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Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle.

Matsuura K, Wada H, Nagai T, Iijima Y, Minamino T, Sano M, Akazawa H, Molkentin JD, Kasanuki H, Komuro I - J. Cell Biol. (2004)

Bottom Line: Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes.Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo.In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

ABSTRACT
The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.

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Cardiomyocytes fused with adult immature somatic cells in vitro. (A) LacZ-expressing cardiomyocytes of neonatal rats were cocultured with GFP+ bone marrow mesenchymal cells. After 4 d of coculture, cells were stained with mouse monoclonal anti-cTnT (red) and rabbit polyclonal anti-β-gal antibodies (blue). Merged image was obtained from the same confocal plane. GFP+ mesenchymal cells (a, arrow) expressed cTnT (b, arrow) and β-gal (c, arrow) in the same cell (merged on d). Arrowheads indicate bone marrow cells fused with noncardiomyocyte. Bars, 50 μm. (B) Fluorescent microscopic images of EPCs cultured 7 d after isolation from peripheral blood. EPCs were identified as double-positive cells of DiI-labeled AcLDL uptake (a, red) and UEA-1 lectin reactivity (b, yellow in merged images). Some adherent cells expressed vWF (c, green). Nuclei were stained with Hoechst 33258 (c, blue). Bars, 50 μm. (C) Human-derived EPCs were cocultured with neonatal mouse cardiomyocytes infected with LacZ adenovirus. After 4 d of coculture, cells were stained with rabbit polyclonal anti-vWF (green), goat polyclonal anti-cTnT (red), and mouse monoclonal anti-β-gal antibodies (blue in top row) and Hoechst 33258 (bottom row, blue). The fluorescent confocal microscopic images (a–d, top row) demonstrate that vWF-expressing cells (a) expressed cTnT (b) and β-gal (c) in the same cell. Note that Cy5-conjugated secondary antibodies were used to visualize β-gal. The images of the same cell were taken by fluorescent microscope (e–h, bottom row). Hoechst staining of the nuclei revealed that homogenously stained nuclei (arrow) were of human cell origin and that mouse nuclei showed a punctate appearance (arrowhead). d and h represent merged images. Bars, 50 μm.
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fig9: Cardiomyocytes fused with adult immature somatic cells in vitro. (A) LacZ-expressing cardiomyocytes of neonatal rats were cocultured with GFP+ bone marrow mesenchymal cells. After 4 d of coculture, cells were stained with mouse monoclonal anti-cTnT (red) and rabbit polyclonal anti-β-gal antibodies (blue). Merged image was obtained from the same confocal plane. GFP+ mesenchymal cells (a, arrow) expressed cTnT (b, arrow) and β-gal (c, arrow) in the same cell (merged on d). Arrowheads indicate bone marrow cells fused with noncardiomyocyte. Bars, 50 μm. (B) Fluorescent microscopic images of EPCs cultured 7 d after isolation from peripheral blood. EPCs were identified as double-positive cells of DiI-labeled AcLDL uptake (a, red) and UEA-1 lectin reactivity (b, yellow in merged images). Some adherent cells expressed vWF (c, green). Nuclei were stained with Hoechst 33258 (c, blue). Bars, 50 μm. (C) Human-derived EPCs were cocultured with neonatal mouse cardiomyocytes infected with LacZ adenovirus. After 4 d of coculture, cells were stained with rabbit polyclonal anti-vWF (green), goat polyclonal anti-cTnT (red), and mouse monoclonal anti-β-gal antibodies (blue in top row) and Hoechst 33258 (bottom row, blue). The fluorescent confocal microscopic images (a–d, top row) demonstrate that vWF-expressing cells (a) expressed cTnT (b) and β-gal (c) in the same cell. Note that Cy5-conjugated secondary antibodies were used to visualize β-gal. The images of the same cell were taken by fluorescent microscope (e–h, bottom row). Hoechst staining of the nuclei revealed that homogenously stained nuclei (arrow) were of human cell origin and that mouse nuclei showed a punctate appearance (arrowhead). d and h represent merged images. Bars, 50 μm.

