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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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Nodal signalling is involved in 1m-induced differentiation of hESCs. (A) Shef-3 hESCs were cultured for a total of 7 days with 2 μM 1m included in the medium for the number of days indicated and RT-PCR analyses were performed using primers selective for the genes specified. 0d, hESCs cultured for 7d without 1m. (B) Shef-1 hESCs were cultured for a total of 7 days and either treated with 2 μM 1m alone or in combination with 10 μM SB43125 (SB) for the number of days indicated and RT-PCR performed. Con, cultures to which inhibitors were not added. (C) Shef-3 hESCs were treated with 1m alone or in combination with 100 ng/ml activin A (Act) for the times indicated and RT-PCR performed. Con, cultures to which factors were not added. (D,E) Shef-3 hESCs were treated with 2 μM 1m or 100 ng/ml activin A (Act) alone or in combination with 1m for 5 days. Control cultures were grown for the same 5-day period without addition of activin A or 1m. (D) Expression of the endodermal markers FOXA2 and HNF4α were analysed by immunofluorescence. Scale bar: 50 μm. The mean percentage of positive cells (±s.e.m.) over three independent experiments is indicated. (E) Expression of CXCR4 was analysed by flow cytometry. The mean percentage of CXCR4-positive cells (±s.e.m.) for eight individual experiments is indicated.
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Figure 4: Nodal signalling is involved in 1m-induced differentiation of hESCs. (A) Shef-3 hESCs were cultured for a total of 7 days with 2 μM 1m included in the medium for the number of days indicated and RT-PCR analyses were performed using primers selective for the genes specified. 0d, hESCs cultured for 7d without 1m. (B) Shef-1 hESCs were cultured for a total of 7 days and either treated with 2 μM 1m alone or in combination with 10 μM SB43125 (SB) for the number of days indicated and RT-PCR performed. Con, cultures to which inhibitors were not added. (C) Shef-3 hESCs were treated with 1m alone or in combination with 100 ng/ml activin A (Act) for the times indicated and RT-PCR performed. Con, cultures to which factors were not added. (D,E) Shef-3 hESCs were treated with 2 μM 1m or 100 ng/ml activin A (Act) alone or in combination with 1m for 5 days. Control cultures were grown for the same 5-day period without addition of activin A or 1m. (D) Expression of the endodermal markers FOXA2 and HNF4α were analysed by immunofluorescence. Scale bar: 50 μm. The mean percentage of positive cells (±s.e.m.) over three independent experiments is indicated. (E) Expression of CXCR4 was analysed by flow cytometry. The mean percentage of CXCR4-positive cells (±s.e.m.) for eight individual experiments is indicated.

Mentions: We were particularly interested in the ability of 1m to induce the generation of DE, as this could represent a novel route to generation of hepatic lineages. Genetic studies in mice have revealed a role for Nodal during initiation of the PS and formation of DE (Conlon et al., 1994; Brennan et al., 2001), with the intensity and duration of Nodal signalling influencing specification of the anterior PS to either mesoderm or, at higher levels, endoderm (Lowe et al., 2001; Vincent et al., 2003). Therefore, we were initially interested in testing whether Nodal signalling was involved in 1m-mediated effects. We found that 1m induced a rapid and transient increase in NODAL gene expression (despite replenishment every 2 days; Fig. 4A), which was partially inhibited by the inhibition of activin receptor-like kinases (ALK4, ALK5 and ALK7) with SB43125 (Fig. 4B). Treatment with SB34125 dramatically reduced 1m-induced GSC and FOXA2 expression, downstream targets of Nodal signalling, and caused a delay in expression of brachyury (T) (Fig. 4B). These data suggest that 1m-induced expression of NODAL and Nodal signalling could be required either for induction of differentiation into PS or later during specification of DE. Owing to the fact that expression of brachyury (T) is delayed, rather than blocked, we favour the latter possibility. Secondly, we compared the effects of 1m or activin A alone and in combination on the generation of DE. Activin A, another member of the TGFβ superfamily, binds to the same receptors as Nodal and is used to mimic Nodal activity in vitro. Generation of DE from hESCs has relied on activation of the activin–Nodal pathway either alone (D'Amour et al., 2005) or in combination with Wnt signalling (D'Amour et al., 2006; Hay et al., 2008; Sumi et al., 2008), which enhances efficiency. Gene expression patterns were similar for all conditions after 3 days (Fig. 4C) and after 7 days SOX17, FOXA2 and HNF4A gene expression were also similar. However, although little or no GSC expression was apparent following treatment with 1m alone for 7 days, indicative of transition through the PS, GSC expression was maintained in the presence of activin A alone or in combination with 1m. Intriguingly, AFP expression was only detected in samples treated with 1m alone for 7 days (Fig. 4C) andFig. 4.


