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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 cardiac cells from Kit+/Cre × R-GFP mice. a, b, c, d, e, f, g, Immunofluorescent images of heart histological sections from Kit+/Cre × R-GFP mice at 4 weeks of age stained with eGFP antibody (green), nuclei in blue and either CD31, von Willebrand factor (vWF), CD34, CD3, CD45, vimentin or smooth muscle α-actin (αSMA) in red. Arrows show cells with overlap in staining. h, i, FACS plot showing lineage markers of heart isolated c-kit derived eGFP+ cells for CD45 (h) and CD31 (i) (representative of n=6 for CD45 at 4 weeks of age, and n=3 for CD31 at 12 weeks of age). j, Immunofluorescent image from heart histological section of a Kit+/Cre × R-GFP mouse at 4 weeks for eGFP fluorescence (green), CD31 antibody staining (blue) and NG2 antibody staining (red). Right panel shows composite with transmitted light.
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Figure 2: Analysis of cardiac cells from Kit+/Cre × R-GFP mice. a, b, c, d, e, f, g, Immunofluorescent images of heart histological sections from Kit+/Cre × R-GFP mice at 4 weeks of age stained with eGFP antibody (green), nuclei in blue and either CD31, von Willebrand factor (vWF), CD34, CD3, CD45, vimentin or smooth muscle α-actin (αSMA) in red. Arrows show cells with overlap in staining. h, i, FACS plot showing lineage markers of heart isolated c-kit derived eGFP+ cells for CD45 (h) and CD31 (i) (representative of n=6 for CD45 at 4 weeks of age, and n=3 for CD31 at 12 weeks of age). j, Immunofluorescent image from heart histological section of a Kit+/Cre × R-GFP mouse at 4 weeks for eGFP fluorescence (green), CD31 antibody staining (blue) and NG2 antibody staining (red). Right panel shows composite with transmitted light.

Mentions: Hearts of Kit+/Cre × R-GFP mice at 4 weeks of age were further examined to identify the remaining eGFP+ non-myocytes. Examples of eGFP labeling co-incident with fibroblasts (vimentin co-labeling), endothelial cells (CD31, CD34, vWF), immune cells (CD3 and CD45), and rarely smooth muscle α-actin (αSMA) expressing cells were identified, although the most prevalent co-localizations were with CD31, CD45 or CD34 positive cells (Fig. 2a–g). Indeed, using a cocktail of antibodies for CD31, CD45, CD34 and CD3, versus sarcomeric α-actin, we were able to account for almost all eGFP+ non-myocytes in the hearts of adult Kit+/Cre × R-GFP mice, either when analyzed from histological sections or as dissociated individual cells (Extended Data Fig. 2a–c). FACS analysis showed that 18% and 77% of the total eGFP+ non-myocytes in the heart were CD45 or CD31 positive, respectively (Fig. 2h and i). Confocal microscopy analysis showed exact co-localization between eGFP+ cells in the heart and CD31 protein expression, but not with NG2 staining for pericytes (Fig. 2j).


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 cardiac cells from Kit+/Cre × R-GFP mice. a, b, c, d, e, f, g, Immunofluorescent images of heart histological sections from Kit+/Cre × R-GFP mice at 4 weeks of age stained with eGFP antibody (green), nuclei in blue and either CD31, von Willebrand factor (vWF), CD34, CD3, CD45, vimentin or smooth muscle α-actin (αSMA) in red. Arrows show cells with overlap in staining. h, i, FACS plot showing lineage markers of heart isolated c-kit derived eGFP+ cells for CD45 (h) and CD31 (i) (representative of n=6 for CD45 at 4 weeks of age, and n=3 for CD31 at 12 weeks of age). j, Immunofluorescent image from heart histological section of a Kit+/Cre × R-GFP mouse at 4 weeks for eGFP fluorescence (green), CD31 antibody staining (blue) and NG2 antibody staining (red). Right panel shows composite with transmitted light.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127035&req=5

Figure 2: Analysis of cardiac cells from Kit+/Cre × R-GFP mice. a, b, c, d, e, f, g, Immunofluorescent images of heart histological sections from Kit+/Cre × R-GFP mice at 4 weeks of age stained with eGFP antibody (green), nuclei in blue and either CD31, von Willebrand factor (vWF), CD34, CD3, CD45, vimentin or smooth muscle α-actin (αSMA) in red. Arrows show cells with overlap in staining. h, i, FACS plot showing lineage markers of heart isolated c-kit derived eGFP+ cells for CD45 (h) and CD31 (i) (representative of n=6 for CD45 at 4 weeks of age, and n=3 for CD31 at 12 weeks of age). j, Immunofluorescent image from heart histological section of a Kit+/Cre × R-GFP mouse at 4 weeks for eGFP fluorescence (green), CD31 antibody staining (blue) and NG2 antibody staining (red). Right panel shows composite with transmitted light.
Mentions: Hearts of Kit+/Cre × R-GFP mice at 4 weeks of age were further examined to identify the remaining eGFP+ non-myocytes. Examples of eGFP labeling co-incident with fibroblasts (vimentin co-labeling), endothelial cells (CD31, CD34, vWF), immune cells (CD3 and CD45), and rarely smooth muscle α-actin (αSMA) expressing cells were identified, although the most prevalent co-localizations were with CD31, CD45 or CD34 positive cells (Fig. 2a–g). Indeed, using a cocktail of antibodies for CD31, CD45, CD34 and CD3, versus sarcomeric α-actin, we were able to account for almost all eGFP+ non-myocytes in the hearts of adult Kit+/Cre × R-GFP mice, either when analyzed from histological sections or as dissociated individual cells (Extended Data Fig. 2a–c). FACS analysis showed that 18% and 77% of the total eGFP+ non-myocytes in the heart were CD45 or CD31 positive, respectively (Fig. 2h and i). Confocal microscopy analysis showed exact co-localization between eGFP+ cells in the heart and CD31 protein expression, but not with NG2 staining for pericytes (Fig. 2j).

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