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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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Additional examination of the Kit-MerCreMer knock-in allele and its potential leakiness in the absence of tamoxifena, Histological analysis of eGFP fluorescent cells from the indicated tissues at day 28 from Kit+/MCM × R-GFP mice that were given tamoxifen from 2 to 28 days. Nuclei are shown in blue and green shows eGFP fluorescing cells in the expected patterns for known regions of c-kit protein expression, such as the distinct pattern of melanocytes in the skin and widespread expression in the spleen and lungs. b, Immunohistochemistry in the testis of Kit+/MCM × R-GFP mice for endogenous c-kit expression (red) versus cells that underwent recombination when tamoxifen was given by intraperitoneal injection (2 mg) for 5 consecutive days (green). The data show that most of the currently c-kit protein expressing cells in testis (only Leydig cells react, red surface staining) are also eGFP+ (intracellular), indicating that recombination only occurs in c-kit expressing cells, and the majority of them. c, Histological sections through the heart showing that the Kit-MerCreMer allele does not leak at baseline or after MI injury (n=3 mice per treatment). Kit+/MCM × R-GFP mice were placed on tamoxifen-laden food or vehicle food beginning at 4 weeks of age and then subjected to MI injury 4 weeks later, followed by harvesting 4 weeks after that. In the presence of tamoxifen histological sections through the MI border zone of the heart show wide-spread eGFP+ cells (green) from the c-kit lineage (left panel), while in the absence of tamoxifen no eGFP+ cells are observed (right panel), hence the Kit-MerCreMer allele does not leak.
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Figure 8: Additional examination of the Kit-MerCreMer knock-in allele and its potential leakiness in the absence of tamoxifena, Histological analysis of eGFP fluorescent cells from the indicated tissues at day 28 from Kit+/MCM × R-GFP mice that were given tamoxifen from 2 to 28 days. Nuclei are shown in blue and green shows eGFP fluorescing cells in the expected patterns for known regions of c-kit protein expression, such as the distinct pattern of melanocytes in the skin and widespread expression in the spleen and lungs. b, Immunohistochemistry in the testis of Kit+/MCM × R-GFP mice for endogenous c-kit expression (red) versus cells that underwent recombination when tamoxifen was given by intraperitoneal injection (2 mg) for 5 consecutive days (green). The data show that most of the currently c-kit protein expressing cells in testis (only Leydig cells react, red surface staining) are also eGFP+ (intracellular), indicating that recombination only occurs in c-kit expressing cells, and the majority of them. c, Histological sections through the heart showing that the Kit-MerCreMer allele does not leak at baseline or after MI injury (n=3 mice per treatment). Kit+/MCM × R-GFP mice were placed on tamoxifen-laden food or vehicle food beginning at 4 weeks of age and then subjected to MI injury 4 weeks later, followed by harvesting 4 weeks after that. In the presence of tamoxifen histological sections through the MI border zone of the heart show wide-spread eGFP+ cells (green) from the c-kit lineage (left panel), while in the absence of tamoxifen no eGFP+ cells are observed (right panel), hence the Kit-MerCreMer allele does not leak.

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)

Additional examination of the Kit-MerCreMer knock-in allele and its potential leakiness in the absence of tamoxifena, Histological analysis of eGFP fluorescent cells from the indicated tissues at day 28 from Kit+/MCM × R-GFP mice that were given tamoxifen from 2 to 28 days. Nuclei are shown in blue and green shows eGFP fluorescing cells in the expected patterns for known regions of c-kit protein expression, such as the distinct pattern of melanocytes in the skin and widespread expression in the spleen and lungs. b, Immunohistochemistry in the testis of Kit+/MCM × R-GFP mice for endogenous c-kit expression (red) versus cells that underwent recombination when tamoxifen was given by intraperitoneal injection (2 mg) for 5 consecutive days (green). The data show that most of the currently c-kit protein expressing cells in testis (only Leydig cells react, red surface staining) are also eGFP+ (intracellular), indicating that recombination only occurs in c-kit expressing cells, and the majority of them. c, Histological sections through the heart showing that the Kit-MerCreMer allele does not leak at baseline or after MI injury (n=3 mice per treatment). Kit+/MCM × R-GFP mice were placed on tamoxifen-laden food or vehicle food beginning at 4 weeks of age and then subjected to MI injury 4 weeks later, followed by harvesting 4 weeks after that. In the presence of tamoxifen histological sections through the MI border zone of the heart show wide-spread eGFP+ cells (green) from the c-kit lineage (left panel), while in the absence of tamoxifen no eGFP+ cells are observed (right panel), hence the Kit-MerCreMer allele does not leak.
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Related In: Results  -  Collection

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Figure 8: Additional examination of the Kit-MerCreMer knock-in allele and its potential leakiness in the absence of tamoxifena, Histological analysis of eGFP fluorescent cells from the indicated tissues at day 28 from Kit+/MCM × R-GFP mice that were given tamoxifen from 2 to 28 days. Nuclei are shown in blue and green shows eGFP fluorescing cells in the expected patterns for known regions of c-kit protein expression, such as the distinct pattern of melanocytes in the skin and widespread expression in the spleen and lungs. b, Immunohistochemistry in the testis of Kit+/MCM × R-GFP mice for endogenous c-kit expression (red) versus cells that underwent recombination when tamoxifen was given by intraperitoneal injection (2 mg) for 5 consecutive days (green). The data show that most of the currently c-kit protein expressing cells in testis (only Leydig cells react, red surface staining) are also eGFP+ (intracellular), indicating that recombination only occurs in c-kit expressing cells, and the majority of them. c, Histological sections through the heart showing that the Kit-MerCreMer allele does not leak at baseline or after MI injury (n=3 mice per treatment). Kit+/MCM × R-GFP mice were placed on tamoxifen-laden food or vehicle food beginning at 4 weeks of age and then subjected to MI injury 4 weeks later, followed by harvesting 4 weeks after that. In the presence of tamoxifen histological sections through the MI border zone of the heart show wide-spread eGFP+ cells (green) from the c-kit lineage (left panel), while in the absence of tamoxifen no eGFP+ cells are observed (right panel), hence the Kit-MerCreMer allele does not leak.
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