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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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Kit-Cre lineage tracinga, The Kit locus was targeted in mice to express Cre recombinase and eGFP with a nuclear localization sequence (eGFPnls) from an internal ribosome entry site (IRES). These mice were crossed with Rosa26 reporter mice (R-GFP) for lineage tracing. b, Diagram of mice used for all experimentation in this figure. c, Representative flow cytometry (FACS) plot of bone marrow from Kit+/Cre × R-GFP mice gated for c-kit antibody, then eGFP fluorescence to reflect recombination of the R-GFP locus (representative of n=6 total). d, Direct imaging cytometry analysis of eGFP expression in bone marrow (n=3, *P<0.05 vs R-GFP). e, same quantitative imaging cytometry analysis as in d except the non-myocytes were isolated from hearts of Kit+/Cre × R-GFP mice (n=3 hearts, *P<0.05 vs R-GFP). f, Immunohistochemistry to show current expression from the Kit-Cre allele (green, eGFPnls) versus endogenous c-kit protein detected by antibody (red). The inset box shows 2 mononuclear c-kit expressing cells. g, Quantitation of average number of c-kit+ cells per longitudinal heart section (n=4 hearts) h, Representative histological section at 2 magnifications (white box) of a Kit+/Cre × R-GFP mouse heart with desmin antibody in red, eGFP antibody in green, and nuclei in blue. The arrow shows an eGFP+ cardiomyocyte. i, Immunohistological image showing a rare area of cardiomyocyte clonal expansion (arrow). j, Image of cells disassociated from the hearts of Kit+/Cre × R-GFP mice. White arrow shows a rare eGFP fluorescing cardiomyocyte, while the black arrowheads show eGFP fluorescent non-myocytes. k, Quantitation of eGFP+ fluorescent cardiomyocytes (81 from 303,264 total cardiomyocytes, 3 hearts,*P<0.05 vs R-GFP). l, DNA electrophoresis after PCR showing Cre-mediated Rosa26 locus recombination in semi-purified cardiomyocytes and spleen (n=2 each). All error bars represent s.e.m.
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Figure 1: Kit-Cre lineage tracinga, The Kit locus was targeted in mice to express Cre recombinase and eGFP with a nuclear localization sequence (eGFPnls) from an internal ribosome entry site (IRES). These mice were crossed with Rosa26 reporter mice (R-GFP) for lineage tracing. b, Diagram of mice used for all experimentation in this figure. c, Representative flow cytometry (FACS) plot of bone marrow from Kit+/Cre × R-GFP mice gated for c-kit antibody, then eGFP fluorescence to reflect recombination of the R-GFP locus (representative of n=6 total). d, Direct imaging cytometry analysis of eGFP expression in bone marrow (n=3, *P<0.05 vs R-GFP). e, same quantitative imaging cytometry analysis as in d except the non-myocytes were isolated from hearts of Kit+/Cre × R-GFP mice (n=3 hearts, *P<0.05 vs R-GFP). f, Immunohistochemistry to show current expression from the Kit-Cre allele (green, eGFPnls) versus endogenous c-kit protein detected by antibody (red). The inset box shows 2 mononuclear c-kit expressing cells. g, Quantitation of average number of c-kit+ cells per longitudinal heart section (n=4 hearts) h, Representative histological section at 2 magnifications (white box) of a Kit+/Cre × R-GFP mouse heart with desmin antibody in red, eGFP antibody in green, and nuclei in blue. The arrow shows an eGFP+ cardiomyocyte. i, Immunohistological image showing a rare area of cardiomyocyte clonal expansion (arrow). j, Image of cells disassociated from the hearts of Kit+/Cre × R-GFP mice. White arrow shows a rare eGFP fluorescing cardiomyocyte, while the black arrowheads show eGFP fluorescent non-myocytes. k, Quantitation of eGFP+ fluorescent cardiomyocytes (81 from 303,264 total cardiomyocytes, 3 hearts,*P<0.05 vs R-GFP). l, DNA electrophoresis after PCR showing Cre-mediated Rosa26 locus recombination in semi-purified cardiomyocytes and spleen (n=2 each). All error bars represent s.e.m.

