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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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Identification of non-myocytes from the hearts of Kit+/Cre × R-GFP miceKit+/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit-Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP+ and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit+/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP+ (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP+ non-myocytes, which in panel c, were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.
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Figure 6: Identification of non-myocytes from the hearts of Kit+/Cre × R-GFP miceKit+/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit-Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP+ and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit+/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP+ (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP+ non-myocytes, which in panel c, were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.

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)

Identification of non-myocytes from the hearts of Kit+/Cre × R-GFP miceKit+/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit-Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP+ and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit+/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP+ (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP+ non-myocytes, which in panel c, were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.
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Related In: Results  -  Collection

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Figure 6: Identification of non-myocytes from the hearts of Kit+/Cre × R-GFP miceKit+/Cre × R-GFP mice were harvested at 6 weeks of age (constitutive lineage labeling the entire time), although MI was performed at week 4 to induce greater vascular remodeling and potentially more c-kit lineage recruitment over the next 2 weeks. a, Hearts were then collected at week 6 and subjected to immunohistochemistry with a pool of antibodies for CD31, CD34, CD45 and CD3 in red, while the green channel was for eGFP expression from the recombined R-GFP reporter allele due to Kit-Cre lineage expression. The white arrowheads show endothelial cells that are not contiguous with the underlying network, although most of the endothelial cells are from the c-kit lineage when the red and green channels are compared. The white arrow shows a cardiomyocyte that lacks red staining, while the yellow arrows show 2 areas with relatively large cells that are eGFP+ and could be mistaken for a cardiomyocyte, although they are also positive for the non-myocyte marker panel of antibodies. b, c, Spread of cells isolated from hearts of 8 week-old Kit+/Cre × R-GFP mice at baseline that were subjected to immunocytochemistry for the indicated markers. The large white arrow in panel b shows an eGFP+ (green) cardiomyocyte that also co-stains with sarcomeric α-actin (red). The smaller arrows show eGFP+ non-myocytes, which in panel c, were subject to staining with a cocktail of antibodies again for CD31, CD34, CD45 and CD3 (all in red). This analysis identifies nearly all of the non-myocytes in these cell spreads. The very last image in panel c shows a fourth channel with higher gain so that the underlying cardiomyocytes (CMs) autofluoresce (in white) to show the mixed nature of the spread cells. Nuclei were stained blue with DAPI in the indicated panels.
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