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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 1m inhibits GSK-3. (A,B) Shef-1 hESCs, cultured on MEFs or Matrigel in mTeSR1 medium, were treated with BIO or 1m for 30 minutes. Immunoblotting was performed to detect phosphorylated forms of β-catenin and ERK1/2. The same immunoblot in each case was re-probed for total β-catenin and ERK1 to assess loading. The bar graph shows the mean relative β-catenin phosphorylation levels (+s.e.m.; n=3). Data were analysed for statistical significance using two-tailed paired t-tests. *P<0.05; **P<0.01. (C) TOPFlash luciferase reporter assay following 24 hours of treatment of Shef-1 hESCs with BIO, 1m or vehicle (DMSO). Luciferase activity is expressed relative to the normalised TOPFlash luciferase activity in untreated (UT) control cells. A control reporter (FOPFlash), containing mutant TCF-binding sites, was also run in parallel. Data are means±s.e.m. for three independent experiments.
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Figure 2: Treatment of hESCs with 1m inhibits GSK-3. (A,B) Shef-1 hESCs, cultured on MEFs or Matrigel in mTeSR1 medium, were treated with BIO or 1m for 30 minutes. Immunoblotting was performed to detect phosphorylated forms of β-catenin and ERK1/2. The same immunoblot in each case was re-probed for total β-catenin and ERK1 to assess loading. The bar graph shows the mean relative β-catenin phosphorylation levels (+s.e.m.; n=3). Data were analysed for statistical significance using two-tailed paired t-tests. *P<0.05; **P<0.01. (C) TOPFlash luciferase reporter assay following 24 hours of treatment of Shef-1 hESCs with BIO, 1m or vehicle (DMSO). Luciferase activity is expressed relative to the normalised TOPFlash luciferase activity in untreated (UT) control cells. A control reporter (FOPFlash), containing mutant TCF-binding sites, was also run in parallel. Data are means±s.e.m. for three independent experiments.

Mentions: 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.


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 1m inhibits GSK-3. (A,B) Shef-1 hESCs, cultured on MEFs or Matrigel in mTeSR1 medium, were treated with BIO or 1m for 30 minutes. Immunoblotting was performed to detect phosphorylated forms of β-catenin and ERK1/2. The same immunoblot in each case was re-probed for total β-catenin and ERK1 to assess loading. The bar graph shows the mean relative β-catenin phosphorylation levels (+s.e.m.; n=3). Data were analysed for statistical significance using two-tailed paired t-tests. *P<0.05; **P<0.01. (C) TOPFlash luciferase reporter assay following 24 hours of treatment of Shef-1 hESCs with BIO, 1m or vehicle (DMSO). Luciferase activity is expressed relative to the normalised TOPFlash luciferase activity in untreated (UT) control cells. A control reporter (FOPFlash), containing mutant TCF-binding sites, was also run in parallel. Data are means±s.e.m. for three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104033&req=5

Figure 2: Treatment of hESCs with 1m inhibits GSK-3. (A,B) Shef-1 hESCs, cultured on MEFs or Matrigel in mTeSR1 medium, were treated with BIO or 1m for 30 minutes. Immunoblotting was performed to detect phosphorylated forms of β-catenin and ERK1/2. The same immunoblot in each case was re-probed for total β-catenin and ERK1 to assess loading. The bar graph shows the mean relative β-catenin phosphorylation levels (+s.e.m.; n=3). Data were analysed for statistical significance using two-tailed paired t-tests. *P<0.05; **P<0.01. (C) TOPFlash luciferase reporter assay following 24 hours of treatment of Shef-1 hESCs with BIO, 1m or vehicle (DMSO). Luciferase activity is expressed relative to the normalised TOPFlash luciferase activity in untreated (UT) control cells. A control reporter (FOPFlash), containing mutant TCF-binding sites, was also run in parallel. Data are means±s.e.m. for three independent experiments.
Mentions: 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.

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