Mentions: The Kit locus was targeted with a cDNA encoding Cre recombinase fused to an internal ribosome entry sequence (IRES) to concurrently express enhanced green fluorescent protein (eGFP) tagged with a nuclear localization signal (nls) (Fig. 1a). These Kit+/Cre mice were bred to LoxP site-dependent Rosa26-CAG-loxP-STOP-loxP-eGFP (R-GFP) reporter mice to irreversibly mark any cell that previously or currently expresses this Kit locus (Fig. 1a). Four to eight weeks after birth the fidelity of the genetic system was assessed in comparison with known domains of c-kit protein expression, such as melanocytes of the skin, Leydig cells in the testis, interstitial cells of the intestine and wide areas of the spleen, all of which showed eGFP cellular labeling (Fig. 1b, Extended Data Fig. 1a) 11–13. In bone marrow, 83% of the c-kit antibody detected cells were eGFP+ by standard FACS analysis (Fig. 1c), while imaging cytometry analysis detected coincident eGFP+ expression and c-kit immunoreactivity in 88% of the bone marrow cells and 76% of the non-myocyte fraction from the heart (Fig. 1d, e). To further verify the specificity of the Kit-Cre allele we examined real time eGFPnls expression in the heart, ileum and skeletal muscle for coexpression of c-kit protein (antibody), which was always coincident (Fig. 1f, g, and Extended Data Fig. 1b, c). In bone marrow, 94% of the eGFP+ cells were Lin+, indicating a high degree of fidelity with the Kit-Cre allele (Extended Data Fig. 1d). In the heart c-kit antibody positive mononuclear cells were predominantly eGFP+ at 4 weeks of age using the Kit+/Cre × R-GFP reporter strategy, while in testis recombination was only observed in Leydig cells, of which >80% were eGFP+ (Extended Data Fig. 1e, f). Thus, the specificity of the Kit-Cre allele appears identical with known regions of c-kit protein expression in vivo.


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)

Kit-Cre lineage tracinga, The Kit locus was targeted in mice to express Cre recombinase and eGFP with a nuclear localization sequence (eGFPnls) from an internal ribosome entry site (IRES). These mice were crossed with Rosa26 reporter mice (R-GFP) for lineage tracing. b, Diagram of mice used for all experimentation in this figure. c, Representative flow cytometry (FACS) plot of bone marrow from Kit+/Cre × R-GFP mice gated for c-kit antibody, then eGFP fluorescence to reflect recombination of the R-GFP locus (representative of n=6 total). d, Direct imaging cytometry analysis of eGFP expression in bone marrow (n=3, *P<0.05 vs R-GFP). e, same quantitative imaging cytometry analysis as in d except the non-myocytes were isolated from hearts of Kit+/Cre × R-GFP mice (n=3 hearts, *P<0.05 vs R-GFP). f, Immunohistochemistry to show current expression from the Kit-Cre allele (green, eGFPnls) versus endogenous c-kit protein detected by antibody (red). The inset box shows 2 mononuclear c-kit expressing cells. g, Quantitation of average number of c-kit+ cells per longitudinal heart section (n=4 hearts) h, Representative histological section at 2 magnifications (white box) of a Kit+/Cre × R-GFP mouse heart with desmin antibody in red, eGFP antibody in green, and nuclei in blue. The arrow shows an eGFP+ cardiomyocyte. i, Immunohistological image showing a rare area of cardiomyocyte clonal expansion (arrow). j, Image of cells disassociated from the hearts of Kit+/Cre × R-GFP mice. White arrow shows a rare eGFP fluorescing cardiomyocyte, while the black arrowheads show eGFP fluorescent non-myocytes. k, Quantitation of eGFP+ fluorescent cardiomyocytes (81 from 303,264 total cardiomyocytes, 3 hearts,*P<0.05 vs R-GFP). l, DNA electrophoresis after PCR showing Cre-mediated Rosa26 locus recombination in semi-purified cardiomyocytes and spleen (n=2 each). All error bars represent s.e.m.
