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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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1m treatment induces differentiation towards definitive endoderm. Shef-3 hESCs were cultured for a total of 7 days, feeder-free on Matrigel, in chemically defined mTeSR1 medium with 2 μM 1m included in the medium for the number of days indicated (0d, hESCs cultured for 7 days without 1m). (A) RT-PCR analyses were performed using primers selective for the genes specified. (B) Shef-3 hESCs cultured under the conditions indicated for the duration of the experiment in Lumox 24-well trays were analysed by immunofluorescence for expression of brachyury, PDGFRβ, FOXA2, SOX17 and HNF4α. Cells were counterstained with DAPI for nuclear localization and the lower panels show the superimposed images. Scale bars: 50 μm. DMSO controls were cultured for the entire 7-day period.
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Figure 3: 1m treatment induces differentiation towards definitive endoderm. Shef-3 hESCs were cultured for a total of 7 days, feeder-free on Matrigel, in chemically defined mTeSR1 medium with 2 μM 1m included in the medium for the number of days indicated (0d, hESCs cultured for 7 days without 1m). (A) RT-PCR analyses were performed using primers selective for the genes specified. (B) Shef-3 hESCs cultured under the conditions indicated for the duration of the experiment in Lumox 24-well trays were analysed by immunofluorescence for expression of brachyury, PDGFRβ, FOXA2, SOX17 and HNF4α. Cells were counterstained with DAPI for nuclear localization and the lower panels show the superimposed images. Scale bars: 50 μm. DMSO controls were cultured for the entire 7-day period.

Mentions: On the basis of the knowledge that Wnt–β-catenin signalling plays a pivotal role in formation of the PS, mesoderm and endoderm in ESCs (Hay et al., 2008; Nakanishi et al., 2009; Sumi et al., 2008), we investigated whether treatment of hESCs with 1m could induce differentiation towards these lineages. The following experiments were all performed on hESCs cultured feeder-free on Matrigel in chemically defined mTeSR1 medium. This system has the advantage of simplicity and reliability, leading to consistent and reproducible cell culture and differentiation. Initially, the effects of 1m treatment on gene expression in hESCs cultured over a 7-day period were examined (Fig. 3A). Induction of differentiation was indicated by loss of OCT4 and NANOG expression. The emergence of the PS and mesendoderm is characterised by expression of MIXL1 (Davis et al., 2008; Pearce and Evans, 1999) and GSC, the gene encoding the homeobox protein goosecoid (Tada et al., 2005), which were both rapidly upregulated following 1m treatment. In addition, T, the gene encoding the brachyury protein, which is expressed in the PS and mesendoderm, and also the early mesoderm (Kubo et al., 2004; Wilkinson et al., 1990), displayed a similar expression profile. Expression of brachyury protein emerged on day 3 and was maintained to day 7 (Fig. 3B). We also observed expression of other early mesodermal markers including KDR (also known as FLK1 and VEGFR2), MSX1 and PDGFRB (Fig. 3A). In addition, we observed some expression of PGDFRβ protein following 7 days of differentiation (Fig. 3B), indicating mesodermal differentiation. Expression of FOXA2 [encoding hepatocyte nuclear factor (HNF) 3β] and CXCR4, expressed in the PS and maintained in definitive endoderm progenitors, were also rapidly induced, followed by the DE marker SOX17. CXCR4 is expressed in the developing mesoderm and definitive endoderm but not the visceral endoderm (VE) (McGrath et al., 1999) and has been used to distinguish DE from VE in both mouse and human ESCs (D'Amour et al., 2005; Yasunaga et al., 2005). Expression of CXCR4 along with SOX17, GSC and FOXA2, is consistent with differentiation towards the DE. Importantly, hESCs expressing CXCR4 are capable of differentiating into endodermal cells with either a pancreatic (D'Amour et al., 2006) or hepatic (Gouon-Evans et al., 2006) phenotype. Following 7 days of treatment with 1m, we observed expression of HNF4A and AFP (encoding α-fetoprotein), the latter marking the earliest stage of specification to the hepatic lineageFig. 3.


