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Induction of Pax3 gene expression impedes cardiac differentiation.

Li Q, Le May M, Lacroix N, Chen J - Sci Rep (2013)

Bottom Line: Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1.Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification.Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada. qiaoli@uottawa.ca

ABSTRACT
Cell-based therapies using pluripotent stem cells hold great promise as regenerative approaches to treat many types of diseases. Nevertheless many challenges remain and, perhaps foremost, is the issue of how to direct and enhance the specification and differentiation of a desired cell type for potential therapeutics. We have examined the molecular basis for the inverse correlation of cardiac and skeletal myogenesis in small molecule-enhanced stem cell differentiation. Our study shows that activation of premyogenic factor Pax3 coincides with inhibiting gene expression of early cardiac factor GATA4. Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1. Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification. Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

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Bexarotene inhibits cardiac differentiation of P19 pluripotent stem cells.(A) P19 cells were differentiated with DMSO in the absence or presence of increasing concentrations of bexarotene (BEX, 10 nM, 100 nM, 1 μM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and costained with specific antibodies for MyoD (red), myosin heavy chain (MyHC, green) and Hoechst for the nuclei (blue). The cells were also harvested at different time points for RT-PCR and Western analyses. (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the fractions of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (D) Gene expression of myogenin and GATA4 and protein on day 4 and day 9 of differentiation was determined by Western analysis in parallel with the use of the same batch of cell extracts. The blots were then stripped and reprobed for β-tubulin as loading controls. Shown are the cropped blot images representing indicated protein. (E) and (F) Real-time RT-PCR was used to determine the transcripts levels of Pax3 and Meox1 on day 4 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
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f2: Bexarotene inhibits cardiac differentiation of P19 pluripotent stem cells.(A) P19 cells were differentiated with DMSO in the absence or presence of increasing concentrations of bexarotene (BEX, 10 nM, 100 nM, 1 μM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and costained with specific antibodies for MyoD (red), myosin heavy chain (MyHC, green) and Hoechst for the nuclei (blue). The cells were also harvested at different time points for RT-PCR and Western analyses. (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the fractions of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (D) Gene expression of myogenin and GATA4 and protein on day 4 and day 9 of differentiation was determined by Western analysis in parallel with the use of the same batch of cell extracts. The blots were then stripped and reprobed for β-tubulin as loading controls. Shown are the cropped blot images representing indicated protein. (E) and (F) Real-time RT-PCR was used to determine the transcripts levels of Pax3 and Meox1 on day 4 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).

Mentions: Pluripotent P19 stem cells can be directed into differentiation to form cell lineages of all three germ layers2728. They have served as a valuable model system to study myogenesis and to identify small molecule inducers for lineage specification2628. Specifically, the differentiation of P19 cells into cardiac and skeletal myocytes reflects the cellular and molecular processes occurring in early embryogenesis and ES cell differentiation2629. In tissue cultures, the P19 cells can be induced into differentiation with an aggregation protocol that involves the formation of early embryo-like cell aggregates also known as EBs (Fig. 2A). Formation of the EBs in the absence of exogenous stimuli results in the expression of markers of mesoderm, but not of cardiac or skeletal myocytes30. Cells treated with DMSO differentiate into small percentages of cardiac and skeletal myocytes along with other mesodermal and endodermal cell types31.


Induction of Pax3 gene expression impedes cardiac differentiation.

Li Q, Le May M, Lacroix N, Chen J - Sci Rep (2013)

Bexarotene inhibits cardiac differentiation of P19 pluripotent stem cells.(A) P19 cells were differentiated with DMSO in the absence or presence of increasing concentrations of bexarotene (BEX, 10 nM, 100 nM, 1 μM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and costained with specific antibodies for MyoD (red), myosin heavy chain (MyHC, green) and Hoechst for the nuclei (blue). The cells were also harvested at different time points for RT-PCR and Western analyses. (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the fractions of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (D) Gene expression of myogenin and GATA4 and protein on day 4 and day 9 of differentiation was determined by Western analysis in parallel with the use of the same batch of cell extracts. The blots were then stripped and reprobed for β-tubulin as loading controls. Shown are the cropped blot images representing indicated protein. (E) and (F) Real-time RT-PCR was used to determine the transcripts levels of Pax3 and Meox1 on day 4 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750538&req=5

f2: Bexarotene inhibits cardiac differentiation of P19 pluripotent stem cells.(A) P19 cells were differentiated with DMSO in the absence or presence of increasing concentrations of bexarotene (BEX, 10 nM, 100 nM, 1 μM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and costained with specific antibodies for MyoD (red), myosin heavy chain (MyHC, green) and Hoechst for the nuclei (blue). The cells were also harvested at different time points for RT-PCR and Western analyses. (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the fractions of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (D) Gene expression of myogenin and GATA4 and protein on day 4 and day 9 of differentiation was determined by Western analysis in parallel with the use of the same batch of cell extracts. The blots were then stripped and reprobed for β-tubulin as loading controls. Shown are the cropped blot images representing indicated protein. (E) and (F) Real-time RT-PCR was used to determine the transcripts levels of Pax3 and Meox1 on day 4 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
Mentions: Pluripotent P19 stem cells can be directed into differentiation to form cell lineages of all three germ layers2728. They have served as a valuable model system to study myogenesis and to identify small molecule inducers for lineage specification2628. Specifically, the differentiation of P19 cells into cardiac and skeletal myocytes reflects the cellular and molecular processes occurring in early embryogenesis and ES cell differentiation2629. In tissue cultures, the P19 cells can be induced into differentiation with an aggregation protocol that involves the formation of early embryo-like cell aggregates also known as EBs (Fig. 2A). Formation of the EBs in the absence of exogenous stimuli results in the expression of markers of mesoderm, but not of cardiac or skeletal myocytes30. Cells treated with DMSO differentiate into small percentages of cardiac and skeletal myocytes along with other mesodermal and endodermal cell types31.

Bottom Line: Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1.Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification.Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada. qiaoli@uottawa.ca

ABSTRACT
Cell-based therapies using pluripotent stem cells hold great promise as regenerative approaches to treat many types of diseases. Nevertheless many challenges remain and, perhaps foremost, is the issue of how to direct and enhance the specification and differentiation of a desired cell type for potential therapeutics. We have examined the molecular basis for the inverse correlation of cardiac and skeletal myogenesis in small molecule-enhanced stem cell differentiation. Our study shows that activation of premyogenic factor Pax3 coincides with inhibiting gene expression of early cardiac factor GATA4. Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1. Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification. Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

Show MeSH
Related in: MedlinePlus