doi:10.1016/j.bbrc.2005.05.202 
Copyright © 2005 Elsevier Inc. All rights reserved.
RXR agonist enhances the differentiation of cardiomyocytes derived from embryonic stem cells in serum-free conditions
Masahiko Hondaa, f, 1, Tatsuo S. Hamazakib, f, 
, 1, 
, Shinji Komazakic, Hiroyuki Kagechikad, Koichi Shudoe and Makoto Asashimaf, g, ,
aDepartment of Biological Science, Graduate School of Sciences, The University of Tokyo, Tokyo, Japan
bDepartment of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan
cDepartment of Anatomy, Saitama Medical School, Saitama, Japan
dLaboratory of Organic and Medicinal Chemistry, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan
eResearch Foundation ITSUU Laboratory, Tokyo, Japan
fDepartment of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, Japan
gICORP/Japan Science and Technology Corporation (JST), Japan
Received 26 May 2005.
Available online 20 June 2005.
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Abstract
Signaling from the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) is essential for cardiovascular morphogenesis in vivo. RAR and/or RXR signaling can also enhance the in vitro induction of cardiomyocytes from murine embryonic stem (ES) cells in the presence of serum. The present study examined the effect of RXR agonist that was specifically bound to RXRs on the differentiation of mouse ES cells into cardiomyocytes in vitro in the absence of serum. The number of beating embryoid body-like spheres (EBSs) derived from the ES cells increased significantly following treatment with PA024, an RXR agonist. In contrast, when EBSs were treated with PA452, which was specifically bound to RXR and worked as an antagonist, the number of beating EBSs was decreased in a dose-dependent manner. These results suggest that RXR signaling regulates cardiomyocyte numbers during the differentiation of ES cells in vitro and probably in normal development.
Keywords: ES cells; Cardiomyocytes; RXR agonist; RXR antagonist; PA024; PA452
Fig. 1. Scheme of the inductive steps in this procedure. (A) The colonies of ES cells were directly isolated and cultured for 3 days in the absence of the serum. Then, they were seeded onto gelatin-coated plates, cultured for a further 2 days, and then treated with retinoid derivatives for 2 days. (B) Formulas of PA024 and PA452 used in the present experiment.
Fig. 2. Efficiency of cardiomyocyte differentiation in EBSs. (A) Each treatment revealed that the percentage of EBSs that contained the beating cardiomyocytes was highest when EBSs were treated with 5 μM of PA024 (RXR agonist). Two and 10 μM of PA024 showed a less pronounced effect on the increment of cardiomyocytes. No significant differences were observed among the EBSs treated with ATRA (5 μM), DMSO (0.02%), and non-treated EBSs. Other RAR agonists, Am80 and Ch55, which specifically bind to RAR, also showed no effect when used in the concentration range of 0.1–10 μM (data not shown). (B) One day of PA024 treatment induced EBSs containing spontaneously beating areas after 6 days in culture. In contrast, the beating area was not observed until day 7 in the control EBSs treated with DMSO. PA024 treatment facilitated and elevated the number of cardiomyocytes (*P < 0.05).
Fig. 3. Immunocytochemical detection of ES cell-derived cardiomyocytes. EBSs were cultured for 10 days (day 10) and stained with each cardiomyocyte-specific antibody. The transcription factors, Nkx 2.5 (B), GATA-4 (D), and MEF2 (F), were observed in beating cell aggregates following treatment with PA024. Phase contrast pictures corresponding to (B), (D), and (F) are shown in (A), (C), and (E), respectively. Structural proteins, cardiac troponin I (G) and cardiac troponin T (H), were also expressed.
Fig. 4. Ultrastructure of PA024-treated ES cell-derived cardiomyocytes. (A) Transmission electron microscopy shows well-developed myofibrils (arrowheads). (B,C) Between adjacent cells, intercalated disks consisting of gap junctions (black arrowhead) and desmosomes (white arrowhead) were also observed. N, nucleus; M, mitochondrion (Scale bar = 1 μm).
Fig. 5. Gene expression analysis and the effect of an RXR antagonist on the number of EBSs containing cardiomyocytes. (A) RT-PCR analysis revealed that both early cardiomyocyte-marker genes, MLC-2a and MLC-2v, were expressed in the PA024-treated EBSs. Other marker genes, α-MHC, β-MHC, and α-cardiac actin, were also detected. All RXR subtypes were expressed in day 6 EBSs. (B) When the EBSs were treated with the RXR antagonist, PA452, the number of EBSs containing spontaneously beating cardiomyocyte decreased in a dose-dependent manner. (*P < 0.05, compared to the control of DMSO treatment.)
Abstract
Purchase PDF (885 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (7)
Purchase PDF (460 K)