Mentions: Our in vitro and in vivo results suggest that cells expressing both cTnT and vWF in the damaged heart are fusion products of cardiomyocytes and endothelial cells. However, it has been reported that bone marrow–derived cells and EPCs differentiate into vascular cells and cardiomyocytes (Jackson et al., 2001; Badorff et al., 2003), leading us to examine whether bone marrow–derived or peripheral blood–derived EPCs may fuse with cardiomyocytes and express cTnT and vWF. Hematopoietic cells and mesenchymal cells from bone marrow of GFP transgenic mouse and human-derived EPCs were cocultured with neonatal cardiomyocytes that were infected with the adenoviral vector carrying the LacZ reporter gene. When GFP+ bone marrow–derived mesenchymal cells were cocultured with LacZ+ neonatal rat cardiomyocytes, ∼0.01% of GFP+ cells expressed cTnT and β-gal, suggesting that bone marrow–derived mesenchymal cells express cardiac-specific protein through cell fusion with cardiomyocytes (Fig. 9 A, a–d, arrow). In contrast, GFP+ hematopoietic cells expressed neither cTnT nor β-gal when cocultured with LacZ+ neonatal rat cardiomyocytes. Next, we isolated human-derived EPCs from the healthy volunteers and cultured them as described previously (Kalka et al., 2000). At 7 d after starting culture, spindle-shaped cells were stained with DiI-AcLDL and FITC-labeled ulex europaeus agglutinin-1 (UEA-1) lectin (Fig. 9 B, a and b), suggesting that these adherent cells were endothelial lineage cells. Immunocytochemical analysis revealed that ∼30% of these cells expressed vWF at this time (Fig. 9 B, c). When human-derived EPCs were cocultured with neonatal mouse cardiomyocytes that were infected with the adenoviral vector carrying the LacZ reporter gene, ∼0.1% of vWF-expressing cells expressed cTnT and β-gal (Fig. 9 C, a–d). Hoechst nuclear staining (Blau et al., 1983) revealed that these cells contained both a human-derived nucleus (smoothly appearance) and a mouse-derived nucleus (punctate appearance) (Fig. 9 C, e–h), suggesting that human-derived EPCs express cardiac-specific proteins through cell fusion with cardiomyocytes. These results suggest that bone marrow–derived mesenchymal cells and circulating EPCs, but not hematopoietic cells, fuse with cardiomyocytes.


Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle.

Matsuura K, Wada H, Nagai T, Iijima Y, Minamino T, Sano M, Akazawa H, Molkentin JD, Kasanuki H, Komuro I - J. Cell Biol. (2004)

