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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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Gene expression analysis of gut endodermal markers at day 16 using the FGF4/RA differentiation protocol.(A) The FGF4/RA differentiation protocol. FBS = fetal bovine serum. Activin = Activin A 100 ng/mL, Wnt3a (25 ng/mL). (B) Relative mRNA expression of PDX1 in RA/FGF4-treated hESC (Day 16) and human islets (C) Relative mRNA expression of PDX1, FOXA2, HNF6, SOX9, and CDX2 at day 12 and 16 with or without (NT = no treatment) addition of RA and Fgf4 (F4) after AA-induction. In these experiments cell line Hues-3 (subclone 52) was used. In Experiment 1, NT day 16 is missing.
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pone-0004794-g003: Gene expression analysis of gut endodermal markers at day 16 using the FGF4/RA differentiation protocol.(A) The FGF4/RA differentiation protocol. FBS = fetal bovine serum. Activin = Activin A 100 ng/mL, Wnt3a (25 ng/mL). (B) Relative mRNA expression of PDX1 in RA/FGF4-treated hESC (Day 16) and human islets (C) Relative mRNA expression of PDX1, FOXA2, HNF6, SOX9, and CDX2 at day 12 and 16 with or without (NT = no treatment) addition of RA and Fgf4 (F4) after AA-induction. In these experiments cell line Hues-3 (subclone 52) was used. In Experiment 1, NT day 16 is missing.

Mentions: To test the reproducibility of the combined action of RA and FGF4 to direct differentiation of PDX1-expressing cells, we repeated our protocol (Fig. 3A) three times using cell line Hues-3 (subclone 52) at different passages. More specifically, passage 68, 75 and 76 were used. In order to get some relevant estimation of the magnitude of PDX1-expression in differentiated hESC at day 16, the expression was compared to PDX1-expression in human islets. PDX1-mRNA levels in differentiated hESC were approximately 50% of the levels detected in human islets (Fig. 3B). In order to analyze the real-time PCR data, the lowest value of each data set was set to one and all other values were related to this. Following this procedure, a mean value was calculated for each of the duplicate or triplicate samples. In some cases, the non-treated cells did not have any measurable level of PDX1-transcripts (Fig. 3C; Experiments 1 and 2) and consequently Ct-values were set to 45. Moreover, to further establish the robustness of this protocol, its cell line specificity was tested. For this purpose, the RA/FGF4 protocol was tested on another hESC line: Hues-15 (Fig. S2). Indeed, RA/FGF4 effectively induced PDX1 expression in Hues-15 (Fig. S2B). Thus, the fact that RA and FGF4 significantly increased PDX1 mRNA expression in Hues-15 and Hues-3 subclone 52, suggests that the ability of these factors to direct differentiation of AA-induced hESC into PDX1+ cells is cell line independent.


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)

Gene expression analysis of gut endodermal markers at day 16 using the FGF4/RA differentiation protocol.(A) The FGF4/RA differentiation protocol. FBS = fetal bovine serum. Activin = Activin A 100 ng/mL, Wnt3a (25 ng/mL). (B) Relative mRNA expression of PDX1 in RA/FGF4-treated hESC (Day 16) and human islets (C) Relative mRNA expression of PDX1, FOXA2, HNF6, SOX9, and CDX2 at day 12 and 16 with or without (NT = no treatment) addition of RA and Fgf4 (F4) after AA-induction. In these experiments cell line Hues-3 (subclone 52) was used. In Experiment 1, NT day 16 is missing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651644&req=5

pone-0004794-g003: Gene expression analysis of gut endodermal markers at day 16 using the FGF4/RA differentiation protocol.(A) The FGF4/RA differentiation protocol. FBS = fetal bovine serum. Activin = Activin A 100 ng/mL, Wnt3a (25 ng/mL). (B) Relative mRNA expression of PDX1 in RA/FGF4-treated hESC (Day 16) and human islets (C) Relative mRNA expression of PDX1, FOXA2, HNF6, SOX9, and CDX2 at day 12 and 16 with or without (NT = no treatment) addition of RA and Fgf4 (F4) after AA-induction. In these experiments cell line Hues-3 (subclone 52) was used. In Experiment 1, NT day 16 is missing.
Mentions: To test the reproducibility of the combined action of RA and FGF4 to direct differentiation of PDX1-expressing cells, we repeated our protocol (Fig. 3A) three times using cell line Hues-3 (subclone 52) at different passages. More specifically, passage 68, 75 and 76 were used. In order to get some relevant estimation of the magnitude of PDX1-expression in differentiated hESC at day 16, the expression was compared to PDX1-expression in human islets. PDX1-mRNA levels in differentiated hESC were approximately 50% of the levels detected in human islets (Fig. 3B). In order to analyze the real-time PCR data, the lowest value of each data set was set to one and all other values were related to this. Following this procedure, a mean value was calculated for each of the duplicate or triplicate samples. In some cases, the non-treated cells did not have any measurable level of PDX1-transcripts (Fig. 3C; Experiments 1 and 2) and consequently Ct-values were set to 45. Moreover, to further establish the robustness of this protocol, its cell line specificity was tested. For this purpose, the RA/FGF4 protocol was tested on another hESC line: Hues-15 (Fig. S2). Indeed, RA/FGF4 effectively induced PDX1 expression in Hues-15 (Fig. S2B). Thus, the fact that RA and FGF4 significantly increased PDX1 mRNA expression in Hues-15 and Hues-3 subclone 52, suggests that the ability of these factors to direct differentiation of AA-induced hESC into PDX1+ cells is cell line independent.

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