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FGF4 and retinoic acid direct differentiation of hESCs into PDX1-expressing foregut endoderm in a time- and concentration-dependent manner.

Johannesson M, Ståhlberg A, Ameri J, Sand FW, Norrman K, Semb H - PLoS ONE (2009)

Bottom Line: Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm.In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner.Part of RA's activity is mediated by FGF signaling.

View Article: PubMed Central - PubMed

Affiliation: Lund Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.

ABSTRACT

Background: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm.

Methodology/principal findings: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm.

Conclusion/significance: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.

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Overview of the different experiments leading up to conditions for obtaining PDX1-positive cells.(A) Medium compositions in the differentiation procotol. (B) Optimal concentration of fibroblast growth factor 4 (FGF4) on days 4–7 in the presence of retinoic acid (RA) on days 8–11. (C) mRNA expression of the retinoic acid receptor beta, RARβ, after activin (AA) induction. (D) The impact of FGF4 and RA on relative PDX1 gene expression (Rel. Expr.) and cell amount. Abbreviations and concentrations used: AA = Activin A (100 ng/mL), Fgf4 (1.1 ng/mL) where not stated otherwise, RA = Retinoic acid (2 µM), NT = no treatment after activin induction. (E) Immunofluorescence staining of Pdx1 using Pdx1-anti-goat (1∶1500) on day 13. Scale bars: E, 100 µm ; inset, 200 µm.
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pone-0004794-g001: Overview of the different experiments leading up to conditions for obtaining PDX1-positive cells.(A) Medium compositions in the differentiation procotol. (B) Optimal concentration of fibroblast growth factor 4 (FGF4) on days 4–7 in the presence of retinoic acid (RA) on days 8–11. (C) mRNA expression of the retinoic acid receptor beta, RARβ, after activin (AA) induction. (D) The impact of FGF4 and RA on relative PDX1 gene expression (Rel. Expr.) and cell amount. Abbreviations and concentrations used: AA = Activin A (100 ng/mL), Fgf4 (1.1 ng/mL) where not stated otherwise, RA = Retinoic acid (2 µM), NT = no treatment after activin induction. (E) Immunofluorescence staining of Pdx1 using Pdx1-anti-goat (1∶1500) on day 13. Scale bars: E, 100 µm ; inset, 200 µm.

Mentions: For differentiation experiments, Hues-3 (subclone 52) cells were seeded at a density of 20,000 cells/cm2 at passages 68–76, and cultured for three to four days until a confluent flat layer of undifferentiated cells was formed. Hues-15 cells were seeded at a density of 17,000 cells/cm2 at passage 23. At the start of each differentiation procedure at high confluence of the cells, phosphate buffered saline (PBS) (Gibco) was used to wash the cell layer once. The medium-composition during the differentiation experiments is described in Fig. 1A. Activin A (100 ng/mL) (R&D systems) and Wingless-type MMTV integration site family, member 3A (Wnt3a) (25 ng/mL) (R&D systems) was used to induce definitive endoderm (DE) in Rosewell Park Memorial Intitute (RPMI) 1640 (Gibco) supplemented with no fetal bovine serum (FBS) (Sigma) the first day and 0.2% FBS the second and third day. As a control for DE-induction, RPMI 1640 was used without addition of substances other than FBS. At day four, samples were taken for real-time polymerase chain reaction (PCR) analysis. On days four to seven, RPMI 1640 was supplemented with 2% FBS and from day eight, Dulbecco's modified Eagle medium (DMEM) (Gibco) was used supplemented with 2% FBS. From day four onward, Fibroblast growth factor 4 (FGF4) (R&D systems), and Retinoic acid (RA) (Sigma) were added in different combinations and concentrations as described. On various different time-points, cyclopamine (Sigma) was used in a concentration of 0.25 µM in order to inhibit shh. Penicillin/Streptomycin (PEST) (1%) was added to the differentiation medias. Non-treated (NT) cells did not get addition of substances other than DE-induction. For the D'Amour protocol, cells were treated as previously described [7]. When the D'Amour protocol was tested on cell line Hues-1, cells died at stage three representing the posterior foregut stage. However, with cell line Hues-3, a small number of PDX1+ cells was obtained at stage three. Still, cells did not survive further treatment onto stage four (pancreatic and endocrine precursors) or five (hormone expressing endocrine cells) (data not shown). Brightfield images of cells were taken on an inverted microscope (Eclipse TE2000-U) (Nicon).


