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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 the differentiation of ES cells into cardiac myocytes.(A) ES cells were grown in hanging drops for 2 days, in suspension with RA (5 nM) or bexarotene (BEX, 50 nM) for 5 days, and maintained on coverslips without any treatments for an additional 10 days. The cells were then harvested for RT-PCR analysis, or costained with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst for the nuclei (blue). (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the percentage of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 3). (D) Real-time RT-PCR was used to determine the transcript levels of Meox1 and Pax3 on day 7 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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f1: Bexarotene inhibits the differentiation of ES cells into cardiac myocytes.(A) ES cells were grown in hanging drops for 2 days, in suspension with RA (5 nM) or bexarotene (BEX, 50 nM) for 5 days, and maintained on coverslips without any treatments for an additional 10 days. The cells were then harvested for RT-PCR analysis, or costained with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst for the nuclei (blue). (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the percentage of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 3). (D) Real-time RT-PCR was used to determine the transcript levels of Meox1 and Pax3 on day 7 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).

Mentions: Since the early events of embryonic myogenesis is recapitulated closely by differentiation of ES cells into cardiac and skeletal muscle lineages2425, we used an embryoid body (EB) formation approach to study the impact of small molecules on myogenic conversion of ES cells. The ES cells were cultivated in the hanging drops for 2 days to form the EBs which were then cultured in suspension for 5 days and maintained as adhering culture for additional 10 days (Fig. 1A). For immunofluorescence microscopy analysis, the cells were co-stained for myosin heavy chain and MyoD to quantify the development of cardiac and skeletal myocytes. Developed cardiac and skeletal myocytes are distinguished by their staining pattern and morphological characteristics. Skeletal myocytes were recognized as elongated bipolar myocytes positively stained for myosin heavy chain and MyoD, while cardiac myocytes are rounded myosin heavy chain positive but MyoD negative cells.


Induction of Pax3 gene expression impedes cardiac differentiation.

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

Bexarotene inhibits the differentiation of ES cells into cardiac myocytes.(A) ES cells were grown in hanging drops for 2 days, in suspension with RA (5 nM) or bexarotene (BEX, 50 nM) for 5 days, and maintained on coverslips without any treatments for an additional 10 days. The cells were then harvested for RT-PCR analysis, or costained with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst for the nuclei (blue). (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the percentage of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 3). (D) Real-time RT-PCR was used to determine the transcript levels of Meox1 and Pax3 on day 7 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

License
Show All Figures
getmorefigures.php?uid=PMC3750538&req=5

f1: Bexarotene inhibits the differentiation of ES cells into cardiac myocytes.(A) ES cells were grown in hanging drops for 2 days, in suspension with RA (5 nM) or bexarotene (BEX, 50 nM) for 5 days, and maintained on coverslips without any treatments for an additional 10 days. The cells were then harvested for RT-PCR analysis, or costained with specific antibodies for microscopic analysis of MyoD (red), myosin heavy chain (MyHC, green), and with Hoechst for the nuclei (blue). (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the percentage of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 3). (D) Real-time RT-PCR was used to determine the transcript levels of Meox1 and Pax3 on day 7 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
Mentions: Since the early events of embryonic myogenesis is recapitulated closely by differentiation of ES cells into cardiac and skeletal muscle lineages2425, we used an embryoid body (EB) formation approach to study the impact of small molecules on myogenic conversion of ES cells. The ES cells were cultivated in the hanging drops for 2 days to form the EBs which were then cultured in suspension for 5 days and maintained as adhering culture for additional 10 days (Fig. 1A). For immunofluorescence microscopy analysis, the cells were co-stained for myosin heavy chain and MyoD to quantify the development of cardiac and skeletal myocytes. Developed cardiac and skeletal myocytes are distinguished by their staining pattern and morphological characteristics. Skeletal myocytes were recognized as elongated bipolar myocytes positively stained for myosin heavy chain and MyoD, while cardiac myocytes are rounded myosin heavy chain positive but MyoD negative cells.

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