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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-induced DE has hepatic potential. Shef-3 hESCs were treated with 2 μM 1m for 7 days to induce differentiation into DE. Hepatic induction and maturation conditions were then applied (see Materials and Methods). (A) Schematic representation of the differentiation protocol. (B) Images of differentiated cells at stage II (day 14) and stage III (day 25). Scale bars: 50 μm. The bottom panel shows a higher magnification of the boxed area of stage III cultures. (C) RT-PCR analyses of RNA prepared from cells at the indicated stages of differentiation. RNA extracted from human liver (hLiver) was used as a positive control. The β-actin-encoding gene was used as a housekeeping gene. (D) Expression of the early hepatic markers HNF4α, AFP and TTR in cells at stage II and stage III analysed by immunofluorescence. Scale bar: 50 μm. (E) Medium (1 μl) from stage III (day 25) cells, conditioned for 48 hours, and from a medium alone (MA) control was analysed by immunoblotting with an antibody against AFP. Purified AFP and medium conditioned from the Huh7 human hepatocarcinoma cell line were run as controls. (F) ELISA assay for human albumin from stage III (day 25) cell medium. The limit of detection is 12.5 ng/ml. Results are mean+s.d. (n=2 assay repeats). Medium conditioned for 24 hours by primary human hepatocytes is included as a positive control.
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Figure 5: 1m-induced DE has hepatic potential. Shef-3 hESCs were treated with 2 μM 1m for 7 days to induce differentiation into DE. Hepatic induction and maturation conditions were then applied (see Materials and Methods). (A) Schematic representation of the differentiation protocol. (B) Images of differentiated cells at stage II (day 14) and stage III (day 25). Scale bars: 50 μm. The bottom panel shows a higher magnification of the boxed area of stage III cultures. (C) RT-PCR analyses of RNA prepared from cells at the indicated stages of differentiation. RNA extracted from human liver (hLiver) was used as a positive control. The β-actin-encoding gene was used as a housekeeping gene. (D) Expression of the early hepatic markers HNF4α, AFP and TTR in cells at stage II and stage III analysed by immunofluorescence. Scale bar: 50 μm. (E) Medium (1 μl) from stage III (day 25) cells, conditioned for 48 hours, and from a medium alone (MA) control was analysed by immunoblotting with an antibody against AFP. Purified AFP and medium conditioned from the Huh7 human hepatocarcinoma cell line were run as controls. (F) ELISA assay for human albumin from stage III (day 25) cell medium. The limit of detection is 12.5 ng/ml. Results are mean+s.d. (n=2 assay repeats). Medium conditioned for 24 hours by primary human hepatocytes is included as a positive control.

Mentions: One of our main interests is in generation of HLCs, so we next tested whether 1m-induced DE was capable of differentiating into HLCs. The ability to generate HLCs provides an indication of the developmental potential of the chemically derived DE, where the ultimate aim is the generation of hepatocytes displaying functional characteristics. To evaluate this potential, we employed a differentiation protocol based on that of Agarwal (Agarwal et al., 2008) (Fig. 5A). Untreated hESCs did not survive the hepatic induction and maturation regimes. However, following hepatic induction of 1m-induced DE, stage II cells began to adopt morphological features characteristic of cells of the hepatic lineage (Fig. 5B) and expressed the genes encoding α-fetoprotein (AFP), albumin (ALB), transferrin (TFN; officially known as TF), transthyrettin (TTR) and α1 anti-trypsin (AAT; officially known as SERPINA1), correlating with initial hepatic differentiation (Fig. 5C). Following hepatic maturation, stage III cells exhibited characteristics of hepatocytes including a polygonal morphology, distinct round nuclei with 1 or 2 prominent nucleoli, and the presence of binucleate cells (Fig. 5B,D). Increased ALB and cytochrome P450 3A family (CYP3A) gene expression (Fig. 5C) and increased HNF4α, AFP and TTR protein expression (Fig. 5D) were observed in stage III compared with that in stage II cells. Equivalent amounts of RNA were used for the reverse transcription reaction that were then subjected to PCR; however, human liver expresses proportionally less β-actin-encoding mRNA than hESCs, which results in the apparent absence of β-actin in the human liver samples. To assess liver-specific function, secretion of the key serum proteins, AFP and albumin, was determined. Both AFP and albumin (Fig. 5E,F) were detected in the culture medium of stage III cells derived from 1m-induced DE. These results indicate that 1m-derived definitive endoderm has the potential to differentiate into cells with hepatocyte characteristics. Similar results were also generated using BIO-derived DE cells (supplementary material Fig. S6).


