Abstract
BackgroundBabies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified.MethodWe investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells.ResultsGlucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca2+ wave and contraction.ConclusionsOur findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0446-5) contains supplementary material, which is available to authorized users.
Highlights
Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD)
Our findings suggest that high glucose inhibits Embryonic stem cells (ESCs) cardiogenesis by suppressing key developmental genes essential for the cardiac program
E14 cells adapted to physiological glucose concentrations retain stemness and have a high expression of glucose transporter 2 Like most embryonic stem (ES) cell lines, the E14 cell is maintained under high glucose conditions (25 mM glucose)
Summary
Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. Infants from pregestational diabetic mothers have a greater risk of structural birth defects including congenital heart defects (CHDs) than those from nondiabetic mothers [2]. The ES differentiation process to cardiomyocytes is a unique in vitro model for studying the effect of high glucose on early cardiac lineage specification and cardiomyocyte maturation. Using the embryoid body (EB) hanging-drop method, a low number of contracting cardiomyocytes can be generated in regular medium without small molecules and kinase inhibitor treatment. We used this method in our experiment
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