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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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Assessing the fidelity and specificity of the Kit-Cre knock-in allelea, Histological sections from the indicated tissues of Kit+/Cre × R-GFP mice at 4 weeks of age. Blue is nuclei and green is eGFP. The data show eGFP expression in regions of each tissue that is often characteristic of endogenous c-kit protein expression. b, Immunohistochemistry for endogenous c-kit expression (red) in the mouse ileum at 4 weeks of age from Kit+/Cre mice that contain the IRES-eGFPnls cassette (but without the × R-GFP reporter allele) so that eGFP expression can be monitored in real time. The inset box and arrows show the co-staining with c-kit antibody and eGFP. c, Immunohistochemistry for endogenous c-kit expression (red) in quadriceps muscle of Kit+/Cre mice at 4 weeks of age versus nuclear eGFP (green) from the Kit+/Cre allele. While lineage tracing in Kit+/Cre × R-GFP mice, which is cumulative, showed abundant endothelial cells throughout the skeletal muscle (a), instantaneous c-kit expressing cells are rare in skeletal muscle, and when identified, are always mononuclear (inset box). d, FACS quantitation of bone marrow from Kit+/Cre × R-GFP mice at 4 weeks of age sorted for eGFP expression, of which 94% are positive for the “lineage” cocktail of differentiation-specific antibodies (n=3 mice). Hence the Kit-Cre allele is properly expressed in bone marrow and traces lineages that arise from c-kit+ progenitors. e, Immunohistochemistry in the hearts of Kit+/Cre × R-GFP mice for endogenous c-kit expression (red) versus all the cells that underwent recombination throughout development and the first 4 weeks of life, shown in green. While cells that are actively expressing c-kit protein are very rare in the heart (≈5 per heart section), the arrow shows such a cell that is also eGFP+ for recombination. All of the currently c-kit expressing cells identified in the heart were eGFP+, further verifying the fidelity of the Kit-Cre allele. f, Same experiment as in e except the testis was examined because of the characteristic pattern of Leydig cells that are known to be actively c-kit expressing cells. The data show that greater than 80% of the currently c-kit antibody reactive Leydig cells (red outline, better observed in the right panel) are also eGFP+ (arrows show clusters of these cells).
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Figure 5: Assessing the fidelity and specificity of the Kit-Cre knock-in allelea, Histological sections from the indicated tissues of Kit+/Cre × R-GFP mice at 4 weeks of age. Blue is nuclei and green is eGFP. The data show eGFP expression in regions of each tissue that is often characteristic of endogenous c-kit protein expression. b, Immunohistochemistry for endogenous c-kit expression (red) in the mouse ileum at 4 weeks of age from Kit+/Cre mice that contain the IRES-eGFPnls cassette (but without the × R-GFP reporter allele) so that eGFP expression can be monitored in real time. The inset box and arrows show the co-staining with c-kit antibody and eGFP. c, Immunohistochemistry for endogenous c-kit expression (red) in quadriceps muscle of Kit+/Cre mice at 4 weeks of age versus nuclear eGFP (green) from the Kit+/Cre allele. While lineage tracing in Kit+/Cre × R-GFP mice, which is cumulative, showed abundant endothelial cells throughout the skeletal muscle (a), instantaneous c-kit expressing cells are rare in skeletal muscle, and when identified, are always mononuclear (inset box). d, FACS quantitation of bone marrow from Kit+/Cre × R-GFP mice at 4 weeks of age sorted for eGFP expression, of which 94% are positive for the “lineage” cocktail of differentiation-specific antibodies (n=3 mice). Hence the Kit-Cre allele is properly expressed in bone marrow and traces lineages that arise from c-kit+ progenitors. e, Immunohistochemistry in the hearts of Kit+/Cre × R-GFP mice for endogenous c-kit expression (red) versus all the cells that underwent recombination throughout development and the first 4 weeks of life, shown in green. While cells that are actively expressing c-kit protein are very rare in the heart (≈5 per heart section), the arrow shows such a cell that is also eGFP+ for recombination. All of the currently c-kit expressing cells identified in the heart were eGFP+, further verifying the fidelity of the Kit-Cre allele. f, Same experiment as in e except the testis was examined because of the characteristic pattern of Leydig cells that are known to be actively c-kit expressing cells. The data show that greater than 80% of the currently c-kit antibody reactive Leydig cells (red outline, better observed in the right panel) are also eGFP+ (arrows show clusters of these cells).