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-induced DE has hepatic potential. Shef-3 hESCs were treated with 2 μM 1m for 7 days to induce differentiation into DE. Hepatic induction and maturation conditions were then applied (see Materials and Methods). (A) Schematic representation of the differentiation protocol. (B) Images of differentiated cells at stage II (day 14) and stage III (day 25). Scale bars: 50 μm. The bottom panel shows a higher magnification of the boxed area of stage III cultures. (C) RT-PCR analyses of RNA prepared from cells at the indicated stages of differentiation. RNA extracted from human liver (hLiver) was used as a positive control. The β-actin-encoding gene was used as a housekeeping gene. (D) Expression of the early hepatic markers HNF4α, AFP and TTR in cells at stage II and stage III analysed by immunofluorescence. Scale bar: 50 μm. (E) Medium (1 μl) from stage III (day 25) cells, conditioned for 48 hours, and from a medium alone (MA) control was analysed by immunoblotting with an antibody against AFP. Purified AFP and medium conditioned from the Huh7 human hepatocarcinoma cell line were run as controls. (F) ELISA assay for human albumin from stage III (day 25) cell medium. The limit of detection is 12.5 ng/ml. Results are mean+s.d. (n=2 assay repeats). Medium conditioned for 24 hours by primary human hepatocytes is included as a positive control.
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Related In: Results  -  Collection

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Figure 5: 1m-induced DE has hepatic potential. Shef-3 hESCs were treated with 2 μM 1m for 7 days to induce differentiation into DE. Hepatic induction and maturation conditions were then applied (see Materials and Methods). (A) Schematic representation of the differentiation protocol. (B) Images of differentiated cells at stage II (day 14) and stage III (day 25). Scale bars: 50 μm. The bottom panel shows a higher magnification of the boxed area of stage III cultures. (C) RT-PCR analyses of RNA prepared from cells at the indicated stages of differentiation. RNA extracted from human liver (hLiver) was used as a positive control. The β-actin-encoding gene was used as a housekeeping gene. (D) Expression of the early hepatic markers HNF4α, AFP and TTR in cells at stage II and stage III analysed by immunofluorescence. Scale bar: 50 μm. (E) Medium (1 μl) from stage III (day 25) cells, conditioned for 48 hours, and from a medium alone (MA) control was analysed by immunoblotting with an antibody against AFP. Purified AFP and medium conditioned from the Huh7 human hepatocarcinoma cell line were run as controls. (F) ELISA assay for human albumin from stage III (day 25) cell medium. The limit of detection is 12.5 ng/ml. Results are mean+s.d. (n=2 assay repeats). Medium conditioned for 24 hours by primary human hepatocytes is included as a positive control.
Mentions: One of our main interests is in generation of HLCs, so we next tested whether 1m-induced DE was capable of differentiating into HLCs. The ability to generate HLCs provides an indication of the developmental potential of the chemically derived DE, where the ultimate aim is the generation of hepatocytes displaying functional characteristics. To evaluate this potential, we employed a differentiation protocol based on that of Agarwal (Agarwal et al., 2008) (Fig. 5A). Untreated hESCs did not survive the hepatic induction and maturation regimes. However, following hepatic induction of 1m-induced DE, stage II cells began to adopt morphological features characteristic of cells of the hepatic lineage (Fig. 5B) and expressed the genes encoding α-fetoprotein (AFP), albumin (ALB), transferrin (TFN; officially known as TF), transthyrettin (TTR) and α1 anti-trypsin (AAT; officially known as SERPINA1), correlating with initial hepatic differentiation (Fig. 5C). Following hepatic maturation, stage III cells exhibited characteristics of hepatocytes including a polygonal morphology, distinct round nuclei with 1 or 2 prominent nucleoli, and the presence of binucleate cells (Fig. 5B,D). Increased ALB and cytochrome P450 3A family (CYP3A) gene expression (Fig. 5C) and increased HNF4α, AFP and TTR protein expression (Fig. 5D) were observed in stage III compared with that in stage II cells. Equivalent amounts of RNA were used for the reverse transcription reaction that were then subjected to PCR; however, human liver expresses proportionally less β-actin-encoding mRNA than hESCs, which results in the apparent absence of β-actin in the human liver samples. To assess liver-specific function, secretion of the key serum proteins, AFP and albumin, was determined. Both AFP and albumin (Fig. 5E,F) were detected in the culture medium of stage III cells derived from 1m-induced DE. These results indicate that 1m-derived definitive endoderm has the potential to differentiate into cells with hepatocyte characteristics. Similar results were also generated using BIO-derived DE cells (supplementary material Fig. S6).

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