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Figure 1: Kit-Cre lineage tracinga, The Kit locus was targeted in mice to express Cre recombinase and eGFP with a nuclear localization sequence (eGFPnls) from an internal ribosome entry site (IRES). These mice were crossed with Rosa26 reporter mice (R-GFP) for lineage tracing. b, Diagram of mice used for all experimentation in this figure. c, Representative flow cytometry (FACS) plot of bone marrow from Kit+/Cre × R-GFP mice gated for c-kit antibody, then eGFP fluorescence to reflect recombination of the R-GFP locus (representative of n=6 total). d, Direct imaging cytometry analysis of eGFP expression in bone marrow (n=3, *P<0.05 vs R-GFP). e, same quantitative imaging cytometry analysis as in d except the non-myocytes were isolated from hearts of Kit+/Cre × R-GFP mice (n=3 hearts, *P<0.05 vs R-GFP). f, Immunohistochemistry to show current expression from the Kit-Cre allele (green, eGFPnls) versus endogenous c-kit protein detected by antibody (red). The inset box shows 2 mononuclear c-kit expressing cells. g, Quantitation of average number of c-kit+ cells per longitudinal heart section (n=4 hearts) h, Representative histological section at 2 magnifications (white box) of a Kit+/Cre × R-GFP mouse heart with desmin antibody in red, eGFP antibody in green, and nuclei in blue. The arrow shows an eGFP+ cardiomyocyte. i, Immunohistological image showing a rare area of cardiomyocyte clonal expansion (arrow). j, Image of cells disassociated from the hearts of Kit+/Cre × R-GFP mice. White arrow shows a rare eGFP fluorescing cardiomyocyte, while the black arrowheads show eGFP fluorescent non-myocytes. k, Quantitation of eGFP+ fluorescent cardiomyocytes (81 from 303,264 total cardiomyocytes, 3 hearts,*P<0.05 vs R-GFP). l, DNA electrophoresis after PCR showing Cre-mediated Rosa26 locus recombination in semi-purified cardiomyocytes and spleen (n=2 each). All error bars represent s.e.m.
Mentions: The Kit locus was targeted with a cDNA encoding Cre recombinase fused to an internal ribosome entry sequence (IRES) to concurrently express enhanced green fluorescent protein (eGFP) tagged with a nuclear localization signal (nls) (Fig. 1a). These Kit+/Cre mice were bred to LoxP site-dependent Rosa26-CAG-loxP-STOP-loxP-eGFP (R-GFP) reporter mice to irreversibly mark any cell that previously or currently expresses this Kit locus (Fig. 1a). Four to eight weeks after birth the fidelity of the genetic system was assessed in comparison with known domains of c-kit protein expression, such as melanocytes of the skin, Leydig cells in the testis, interstitial cells of the intestine and wide areas of the spleen, all of which showed eGFP cellular labeling (Fig. 1b, Extended Data Fig. 1a) 11–13. In bone marrow, 83% of the c-kit antibody detected cells were eGFP+ by standard FACS analysis (Fig. 1c), while imaging cytometry analysis detected coincident eGFP+ expression and c-kit immunoreactivity in 88% of the bone marrow cells and 76% of the non-myocyte fraction from the heart (Fig. 1d, e). To further verify the specificity of the Kit-Cre allele we examined real time eGFPnls expression in the heart, ileum and skeletal muscle for coexpression of c-kit protein (antibody), which was always coincident (Fig. 1f, g, and Extended Data Fig. 1b, c). In bone marrow, 94% of the eGFP+ cells were Lin+, indicating a high degree of fidelity with the Kit-Cre allele (Extended Data Fig. 1d). In the heart c-kit antibody positive mononuclear cells were predominantly eGFP+ at 4 weeks of age using the Kit+/Cre × R-GFP reporter strategy, while in testis recombination was only observed in Leydig cells, of which >80% were eGFP+ (Extended Data Fig. 1e, f). Thus, the specificity of the Kit-Cre allele appears identical with known regions of c-kit protein expression in vivo.

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