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c-kit+ cells minimally contribute cardiomyocytes to the heart.

van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD - Nature (2014)

Bottom Line: Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008.By contrast, c-kit(+) cells amply generated cardiac endothelial cells.Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA [2] Department of Medicine, division of Cardiology, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA [3].

ABSTRACT
If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

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Analysis of eGFP+ non-myocytes in the hearts of Kit+/MCM × R-GFP mice at baseline or after MI injurya–g,Tamoxifen was given to Kit+/MCM × R-GFP mice for 1 day – 6 months of age (a,e,f) or in mice given tamoxifen and MI injury (b,c,d,g), followed by harvesting the hearts for immunohistochemistry with antibodies for GFP (green), or the indicated antibodies in red; (a) CD45, (b) CD3, (c) smooth muscle α-actin (αSMA), (d) vimentin, (e) CD34, (f) CD31, (g) von Willebrand factor (vWF). Nuclei are shown in blue. The white arrows show cells with coincident green and red reactivity for each of the markers, although sometimes the red marker is membrane localized while the green (eGFP) is always cytoplasmic. The most overlapping activity with GFP expression was observed for CD31 (endothelial cells), then CD34, followed by CD45 (hematopoietic cells). h, Quantitation from FACS plots of total CD31 cells (antibody) in the heart that are also eGFP+ from Kit+/MCM × R-GFP mice (Pre-MI, n=3) after 8 weeks of tamoxifen in early adulthood at either baseline or 4 weeks after MI injury (Post-MI, n=3). The data show about a doubling in the number of CD31 cells that are eGFP+ after MI (*P<0.05 vs pre-MI).
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Figure 9: Analysis of eGFP+ non-myocytes in the hearts of Kit+/MCM × R-GFP mice at baseline or after MI injurya–g,Tamoxifen was given to Kit+/MCM × R-GFP mice for 1 day – 6 months of age (a,e,f) or in mice given tamoxifen and MI injury (b,c,d,g), followed by harvesting the hearts for immunohistochemistry with antibodies for GFP (green), or the indicated antibodies in red; (a) CD45, (b) CD3, (c) smooth muscle α-actin (αSMA), (d) vimentin, (e) CD34, (f) CD31, (g) von Willebrand factor (vWF). Nuclei are shown in blue. The white arrows show cells with coincident green and red reactivity for each of the markers, although sometimes the red marker is membrane localized while the green (eGFP) is always cytoplasmic. The most overlapping activity with GFP expression was observed for CD31 (endothelial cells), then CD34, followed by CD45 (hematopoietic cells). h, Quantitation from FACS plots of total CD31 cells (antibody) in the heart that are also eGFP+ from Kit+/MCM × R-GFP mice (Pre-MI, n=3) after 8 weeks of tamoxifen in early adulthood at either baseline or 4 weeks after MI injury (Post-MI, n=3). The data show about a doubling in the number of CD31 cells that are eGFP+ after MI (*P<0.05 vs pre-MI).

Mentions: To specifically address the question of new cardiomyocyte formation within the adult heart, we generated a mouse model in which the tamoxifen inducible MerCreMer protein was targeted to the Kit locus (Kit+/MCM), followed by cross breeding with the R-GFP reporter line (Fig. 3a). To verify the fidelity of this system, Kit+/MCM × R-GFP mice were given tamoxifen during postnatal maturation for approximately 4 weeks followed by harvesting of tissues with known sites of c-kit expression (Extended Data Fig. 4a). Kit+/MCM × R-GFP mice showed ≈70% overlap in recombination-dependent eGFP expression and endogenous c-kit protein in Leydig cells of the testis (Extended Data Fig. 4b). Importantly, no eGFP+ cells were observed in the absence of tamoxifen at any age examined or after myocardial infarction (MI) injury, demonstrating that the MerCreMer system does not “leak” (Extended Data Fig. 4c). Kit+/MCM × R-GFP mice were also given tamoxifen from day 1 through 6 months of age for continuous labeling (Fig. 3b), which produced eGFP expression in greater than 60% of bone marrow cells, but again no signal in the absence of tamoxifen (Fig. 3c–e). Histological analysis of the heart after 6 months of labeling showed rare examples of eGFP+ adult cardiomyocytes and a relatively large number of non-myocytes (Fig. 3f, g). Careful analysis of the non-myocyte fraction in these hearts showed fibroblasts (rarely), smooth muscle cells (rarely), endothelial cells and immune cells, with the majority again being CD31+ (Extended Data Fig. 5a–h). MI injury also doubled the number of CD31 cells that were eGFP+ in the adult heart with 8 weeks of prior tamoxifen labeling (Extended Data Fig. 5h). We also conducted c-kit lineage labeling from 6–12 weeks of age, just after the postnatal developmental period (Fig. 3h). Upon disassociation of these hearts we observed 0.0055% eGFP+ adult cardiomyocytes (Fig. 3i, j), confirmed as extremely low by PCR and qPCR for Rosa26 locus recombination (Extended Data Fig. 6a, b, c).


