Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

Identification of factors that direct embryonic stem (ES) cell (ESC) differentiation into functional cardiomyocytes is essential for successful use of ESC-based therapy for cardiac repair. Neuregulin-1 (NRG1) and microRNA play important roles in the cardiac differentiation of ESCs. Understanding how NRG1 regulates microRNA will provide new mechanistic insights into the role of NRG1 on ESCs. It may also lead to the discovery of novel microRNAs that are important for ESC cardiac differentiation. The objective of this study was to assess the microRNA expression profile during NRG1-induced ESC cardiac differentiation. Murine ESCs were incubated with a recombinant NRG1β or an inhibitor of ErbB2 or ErbB4 during hanging drop-induced cardiac differentiation. The expression of cardiac-specific markers and microRNAs was analyzed by RT-PCR and microRNA array, respectively. We found that the expression of NRG1 and the ErbB receptors was increased during hanging drop-induced cardiac differentiation of ESCs. NRG1 stimulation during a specific developmental window enhanced, while inhibition of the ErbB2 or ErbB4 receptor inhibited, cardiac differentiation of ESCs. NRG1 increased the expression of mmu-miR-296–3p and mmu-miR-200c*, and decreased mmu-miR-465b-5p. Inhibition of mmu-miR-296–3p or mmu-miR-200c* decreased, while inhibition of mmu-miR-465–5p increased, the differentiation of ESCs into the cardiac lineage. This is the first report demonstrating that microRNAs are differentially regulated by NRG1-ErbB signaling during cardiac differentiation of ESCs. This study has also identified new microRNAs that are important for ESC cardiac differentiation.

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Paving new paths for neuregulin-1-assisted cardiac regenerative medicine. Focus on “Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA”

AJP Cell Physiology ◽

10.1152/ajpcell.00137.2011 ◽

2011 ◽

Vol 301 (1) ◽

pp. C16-C17 ◽

Cited By ~ 4

Author(s):

Katrien Lemmens ◽

Gilles W. De Keulenaer

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Regenerative Medicine ◽

Embryonic Stem Cell ◽

Differential Expression ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryonic Stem Cell Differentiation ◽

Neuregulin 1 ◽

Murine Embryonic Stem Cell

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Abstract 1463: Neuregulin-1 Promotes Embryonic Stem Cell Differentiation into the Cardiac Lineage in vitro

Circulation ◽

10.1161/circ.118.suppl_18.s_330-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Maoyun Sun ◽

Yun Bian ◽

Anthony O. Caggiano ◽

Xinhua Yan ◽

James P. Morgan

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cardiac Troponin ◽

Mrna Level ◽

Embryonic Stem ◽

Erbb Receptors ◽

Hanging Drop ◽

Neuregulin 1 ◽

Cardiac Lineage

Our previous studies demonstrated that transplantation of embryonic stem cell (ESCs) derived cardiomyocytes improved the cardiac function in myocardial infarction injured mice. However, significant challenges remain to be solved, such as how to produce sufficient amount and mature differentiated cardiomyocytes for the therapy. Neuregulin-1 (NRG-1) is essential for the development of the heart. Here, we tested whether NRG-1 could promote the differentiation of ESCs into cardiomyocytes. Differentiation of murine ESCs was induced by the hanging-drop method. NRG-1 was added at different points of ESC differentiation. The expression of cardiac specific genes, as well as NRG-1 and its erbB receptors were measured by quantitative RT-PCR and Western blot analysis. Our results showed that during the course of hanging-drop induced ESC differentiation, the mRNA level of cardiac-specific structure proteins cardiac troponin T (cTNT), cardiac troponin I (cTNI), myosin light chain 2a (MLC2a) and alpha-sarcomeric actin was elevated. The mRNA level of cardiac transcription factors NKX2.5 and GATA4 was also increased. In addition, beating EBs first appeared on day7 and about 90% of beating EBs was observed on day12. Further, the mRNA level of NRG-1α and NRG-1β, and both mRNA and protein levels of erbB receptors were elevated during the differentiation of ESCs. When NRG-1 was added during a certain developmental window, the mRNA level of cTNT, MLC2a and MEF2c, as well as the protein level of cTNT, CXN40, a marker for cardiac conduction system, and transcriptional factors NKX2.5, GATA4 were increased in NRG-1 treated ESCs compared to untreated cells. These results demonstrated that NRG-erbB signaling was activated during the course of hang-drop induced ESC differentiation; NRG-1 treatment further promoted ESC differentiation into the cardiac lineage in vitro. Our results suggest that NRG-1 might be used to produce more and/or more mature ESC derived cardiomyocytes for the treatment of cardiovascular diseases in the clinical setting.

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