An example of a histogram plot from a representative experiment is shown in supplementary material Fig. S2A. (C) RNA was extracted from the cells and RT-PCR analyses were performed using primers specific to the pluripotency genes OCT4 and NANOG and to the house-keeping β-actin-encoding gene. (D) Cell lysates (20 μg) were separated by SDS-PAGE and immunoblotting was performed using an antibody against OCT4. Blots were stripped and re-probed with anti-GAPDH antibodies to assess equal loading.

Mentions: We previously synthesised and characterised a panel of compounds that robustly inhibit GSK-3 in mouse ESCs and that enhance ESC self-renewal (Bone et al., 2009). Given the conflicting evidence regarding the role of GSK-3 in hESCs, we examined the effects of our most potent and specific GSK-3 inhibitor on hESC fate. Treatment of hESCs with a dose of 2 μM 1m (structure shown in supplementary material Fig. S1) did not maintain self-renewal but instead appeared to induce differentiation of the Shef-3 hESC line cultured either on mouse embryonic fibroblasts (MEFs) or in a feeder-free chemically defined system on Matrigel in mTeSR1 medium (Fig. 1A). Expression of surface markers of pluripotency (Tra-1-60 and SSEA4) decreased following 1m treatment (Fig. 1B and supplementary material Fig. S2A), whereas vehicle alone (DMSO) had no effect (Fig. 1A,B). The decrease in SSEA4 expression observed with 1m-treated Shef-3 cells co-cultured on MEFs was less than that observed with mTeSR1 (supplementary material Fig. S2A), but was nonetheless consistent (Fig. 1B). It is possible that factors produced by MEFs slow the loss of SSEA4, accounting for this observation. The reduction in Tra-1-60 and SSEA4 expression was accompanied by loss of OCT4 and NANOG gene expression (Fig. 1C) and OCT4 protein expression (Fig. 1D), and similar results were observed with Shef-1 hESCs cultured on feeders (supplementary material Fig. S3). Importantly, under chemically defined feeder-free conditions, treatment with 2 μM 1m led to a modest (~twofold) enhancement in hESC viability and proliferation (supplementary material Fig. S2B). For comparison, we also investigated the influence of the structurally unrelated GSK-3 inhibitor BIO and interestingly discovered its effects were dependent on culture conditions (Fig. 1). hESCs retained pluripotency when cultured on MEFs in the presence of BIO. However, when cultured in mTeSR1, 2 μM BIO, as for 1m, also induced differentiation.


A novel chemically directed route for the generation of definitive endoderm from human embryonic stem cells based on inhibition of GSK-3.

Bone HK, Nelson AS, Goldring CE, Tosh D, Welham MJ - J. Cell. Sci. (2011)