FGF4 and retinoic acid direct differentiation of hESCs into PDX1-expressing foregut endoderm in a time- and concentration-dependent manner.

Johannesson M, Ståhlberg A, Ameri J, Sand FW, Norrman K, Semb H - PLoS ONE (2009)

Overview of the different experiments leading up to conditions for obtaining PDX1-positive cells.(A) Medium compositions in the differentiation procotol. (B) Optimal concentration of fibroblast growth factor 4 (FGF4) on days 4–7 in the presence of retinoic acid (RA) on days 8–11. (C) mRNA expression of the retinoic acid receptor beta, RARβ, after activin (AA) induction. (D) The impact of FGF4 and RA on relative PDX1 gene expression (Rel. Expr.) and cell amount. Abbreviations and concentrations used: AA = Activin A (100 ng/mL), Fgf4 (1.1 ng/mL) where not stated otherwise, RA = Retinoic acid (2 µM), NT = no treatment after activin induction. (E) Immunofluorescence staining of Pdx1 using Pdx1-anti-goat (1∶1500) on day 13. Scale bars: E, 100 µm ; inset, 200 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651644&req=5

pone-0004794-g001: Overview of the different experiments leading up to conditions for obtaining PDX1-positive cells.(A) Medium compositions in the differentiation procotol. (B) Optimal concentration of fibroblast growth factor 4 (FGF4) on days 4–7 in the presence of retinoic acid (RA) on days 8–11. (C) mRNA expression of the retinoic acid receptor beta, RARβ, after activin (AA) induction. (D) The impact of FGF4 and RA on relative PDX1 gene expression (Rel. Expr.) and cell amount. Abbreviations and concentrations used: AA = Activin A (100 ng/mL), Fgf4 (1.1 ng/mL) where not stated otherwise, RA = Retinoic acid (2 µM), NT = no treatment after activin induction. (E) Immunofluorescence staining of Pdx1 using Pdx1-anti-goat (1∶1500) on day 13. Scale bars: E, 100 µm ; inset, 200 µm.
Mentions: For differentiation experiments, Hues-3 (subclone 52) cells were seeded at a density of 20,000 cells/cm2 at passages 68–76, and cultured for three to four days until a confluent flat layer of undifferentiated cells was formed. Hues-15 cells were seeded at a density of 17,000 cells/cm2 at passage 23. At the start of each differentiation procedure at high confluence of the cells, phosphate buffered saline (PBS) (Gibco) was used to wash the cell layer once. The medium-composition during the differentiation experiments is described in Fig. 1A. Activin A (100 ng/mL) (R&D systems) and Wingless-type MMTV integration site family, member 3A (Wnt3a) (25 ng/mL) (R&D systems) was used to induce definitive endoderm (DE) in Rosewell Park Memorial Intitute (RPMI) 1640 (Gibco) supplemented with no fetal bovine serum (FBS) (Sigma) the first day and 0.2% FBS the second and third day. As a control for DE-induction, RPMI 1640 was used without addition of substances other than FBS. At day four, samples were taken for real-time polymerase chain reaction (PCR) analysis. On days four to seven, RPMI 1640 was supplemented with 2% FBS and from day eight, Dulbecco's modified Eagle medium (DMEM) (Gibco) was used supplemented with 2% FBS. From day four onward, Fibroblast growth factor 4 (FGF4) (R&D systems), and Retinoic acid (RA) (Sigma) were added in different combinations and concentrations as described. On various different time-points, cyclopamine (Sigma) was used in a concentration of 0.25 µM in order to inhibit shh. Penicillin/Streptomycin (PEST) (1%) was added to the differentiation medias. Non-treated (NT) cells did not get addition of substances other than DE-induction. For the D'Amour protocol, cells were treated as previously described [7]. When the D'Amour protocol was tested on cell line Hues-1, cells died at stage three representing the posterior foregut stage. However, with cell line Hues-3, a small number of PDX1+ cells was obtained at stage three. Still, cells did not survive further treatment onto stage four (pancreatic and endocrine precursors) or five (hormone expressing endocrine cells) (data not shown). Brightfield images of cells were taken on an inverted microscope (Eclipse TE2000-U) (Nicon).

Bottom Line: Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm.In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner.Part of RA's activity is mediated by FGF signaling.

View Article: PubMed Central - PubMed

Affiliation: Lund Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.

ABSTRACT

Background: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm.

Methodology/principal findings: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm.

Conclusion/significance: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.

Show MeSH
Related in: MedlinePlus