Cardiomyocytes fused with adult immature somatic cells in vitro. (A) LacZ-expressing cardiomyocytes of neonatal rats were cocultured with GFP+ bone marrow mesenchymal cells. After 4 d of coculture, cells were stained with mouse monoclonal anti-cTnT (red) and rabbit polyclonal anti-β-gal antibodies (blue). Merged image was obtained from the same confocal plane. GFP+ mesenchymal cells (a, arrow) expressed cTnT (b, arrow) and β-gal (c, arrow) in the same cell (merged on d). Arrowheads indicate bone marrow cells fused with noncardiomyocyte. Bars, 50 μm. (B) Fluorescent microscopic images of EPCs cultured 7 d after isolation from peripheral blood. EPCs were identified as double-positive cells of DiI-labeled AcLDL uptake (a, red) and UEA-1 lectin reactivity (b, yellow in merged images). Some adherent cells expressed vWF (c, green). Nuclei were stained with Hoechst 33258 (c, blue). Bars, 50 μm. (C) Human-derived EPCs were cocultured with neonatal mouse cardiomyocytes infected with LacZ adenovirus. After 4 d of coculture, cells were stained with rabbit polyclonal anti-vWF (green), goat polyclonal anti-cTnT (red), and mouse monoclonal anti-β-gal antibodies (blue in top row) and Hoechst 33258 (bottom row, blue). The fluorescent confocal microscopic images (a–d, top row) demonstrate that vWF-expressing cells (a) expressed cTnT (b) and β-gal (c) in the same cell. Note that Cy5-conjugated secondary antibodies were used to visualize β-gal. The images of the same cell were taken by fluorescent microscope (e–h, bottom row). Hoechst staining of the nuclei revealed that homogenously stained nuclei (arrow) were of human cell origin and that mouse nuclei showed a punctate appearance (arrowhead). d and h represent merged images. Bars, 50 μm.
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fig9: Cardiomyocytes fused with adult immature somatic cells in vitro. (A) LacZ-expressing cardiomyocytes of neonatal rats were cocultured with GFP+ bone marrow mesenchymal cells. After 4 d of coculture, cells were stained with mouse monoclonal anti-cTnT (red) and rabbit polyclonal anti-β-gal antibodies (blue). Merged image was obtained from the same confocal plane. GFP+ mesenchymal cells (a, arrow) expressed cTnT (b, arrow) and β-gal (c, arrow) in the same cell (merged on d). Arrowheads indicate bone marrow cells fused with noncardiomyocyte. Bars, 50 μm. (B) Fluorescent microscopic images of EPCs cultured 7 d after isolation from peripheral blood. EPCs were identified as double-positive cells of DiI-labeled AcLDL uptake (a, red) and UEA-1 lectin reactivity (b, yellow in merged images). Some adherent cells expressed vWF (c, green). Nuclei were stained with Hoechst 33258 (c, blue). Bars, 50 μm. (C) Human-derived EPCs were cocultured with neonatal mouse cardiomyocytes infected with LacZ adenovirus. After 4 d of coculture, cells were stained with rabbit polyclonal anti-vWF (green), goat polyclonal anti-cTnT (red), and mouse monoclonal anti-β-gal antibodies (blue in top row) and Hoechst 33258 (bottom row, blue). The fluorescent confocal microscopic images (a–d, top row) demonstrate that vWF-expressing cells (a) expressed cTnT (b) and β-gal (c) in the same cell. Note that Cy5-conjugated secondary antibodies were used to visualize β-gal. The images of the same cell were taken by fluorescent microscope (e–h, bottom row). Hoechst staining of the nuclei revealed that homogenously stained nuclei (arrow) were of human cell origin and that mouse nuclei showed a punctate appearance (arrowhead). d and h represent merged images. Bars, 50 μm.
Mentions: Our in vitro and in vivo results suggest that cells expressing both cTnT and vWF in the damaged heart are fusion products of cardiomyocytes and endothelial cells. However, it has been reported that bone marrow–derived cells and EPCs differentiate into vascular cells and cardiomyocytes (Jackson et al., 2001; Badorff et al., 2003), leading us to examine whether bone marrow–derived or peripheral blood–derived EPCs may fuse with cardiomyocytes and express cTnT and vWF. Hematopoietic cells and mesenchymal cells from bone marrow of GFP transgenic mouse and human-derived EPCs were cocultured with neonatal cardiomyocytes that were infected with the adenoviral vector carrying the LacZ reporter gene. When GFP+ bone marrow–derived mesenchymal cells were cocultured with LacZ+ neonatal rat cardiomyocytes, ∼0.01% of GFP+ cells expressed cTnT and β-gal, suggesting that bone marrow–derived mesenchymal cells express cardiac-specific protein through cell fusion with cardiomyocytes (Fig. 9 A, a–d, arrow). In contrast, GFP+ hematopoietic cells expressed neither cTnT nor β-gal when cocultured with LacZ+ neonatal rat cardiomyocytes. Next, we isolated human-derived EPCs from the healthy volunteers and cultured them as described previously (Kalka et al., 2000). At 7 d after starting culture, spindle-shaped cells were stained with DiI-AcLDL and FITC-labeled ulex europaeus agglutinin-1 (UEA-1) lectin (Fig. 9 B, a and b), suggesting that these adherent cells were endothelial lineage cells. Immunocytochemical analysis revealed that ∼30% of these cells expressed vWF at this time (Fig. 9 B, c). When human-derived EPCs were cocultured with neonatal mouse cardiomyocytes that were infected with the adenoviral vector carrying the LacZ reporter gene, ∼0.1% of vWF-expressing cells expressed cTnT and β-gal (Fig. 9 C, a–d). Hoechst nuclear staining (Blau et al., 1983) revealed that these cells contained both a human-derived nucleus (smoothly appearance) and a mouse-derived nucleus (punctate appearance) (Fig. 9 C, e–h), suggesting that human-derived EPCs express cardiac-specific proteins through cell fusion with cardiomyocytes. These results suggest that bone marrow–derived mesenchymal cells and circulating EPCs, but not hematopoietic cells, fuse with cardiomyocytes.

Bottom Line: Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes.Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo.In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

ABSTRACT
The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.

Show MeSH
Related in: MedlinePlus