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)

1m treatment induces differentiation towards definitive endoderm. Shef-3 hESCs were cultured for a total of 7 days, feeder-free on Matrigel, in chemically defined mTeSR1 medium with 2 μM 1m included in the medium for the number of days indicated (0d, hESCs cultured for 7 days without 1m). (A) RT-PCR analyses were performed using primers selective for the genes specified. (B) Shef-3 hESCs cultured under the conditions indicated for the duration of the experiment in Lumox 24-well trays were analysed by immunofluorescence for expression of brachyury, PDGFRβ, FOXA2, SOX17 and HNF4α. Cells were counterstained with DAPI for nuclear localization and the lower panels show the superimposed images. Scale bars: 50 μm. DMSO controls were cultured for the entire 7-day period.
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Related In: Results  -  Collection

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Figure 3: 1m treatment induces differentiation towards definitive endoderm. Shef-3 hESCs were cultured for a total of 7 days, feeder-free on Matrigel, in chemically defined mTeSR1 medium with 2 μM 1m included in the medium for the number of days indicated (0d, hESCs cultured for 7 days without 1m). (A) RT-PCR analyses were performed using primers selective for the genes specified. (B) Shef-3 hESCs cultured under the conditions indicated for the duration of the experiment in Lumox 24-well trays were analysed by immunofluorescence for expression of brachyury, PDGFRβ, FOXA2, SOX17 and HNF4α. Cells were counterstained with DAPI for nuclear localization and the lower panels show the superimposed images. Scale bars: 50 μm. DMSO controls were cultured for the entire 7-day period.
Mentions: On the basis of the knowledge that Wnt–β-catenin signalling plays a pivotal role in formation of the PS, mesoderm and endoderm in ESCs (Hay et al., 2008; Nakanishi et al., 2009; Sumi et al., 2008), we investigated whether treatment of hESCs with 1m could induce differentiation towards these lineages. The following experiments were all performed on hESCs cultured feeder-free on Matrigel in chemically defined mTeSR1 medium. This system has the advantage of simplicity and reliability, leading to consistent and reproducible cell culture and differentiation. Initially, the effects of 1m treatment on gene expression in hESCs cultured over a 7-day period were examined (Fig. 3A). Induction of differentiation was indicated by loss of OCT4 and NANOG expression. The emergence of the PS and mesendoderm is characterised by expression of MIXL1 (Davis et al., 2008; Pearce and Evans, 1999) and GSC, the gene encoding the homeobox protein goosecoid (Tada et al., 2005), which were both rapidly upregulated following 1m treatment. In addition, T, the gene encoding the brachyury protein, which is expressed in the PS and mesendoderm, and also the early mesoderm (Kubo et al., 2004; Wilkinson et al., 1990), displayed a similar expression profile. Expression of brachyury protein emerged on day 3 and was maintained to day 7 (Fig. 3B). We also observed expression of other early mesodermal markers including KDR (also known as FLK1 and VEGFR2), MSX1 and PDGFRB (Fig. 3A). In addition, we observed some expression of PGDFRβ protein following 7 days of differentiation (Fig. 3B), indicating mesodermal differentiation. Expression of FOXA2 [encoding hepatocyte nuclear factor (HNF) 3β] and CXCR4, expressed in the PS and maintained in definitive endoderm progenitors, were also rapidly induced, followed by the DE marker SOX17. CXCR4 is expressed in the developing mesoderm and definitive endoderm but not the visceral endoderm (VE) (McGrath et al., 1999) and has been used to distinguish DE from VE in both mouse and human ESCs (D'Amour et al., 2005; Yasunaga et al., 2005). Expression of CXCR4 along with SOX17, GSC and FOXA2, is consistent with differentiation towards the DE. Importantly, hESCs expressing CXCR4 are capable of differentiating into endodermal cells with either a pancreatic (D'Amour et al., 2006) or hepatic (Gouon-Evans et al., 2006) phenotype. Following 7 days of treatment with 1m, we observed expression of HNF4A and AFP (encoding α-fetoprotein), the latter marking the earliest stage of specification to the hepatic lineageFig. 3.

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