Cardiomyocytes can be generated from marrow stromal cells in vitro
Shinji Makino, … , Akihiro Umezawa, Satoshi Ogawa
Shinji Makino, … , Akihiro Umezawa, Satoshi Ogawa
Published March 1, 1999
Citation Information: J Clin Invest. 1999;103(5):697-705. https://doi.org/10.1172/JCI5298.
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Article

Cardiomyocytes can be generated from marrow stromal cells in vitro

Abstract

We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in ∼30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node–like and ventricular cell–like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and α-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

Authors

Shinji Makino, Keiichi Fukuda, Shunichirou Miyoshi, Fusako Konishi, Hiroaki Kodama, Jing Pan, Motoaki Sano, Toshiyuki Takahashi, Shingo Hori, Hitoshi Abe, Jun-ichi Hata, Akihiro Umezawa, Satoshi Ogawa

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Expression of cardiomyocyte-specific genes in and phenotype analysis of ...
Expression of cardiomyocyte-specific genes in and phenotype analysis of CMG cells. (a) reverse transcriptase (RT)-PCR Southern analysis of natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in CMG cells. Total RNA was isolated from mouse heart (H), skeletal muscle (Sk), and differentiated CMG cells. After DNase I treatment, RT-PCR was performed, as described in Methods, for ANP and BNP. Each PCR product was identified by Southern blot using 32P-labeled synthetic oligonucleotides. Both ANP and BNP are specifically expressed in cardiac muscle and CMG myotubes. (b) RT-PCR analysis of α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC), α-cardiac actin, and α-skeletal actin expression in CMG cells. Heart (H) and liver (L) were used as positive and negative controls. M represents ΦXHaeIII molecular size marker. CMG myotubes expressed both α-cardiac actin and α-skeletal actin, but the expression of α-skeletal actin was much stronger than that of α-cardiac actin. Note that α-skeletal actin expression was observed before the final 5-azacytidine treatment, although expression was weak. CMG myotubes expressed both α- and β-MHC, but the expression of β-MHC was much stronger than that of α-MHC. (c) Northern blot analysis of α-cardiac actin and α-skeletal actin expression in CMG cells. Adult heart (H) was used as a positive control. α-cardiac actin was more abundantly expressed in adult heart. On the other hand, α-skeletal actin was more abundantly expressed in CMG cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading internal control. (d) RT-PCR analysis of MLC-2a and -2v expression in CMG cells. Adult atrial muscle (A) was used as a positive control for MLC-2a, and adult ventricular muscle (V) was used as a positive control for MLC-2v. M represents ΦXHaeIII molecular size marker. CMG myotubes expressed MLC-2v, but not MLC-2a. These patterns of gene expression in the CMG myotubes corresponded to the phenotype specific to fetal ventricular cardiomyocytes.