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)

Nodal signalling is involved in 1m-induced differentiation of hESCs. (A) Shef-3 hESCs were cultured for a total of 7 days with 2 μM 1m included in the medium for the number of days indicated and RT-PCR analyses were performed using primers selective for the genes specified. 0d, hESCs cultured for 7d without 1m. (B) Shef-1 hESCs were cultured for a total of 7 days and either treated with 2 μM 1m alone or in combination with 10 μM SB43125 (SB) for the number of days indicated and RT-PCR performed. Con, cultures to which inhibitors were not added. (C) Shef-3 hESCs were treated with 1m alone or in combination with 100 ng/ml activin A (Act) for the times indicated and RT-PCR performed. Con, cultures to which factors were not added. (D,E) Shef-3 hESCs were treated with 2 μM 1m or 100 ng/ml activin A (Act) alone or in combination with 1m for 5 days. Control cultures were grown for the same 5-day period without addition of activin A or 1m. (D) Expression of the endodermal markers FOXA2 and HNF4α were analysed by immunofluorescence. Scale bar: 50 μm. The mean percentage of positive cells (±s.e.m.) over three independent experiments is indicated. (E) Expression of CXCR4 was analysed by flow cytometry. The mean percentage of CXCR4-positive cells (±s.e.m.) for eight individual experiments is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Nodal signalling is involved in 1m-induced differentiation of hESCs. (A) Shef-3 hESCs were cultured for a total of 7 days with 2 μM 1m included in the medium for the number of days indicated and RT-PCR analyses were performed using primers selective for the genes specified. 0d, hESCs cultured for 7d without 1m. (B) Shef-1 hESCs were cultured for a total of 7 days and either treated with 2 μM 1m alone or in combination with 10 μM SB43125 (SB) for the number of days indicated and RT-PCR performed. Con, cultures to which inhibitors were not added. (C) Shef-3 hESCs were treated with 1m alone or in combination with 100 ng/ml activin A (Act) for the times indicated and RT-PCR performed. Con, cultures to which factors were not added. (D,E) Shef-3 hESCs were treated with 2 μM 1m or 100 ng/ml activin A (Act) alone or in combination with 1m for 5 days. Control cultures were grown for the same 5-day period without addition of activin A or 1m. (D) Expression of the endodermal markers FOXA2 and HNF4α were analysed by immunofluorescence. Scale bar: 50 μm. The mean percentage of positive cells (±s.e.m.) over three independent experiments is indicated. (E) Expression of CXCR4 was analysed by flow cytometry. The mean percentage of CXCR4-positive cells (±s.e.m.) for eight individual experiments is indicated.
Mentions: We were particularly interested in the ability of 1m to induce the generation of DE, as this could represent a novel route to generation of hepatic lineages. Genetic studies in mice have revealed a role for Nodal during initiation of the PS and formation of DE (Conlon et al., 1994; Brennan et al., 2001), with the intensity and duration of Nodal signalling influencing specification of the anterior PS to either mesoderm or, at higher levels, endoderm (Lowe et al., 2001; Vincent et al., 2003). Therefore, we were initially interested in testing whether Nodal signalling was involved in 1m-mediated effects. We found that 1m induced a rapid and transient increase in NODAL gene expression (despite replenishment every 2 days; Fig. 4A), which was partially inhibited by the inhibition of activin receptor-like kinases (ALK4, ALK5 and ALK7) with SB43125 (Fig. 4B). Treatment with SB34125 dramatically reduced 1m-induced GSC and FOXA2 expression, downstream targets of Nodal signalling, and caused a delay in expression of brachyury (T) (Fig. 4B). These data suggest that 1m-induced expression of NODAL and Nodal signalling could be required either for induction of differentiation into PS or later during specification of DE. Owing to the fact that expression of brachyury (T) is delayed, rather than blocked, we favour the latter possibility. Secondly, we compared the effects of 1m or activin A alone and in combination on the generation of DE. Activin A, another member of the TGFβ superfamily, binds to the same receptors as Nodal and is used to mimic Nodal activity in vitro. Generation of DE from hESCs has relied on activation of the activin–Nodal pathway either alone (D'Amour et al., 2005) or in combination with Wnt signalling (D'Amour et al., 2006; Hay et al., 2008; Sumi et al., 2008), which enhances efficiency. Gene expression patterns were similar for all conditions after 3 days (Fig. 4C) and after 7 days SOX17, FOXA2 and HNF4A gene expression were also similar. However, although little or no GSC expression was apparent following treatment with 1m alone for 7 days, indicative of transition through the PS, GSC expression was maintained in the presence of activin A alone or in combination with 1m. Intriguingly, AFP expression was only detected in samples treated with 1m alone for 7 days (Fig. 4C) andFig. 4.

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