Treatment of hESCs with GSK-3 inhibitors induces differentiation. Shef-3 hESCs were treated with BIO, 1m or vehicle (DMSO) or left untreated (UT) and cultured for 7 days on either MEFs or Matrigel in mTeSR1 medium. (A) Images show the typical colonies that are formed. Scale bar: 1 mm. (B) hESCs were analysed by flow cytometry following immunostaining with antibodies against the pluripotency markers Tra-1-60 and SSEA4. Data show the mean percentage of positive cells (±s.e.m.) from at least three independent experiments. Statistical analysis was conducted using ANOVA and Dunnett's post hoc test to compare each treatment with the untreated control cells. *P<0.05; **P<0.01. An example of a histogram plot from a representative experiment is shown in supplementary material Fig. S2A. (C) RNA was extracted from the cells and RT-PCR analyses were performed using primers specific to the pluripotency genes OCT4 and NANOG and to the house-keeping β-actin-encoding gene. (D) Cell lysates (20 μg) were separated by SDS-PAGE and immunoblotting was performed using an antibody against OCT4. Blots were stripped and re-probed with anti-GAPDH antibodies to assess equal loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Treatment of hESCs with GSK-3 inhibitors induces differentiation. Shef-3 hESCs were treated with BIO, 1m or vehicle (DMSO) or left untreated (UT) and cultured for 7 days on either MEFs or Matrigel in mTeSR1 medium. (A) Images show the typical colonies that are formed. Scale bar: 1 mm. (B) hESCs were analysed by flow cytometry following immunostaining with antibodies against the pluripotency markers Tra-1-60 and SSEA4. Data show the mean percentage of positive cells (±s.e.m.) from at least three independent experiments. Statistical analysis was conducted using ANOVA and Dunnett's post hoc test to compare each treatment with the untreated control cells. *P<0.05; **P<0.01. An example of a histogram plot from a representative experiment is shown in supplementary material Fig. S2A. (C) RNA was extracted from the cells and RT-PCR analyses were performed using primers specific to the pluripotency genes OCT4 and NANOG and to the house-keeping β-actin-encoding gene. (D) Cell lysates (20 μg) were separated by SDS-PAGE and immunoblotting was performed using an antibody against OCT4. Blots were stripped and re-probed with anti-GAPDH antibodies to assess equal loading.
Mentions: We previously synthesised and characterised a panel of compounds that robustly inhibit GSK-3 in mouse ESCs and that enhance ESC self-renewal (Bone et al., 2009). Given the conflicting evidence regarding the role of GSK-3 in hESCs, we examined the effects of our most potent and specific GSK-3 inhibitor on hESC fate. Treatment of hESCs with a dose of 2 μM 1m (structure shown in supplementary material Fig. S1) did not maintain self-renewal but instead appeared to induce differentiation of the Shef-3 hESC line cultured either on mouse embryonic fibroblasts (MEFs) or in a feeder-free chemically defined system on Matrigel in mTeSR1 medium (Fig. 1A). Expression of surface markers of pluripotency (Tra-1-60 and SSEA4) decreased following 1m treatment (Fig. 1B and supplementary material Fig. S2A), whereas vehicle alone (DMSO) had no effect (Fig. 1A,B). The decrease in SSEA4 expression observed with 1m-treated Shef-3 cells co-cultured on MEFs was less than that observed with mTeSR1 (supplementary material Fig. S2A), but was nonetheless consistent (Fig. 1B). It is possible that factors produced by MEFs slow the loss of SSEA4, accounting for this observation. The reduction in Tra-1-60 and SSEA4 expression was accompanied by loss of OCT4 and NANOG gene expression (Fig. 1C) and OCT4 protein expression (Fig. 1D), and similar results were observed with Shef-1 hESCs cultured on feeders (supplementary material Fig. S3). Importantly, under chemically defined feeder-free conditions, treatment with 2 μM 1m led to a modest (~twofold) enhancement in hESC viability and proliferation (supplementary material Fig. S2B). For comparison, we also investigated the influence of the structurally unrelated GSK-3 inhibitor BIO and interestingly discovered its effects were dependent on culture conditions (Fig. 1). hESCs retained pluripotency when cultured on MEFs in the presence of BIO. However, when cultured in mTeSR1, 2 μM BIO, as for 1m, also induced differentiation.

Bottom Line: Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics, that is expressing α-fetoprotein and HNF4α.Furthermore, 1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin.These findings describe, for the first time, the utility of GSK-3 inhibition, in a chemically directed approach, to a method of DE generation that is robust, potentially scalable and applicable to different hESC lines.

View Article: PubMed Central - PubMed

Affiliation: Centre for Regenerative Medicine and Department of Pharmacy and Pharmacology, University of Bath, Bath BA27AY, UK.

ABSTRACT
The use of small molecules to 'chemically direct' differentiation represents a powerful approach to promote specification of embryonic stem cells (ESCs) towards particular functional cell types for use in regenerative medicine and pharmaceutical applications. Here, we demonstrate a novel route for chemically directed differentiation of human ESCs (hESCs) into definitive endoderm (DE) exploiting a selective small-molecule inhibitor of glycogen synthase kinase 3 (GSK-3). This GSK-3 inhibitor, termed 1m, when used as the only supplement to a chemically defined feeder-free culture system, effectively promoted differentiation of ESC lines towards primitive streak (PS), mesoderm and DE. This contrasts with the role of GSK-3 in murine ESCs, where GSK-3 inhibition promotes pluripotency. Interestingly, 1m-mediated induction of differentiation involved transient NODAL expression and Nodal signalling. Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics, that is expressing α-fetoprotein and HNF4α. Furthermore, 1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin. These findings describe, for the first time, the utility of GSK-3 inhibition, in a chemically directed approach, to a method of DE generation that is robust, potentially scalable and applicable to different hESC lines.

Show MeSH
Related in: MedlinePlus