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)

Assessing the fidelity and specificity of the Kit-Cre knock-in allelea, Histological sections from the indicated tissues of Kit+/Cre × R-GFP mice at 4 weeks of age. Blue is nuclei and green is eGFP. The data show eGFP expression in regions of each tissue that is often characteristic of endogenous c-kit protein expression. b, Immunohistochemistry for endogenous c-kit expression (red) in the mouse ileum at 4 weeks of age from Kit+/Cre mice that contain the IRES-eGFPnls cassette (but without the × R-GFP reporter allele) so that eGFP expression can be monitored in real time. The inset box and arrows show the co-staining with c-kit antibody and eGFP. c, Immunohistochemistry for endogenous c-kit expression (red) in quadriceps muscle of Kit+/Cre mice at 4 weeks of age versus nuclear eGFP (green) from the Kit+/Cre allele. While lineage tracing in Kit+/Cre × R-GFP mice, which is cumulative, showed abundant endothelial cells throughout the skeletal muscle (a), instantaneous c-kit expressing cells are rare in skeletal muscle, and when identified, are always mononuclear (inset box). d, FACS quantitation of bone marrow from Kit+/Cre × R-GFP mice at 4 weeks of age sorted for eGFP expression, of which 94% are positive for the “lineage” cocktail of differentiation-specific antibodies (n=3 mice). Hence the Kit-Cre allele is properly expressed in bone marrow and traces lineages that arise from c-kit+ progenitors. e, Immunohistochemistry in the hearts of Kit+/Cre × R-GFP mice for endogenous c-kit expression (red) versus all the cells that underwent recombination throughout development and the first 4 weeks of life, shown in green. While cells that are actively expressing c-kit protein are very rare in the heart (≈5 per heart section), the arrow shows such a cell that is also eGFP+ for recombination. All of the currently c-kit expressing cells identified in the heart were eGFP+, further verifying the fidelity of the Kit-Cre allele. f, Same experiment as in e except the testis was examined because of the characteristic pattern of Leydig cells that are known to be actively c-kit expressing cells. The data show that greater than 80% of the currently c-kit antibody reactive Leydig cells (red outline, better observed in the right panel) are also eGFP+ (arrows show clusters of these cells).
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Figure 5: Assessing the fidelity and specificity of the Kit-Cre knock-in allelea, Histological sections from the indicated tissues of Kit+/Cre × R-GFP mice at 4 weeks of age. Blue is nuclei and green is eGFP. The data show eGFP expression in regions of each tissue that is often characteristic of endogenous c-kit protein expression. b, Immunohistochemistry for endogenous c-kit expression (red) in the mouse ileum at 4 weeks of age from Kit+/Cre mice that contain the IRES-eGFPnls cassette (but without the × R-GFP reporter allele) so that eGFP expression can be monitored in real time. The inset box and arrows show the co-staining with c-kit antibody and eGFP. c, Immunohistochemistry for endogenous c-kit expression (red) in quadriceps muscle of Kit+/Cre mice at 4 weeks of age versus nuclear eGFP (green) from the Kit+/Cre allele. While lineage tracing in Kit+/Cre × R-GFP mice, which is cumulative, showed abundant endothelial cells throughout the skeletal muscle (a), instantaneous c-kit expressing cells are rare in skeletal muscle, and when identified, are always mononuclear (inset box). d, FACS quantitation of bone marrow from Kit+/Cre × R-GFP mice at 4 weeks of age sorted for eGFP expression, of which 94% are positive for the “lineage” cocktail of differentiation-specific antibodies (n=3 mice). Hence the Kit-Cre allele is properly expressed in bone marrow and traces lineages that arise from c-kit+ progenitors. e, Immunohistochemistry in the hearts of Kit+/Cre × R-GFP mice for endogenous c-kit expression (red) versus all the cells that underwent recombination throughout development and the first 4 weeks of life, shown in green. While cells that are actively expressing c-kit protein are very rare in the heart (≈5 per heart section), the arrow shows such a cell that is also eGFP+ for recombination. All of the currently c-kit expressing cells identified in the heart were eGFP+, further verifying the fidelity of the Kit-Cre allele. f, Same experiment as in e except the testis was examined because of the characteristic pattern of Leydig cells that are known to be actively c-kit expressing cells. The data show that greater than 80% of the currently c-kit antibody reactive Leydig cells (red outline, better observed in the right panel) are also eGFP+ (arrows show clusters of these cells).
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