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PAX4 enhances beta-cell differentiation of human embryonic stem cells.

Liew CG, Shah NN, Briston SJ, Shepherd RM, Khoo CP, Dunne MJ, Moore HD, Cosgrove KE, Andrews PW - PLoS ONE (2008)

Bottom Line: Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green.Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells.Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, United Kingdom.

ABSTRACT

Background: Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of beta-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages.

Methods and findings: Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+) microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+) homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green.

Conclusion: Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.

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The effect of Pax4 expression upon EB differentiation of HESC.(A) RT-PCR analysis of gene expression in EBs produced from untransfected H7 cells and from two independent clones transfected with Pax4 during a 16 day in vitro differentiation. (B) Q-PCR analysis of Ins and Pdx1 expression in EBs produced from untransfected H7 and H7.Px4 cells. These experiments were performed on two independent H7.Px4 clones and control data came from 3 independent experiments using untransfected H7 cells. Data were collected on Day 0 and at 7 (Early), 14 (Mid) and 21 (Late) days following EB formation. Gene expression levels were calculated as 2−ΔΔCt values, relative to day 7 (Early) samples. Error bars represent standard deviation, and an asterisk denotes p<0.05. (C) Differentiation of H7.Px4 EBs was also associated with the up-regulated expression of gene encoding enzyme prohormone convertase 1/3 (PC1/3).
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pone-0001783-g002: The effect of Pax4 expression upon EB differentiation of HESC.(A) RT-PCR analysis of gene expression in EBs produced from untransfected H7 cells and from two independent clones transfected with Pax4 during a 16 day in vitro differentiation. (B) Q-PCR analysis of Ins and Pdx1 expression in EBs produced from untransfected H7 and H7.Px4 cells. These experiments were performed on two independent H7.Px4 clones and control data came from 3 independent experiments using untransfected H7 cells. Data were collected on Day 0 and at 7 (Early), 14 (Mid) and 21 (Late) days following EB formation. Gene expression levels were calculated as 2−ΔΔCt values, relative to day 7 (Early) samples. Error bars represent standard deviation, and an asterisk denotes p<0.05. (C) Differentiation of H7.Px4 EBs was also associated with the up-regulated expression of gene encoding enzyme prohormone convertase 1/3 (PC1/3).

Mentions: To determine the behaviour of H7.Px4 cells during their growth as EBs, we next examined the expression of a panel of cell-specific genes and proteins during EB differentiation (Figure 2A). In untransfected and H7.Px4 cells, the expression of Oct4 was down-regulated during EB growth over a 16 day period, though significant expression of Oct4 was retained, and indeed increased at later time points in the untransfected cells. This retention of Oct4 in EBs has been noted in other studies [28] and may reflect the persistence of undifferentiated cells and/or the appearance of cells that also express Oct4, such as those of the germ line [29]. In the H7.Px4 cells, there was a greater down-regulation of Oct4, and delay in disappearance of Sox2, which is expressed by cells of the neural lineage [30]. In addition, we observed strong expression of the endodermal transcription factor forkhead box A2 (Foxa2) in the EBs, indicating HESC could efficiently contribute to definitive endoderm germ layer during spontaneous differentiation [12], Supplementary Information, Figure S2.


PAX4 enhances beta-cell differentiation of human embryonic stem cells.

Liew CG, Shah NN, Briston SJ, Shepherd RM, Khoo CP, Dunne MJ, Moore HD, Cosgrove KE, Andrews PW - PLoS ONE (2008)

The effect of Pax4 expression upon EB differentiation of HESC.(A) RT-PCR analysis of gene expression in EBs produced from untransfected H7 cells and from two independent clones transfected with Pax4 during a 16 day in vitro differentiation. (B) Q-PCR analysis of Ins and Pdx1 expression in EBs produced from untransfected H7 and H7.Px4 cells. These experiments were performed on two independent H7.Px4 clones and control data came from 3 independent experiments using untransfected H7 cells. Data were collected on Day 0 and at 7 (Early), 14 (Mid) and 21 (Late) days following EB formation. Gene expression levels were calculated as 2−ΔΔCt values, relative to day 7 (Early) samples. Error bars represent standard deviation, and an asterisk denotes p<0.05. (C) Differentiation of H7.Px4 EBs was also associated with the up-regulated expression of gene encoding enzyme prohormone convertase 1/3 (PC1/3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2262135&req=5

pone-0001783-g002: The effect of Pax4 expression upon EB differentiation of HESC.(A) RT-PCR analysis of gene expression in EBs produced from untransfected H7 cells and from two independent clones transfected with Pax4 during a 16 day in vitro differentiation. (B) Q-PCR analysis of Ins and Pdx1 expression in EBs produced from untransfected H7 and H7.Px4 cells. These experiments were performed on two independent H7.Px4 clones and control data came from 3 independent experiments using untransfected H7 cells. Data were collected on Day 0 and at 7 (Early), 14 (Mid) and 21 (Late) days following EB formation. Gene expression levels were calculated as 2−ΔΔCt values, relative to day 7 (Early) samples. Error bars represent standard deviation, and an asterisk denotes p<0.05. (C) Differentiation of H7.Px4 EBs was also associated with the up-regulated expression of gene encoding enzyme prohormone convertase 1/3 (PC1/3).
Mentions: To determine the behaviour of H7.Px4 cells during their growth as EBs, we next examined the expression of a panel of cell-specific genes and proteins during EB differentiation (Figure 2A). In untransfected and H7.Px4 cells, the expression of Oct4 was down-regulated during EB growth over a 16 day period, though significant expression of Oct4 was retained, and indeed increased at later time points in the untransfected cells. This retention of Oct4 in EBs has been noted in other studies [28] and may reflect the persistence of undifferentiated cells and/or the appearance of cells that also express Oct4, such as those of the germ line [29]. In the H7.Px4 cells, there was a greater down-regulation of Oct4, and delay in disappearance of Sox2, which is expressed by cells of the neural lineage [30]. In addition, we observed strong expression of the endodermal transcription factor forkhead box A2 (Foxa2) in the EBs, indicating HESC could efficiently contribute to definitive endoderm germ layer during spontaneous differentiation [12], Supplementary Information, Figure S2.

Bottom Line: Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green.Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells.Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, United Kingdom.

ABSTRACT

Background: Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of beta-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages.

Methods and findings: Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+) microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+) homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green.

Conclusion: Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.

Show MeSH
Related in: MedlinePlus