c-kit+ cells minimally contribute cardiomyocytes to the heart.

van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD - Nature (2014)

Analysis of eGFP+ non-myocytes in the hearts of Kit+/MCM × R-GFP mice at baseline or after MI injurya–g,Tamoxifen was given to Kit+/MCM × R-GFP mice for 1 day – 6 months of age (a,e,f) or in mice given tamoxifen and MI injury (b,c,d,g), followed by harvesting the hearts for immunohistochemistry with antibodies for GFP (green), or the indicated antibodies in red; (a) CD45, (b) CD3, (c) smooth muscle α-actin (αSMA), (d) vimentin, (e) CD34, (f) CD31, (g) von Willebrand factor (vWF). Nuclei are shown in blue. The white arrows show cells with coincident green and red reactivity for each of the markers, although sometimes the red marker is membrane localized while the green (eGFP) is always cytoplasmic. The most overlapping activity with GFP expression was observed for CD31 (endothelial cells), then CD34, followed by CD45 (hematopoietic cells). h, Quantitation from FACS plots of total CD31 cells (antibody) in the heart that are also eGFP+ from Kit+/MCM × R-GFP mice (Pre-MI, n=3) after 8 weeks of tamoxifen in early adulthood at either baseline or 4 weeks after MI injury (Post-MI, n=3). The data show about a doubling in the number of CD31 cells that are eGFP+ after MI (*P<0.05 vs pre-MI).
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Figure 9: Analysis of eGFP+ non-myocytes in the hearts of Kit+/MCM × R-GFP mice at baseline or after MI injurya–g,Tamoxifen was given to Kit+/MCM × R-GFP mice for 1 day – 6 months of age (a,e,f) or in mice given tamoxifen and MI injury (b,c,d,g), followed by harvesting the hearts for immunohistochemistry with antibodies for GFP (green), or the indicated antibodies in red; (a) CD45, (b) CD3, (c) smooth muscle α-actin (αSMA), (d) vimentin, (e) CD34, (f) CD31, (g) von Willebrand factor (vWF). Nuclei are shown in blue. The white arrows show cells with coincident green and red reactivity for each of the markers, although sometimes the red marker is membrane localized while the green (eGFP) is always cytoplasmic. The most overlapping activity with GFP expression was observed for CD31 (endothelial cells), then CD34, followed by CD45 (hematopoietic cells). h, Quantitation from FACS plots of total CD31 cells (antibody) in the heart that are also eGFP+ from Kit+/MCM × R-GFP mice (Pre-MI, n=3) after 8 weeks of tamoxifen in early adulthood at either baseline or 4 weeks after MI injury (Post-MI, n=3). The data show about a doubling in the number of CD31 cells that are eGFP+ after MI (*P<0.05 vs pre-MI).
Mentions: To specifically address the question of new cardiomyocyte formation within the adult heart, we generated a mouse model in which the tamoxifen inducible MerCreMer protein was targeted to the Kit locus (Kit+/MCM), followed by cross breeding with the R-GFP reporter line (Fig. 3a). To verify the fidelity of this system, Kit+/MCM × R-GFP mice were given tamoxifen during postnatal maturation for approximately 4 weeks followed by harvesting of tissues with known sites of c-kit expression (Extended Data Fig. 4a). Kit+/MCM × R-GFP mice showed ≈70% overlap in recombination-dependent eGFP expression and endogenous c-kit protein in Leydig cells of the testis (Extended Data Fig. 4b). Importantly, no eGFP+ cells were observed in the absence of tamoxifen at any age examined or after myocardial infarction (MI) injury, demonstrating that the MerCreMer system does not “leak” (Extended Data Fig. 4c). Kit+/MCM × R-GFP mice were also given tamoxifen from day 1 through 6 months of age for continuous labeling (Fig. 3b), which produced eGFP expression in greater than 60% of bone marrow cells, but again no signal in the absence of tamoxifen (Fig. 3c–e). Histological analysis of the heart after 6 months of labeling showed rare examples of eGFP+ adult cardiomyocytes and a relatively large number of non-myocytes (Fig. 3f, g). Careful analysis of the non-myocyte fraction in these hearts showed fibroblasts (rarely), smooth muscle cells (rarely), endothelial cells and immune cells, with the majority again being CD31+ (Extended Data Fig. 5a–h). MI injury also doubled the number of CD31 cells that were eGFP+ in the adult heart with 8 weeks of prior tamoxifen labeling (Extended Data Fig. 5h). We also conducted c-kit lineage labeling from 6–12 weeks of age, just after the postnatal developmental period (Fig. 3h). Upon disassociation of these hearts we observed 0.0055% eGFP+ adult cardiomyocytes (Fig. 3i, j), confirmed as extremely low by PCR and qPCR for Rosa26 locus recombination (Extended Data Fig. 6a, b, c).

Bottom Line: Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008.By contrast, c-kit(+) cells amply generated cardiac endothelial cells.Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA [2] Department of Medicine, division of Cardiology, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA [3].

ABSTRACT
If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

Show MeSH
Related in: MedlinePlus