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Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency
Published on: April 22, 2011
Effects of oxytocin on cardiomyocyte differentiation from mouse embryonic stem cells
Leili Hatami1, Mojtaba Rezazadeh Valojerdi, Seyed Javad Mowla
1Department of Anatomy, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran.
Abstract:
This study sought to investigate the presence of oxytocin receptors and the possible biological role of oxytocin as an effective factor in the differentiation of embryonic stem cells (ESCs) into cardiomyocytes. Mouse ESCs were cultivated in hanging drops to form embryoid bodies (EBs). The EBs were then treated with and without oxytocin (experimental and control groups). Up to 30 days after plating, contraction and beating frequency were monitored and evaluated daily. The growth characteristics of the ESC-derived cardiomyocytes were assessed by cardioactive drugs, immunocytochemistry, transmission electron microscopy (TEM) and reverse transcription-polymerase chain reaction (RT-PCR). In the experimental group, the percentage of the EBs with spontaneous contraction was significantly increased from 17th day onward. The spontaneous beating frequency of each EB in both groups was also changed with cardioactive drugs such as Bay K, carbachol, isopernaline and phenylephrine. However, in the experimental group, changes with isopernaline were more pronounced at the early and intermediate stages of cardiomyocyte development. The beating cells of both groups, stained positive with anti alpha-actinin, desmin, cardiac troponin I and connexin antibodies, and revealed similar ultrastructural features. Oxytocin receptors were detected on the ESCs and derived-differentiated cells. In addition, cardiac-specific genes such as cardiac alpha- and beta-myosin heavy chain, myosin light chain-2v, and atrial natriuretic factor were also detected in the ESC-derived differentiated cells of both groups. In the experimental group, all the specific genes, with the exception of alpha-myosin heavy chain, were more pronounced at the early stage of cardiomyocyte development. In conclusion, oxytocin has receptors on undifferentiated ESCs and derived differentiated cells, and in spite of better improvement of the EBs with spontaneous contraction, it can only promote the early maturation of ESC-derived cardiomyocytes in terms of chronotropic responses and expression of cardiac-specific genes, and have no effect on ultrastructural characteristics of cardiomyocytes in any stage of development.
Insights
Oxytocin receptors are present on embryonic stem cells (ESCs). Oxytocin promotes early maturation of ESC-derived cardiomyocytes, enhancing spontaneous contraction and gene expression but not ultrastructure.
Area of Science:
- Cardiovascular Research
- Stem Cell Biology
- Developmental Biology
Background:
- Embryonic stem cells (ESCs) offer a promising source for generating cardiomyocytes for regenerative medicine.
- Understanding factors that influence ESC differentiation into cardiomyocytes is crucial for optimizing cell-based therapies.
- Oxytocin's role in cardiac development and function is increasingly recognized, necessitating investigation into its effects on ESC differentiation.
Purpose of the Study:
- To investigate the presence of oxytocin receptors on mouse ESCs and their differentiated progeny.
- To determine the biological role of oxytocin in promoting the differentiation of ESCs into functional cardiomyocytes.
- To assess the impact of oxytocin on the maturation, electrophysiological properties, and gene expression of ESC-derived cardiomyocytes.
Main Methods:
- Mouse ESCs were cultured as embryoid bodies (EBs) and treated with or without oxytocin.
- Contraction and beating frequency were monitored daily for 30 days.
- Cardioactive drugs, immunocytochemistry, transmission electron microscopy (TEM), and RT-PCR were used to assess cardiomyocyte characteristics and gene expression.
Main Results:
- Oxytocin treatment significantly increased the percentage of EBs exhibiting spontaneous contraction from day 17 onwards.
- Oxytocin enhanced the chronotropic responses to isopernaline in early and intermediate stages of cardiomyocyte development.
- Oxytocin receptors were detected on ESCs and differentiated cells; cardiac-specific genes were upregulated in oxytocin-treated groups, particularly at early developmental stages.
Conclusions:
- Oxytocin receptors are expressed on both undifferentiated ESCs and their differentiated cardiac cells.
- Oxytocin promotes early maturation of ESC-derived cardiomyocytes, evidenced by improved spontaneous contraction and enhanced expression of cardiac-specific genes.
- Oxytocin does not influence the ultrastructural characteristics of cardiomyocytes at any developmental stage.
