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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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Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formationa, Genetic cross between Kit+/MCM and R-GFP reporter mice to lineage trace c-kit expressing cells when tamoxifen is present. b, Schematic showing tamoxifen treatment between day 1 and 6 months of age (panels c–g). c, d, Representative FACS plots with c-kit antibody (APC) vs eGFP from bone marrow of Kit+/MCM × R-GFP mice without (c) or with tamoxifen (d). e, FACS quantification of eGFP+ cells from bone marrow of these mice (n=2 mice for R-GFP and n=4 for Kit+/MCM × R-GFP). *p<0.05 vs R-GFP. f, g, Representative heart sections from Kit+MCM × R-GFP mice showing c-kit+ lineage cells in green and cardiomyocytes in red (desmin antibody). White arrow indicates eGFP+ adult cardiomyocyte. h,i,j, Tamoxifen treatment of Kit+/MCM × R-GFP mice between 6 – 12 weeks of age followed by disassociation of cells from the hearts of these mice in h (white arrow shows rare cardiomyocyte) that is quantified in j (127,284 cardiomyocytes across 2 hearts, 7 were eGFP+,*P<0.05 vs R-GFP). k,l,m,n, Tamoxifen treatment of Kit+MCM × R-GFP mice between 8 and 14 weeks of age with myocardial infarction (MI) on week 10. l, Immunohistological heart section for desmin (red) and eGFP (green) with nuclei in blue (arrow shows a cardiomyocyte from the c-kit+ lineage). m, n, Disassociated cardiomyocytes show rare but definitive myocyte labeling (white arrow), which was quantified in n (225,760 cardiomyocytes from 2 MI hearts, 37 were eGFP+,*P<0.05 vs R-GFP). o,p, Tamoxifen treatment between 8 – 12 weeks of age with MI injury occurring 3 days after tamoxifen cessation. p, Quantitation of eGFP+ cardiomyocytes from histological images taken from 2 hearts of these mice. eGFP+ cardiomyocytes were quantified as a percentage of the total cardiomyocyte fraction in >50 histological sections across the entire heart. *P<0.05 vs R-GFP. All error bars represent s.e.m.
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Figure 3: Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formationa, Genetic cross between Kit+/MCM and R-GFP reporter mice to lineage trace c-kit expressing cells when tamoxifen is present. b, Schematic showing tamoxifen treatment between day 1 and 6 months of age (panels c–g). c, d, Representative FACS plots with c-kit antibody (APC) vs eGFP from bone marrow of Kit+/MCM × R-GFP mice without (c) or with tamoxifen (d). e, FACS quantification of eGFP+ cells from bone marrow of these mice (n=2 mice for R-GFP and n=4 for Kit+/MCM × R-GFP). *p<0.05 vs R-GFP. f, g, Representative heart sections from Kit+MCM × R-GFP mice showing c-kit+ lineage cells in green and cardiomyocytes in red (desmin antibody). White arrow indicates eGFP+ adult cardiomyocyte. h,i,j, Tamoxifen treatment of Kit+/MCM × R-GFP mice between 6 – 12 weeks of age followed by disassociation of cells from the hearts of these mice in h (white arrow shows rare cardiomyocyte) that is quantified in j (127,284 cardiomyocytes across 2 hearts, 7 were eGFP+,*P<0.05 vs R-GFP). k,l,m,n, Tamoxifen treatment of Kit+MCM × R-GFP mice between 8 and 14 weeks of age with myocardial infarction (MI) on week 10. l, Immunohistological heart section for desmin (red) and eGFP (green) with nuclei in blue (arrow shows a cardiomyocyte from the c-kit+ lineage). m, n, Disassociated cardiomyocytes show rare but definitive myocyte labeling (white arrow), which was quantified in n (225,760 cardiomyocytes from 2 MI hearts, 37 were eGFP+,*P<0.05 vs R-GFP). o,p, Tamoxifen treatment between 8 – 12 weeks of age with MI injury occurring 3 days after tamoxifen cessation. p, Quantitation of eGFP+ cardiomyocytes from histological images taken from 2 hearts of these mice. eGFP+ cardiomyocytes were quantified as a percentage of the total cardiomyocyte fraction in >50 histological sections across the entire heart. *P<0.05 vs R-GFP. All error bars represent s.e.m.

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)

Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formationa, Genetic cross between Kit+/MCM and R-GFP reporter mice to lineage trace c-kit expressing cells when tamoxifen is present. b, Schematic showing tamoxifen treatment between day 1 and 6 months of age (panels c–g). c, d, Representative FACS plots with c-kit antibody (APC) vs eGFP from bone marrow of Kit+/MCM × R-GFP mice without (c) or with tamoxifen (d). e, FACS quantification of eGFP+ cells from bone marrow of these mice (n=2 mice for R-GFP and n=4 for Kit+/MCM × R-GFP). *p<0.05 vs R-GFP. f, g, Representative heart sections from Kit+MCM × R-GFP mice showing c-kit+ lineage cells in green and cardiomyocytes in red (desmin antibody). White arrow indicates eGFP+ adult cardiomyocyte. h,i,j, Tamoxifen treatment of Kit+/MCM × R-GFP mice between 6 – 12 weeks of age followed by disassociation of cells from the hearts of these mice in h (white arrow shows rare cardiomyocyte) that is quantified in j (127,284 cardiomyocytes across 2 hearts, 7 were eGFP+,*P<0.05 vs R-GFP). k,l,m,n, Tamoxifen treatment of Kit+MCM × R-GFP mice between 8 and 14 weeks of age with myocardial infarction (MI) on week 10. l, Immunohistological heart section for desmin (red) and eGFP (green) with nuclei in blue (arrow shows a cardiomyocyte from the c-kit+ lineage). m, n, Disassociated cardiomyocytes show rare but definitive myocyte labeling (white arrow), which was quantified in n (225,760 cardiomyocytes from 2 MI hearts, 37 were eGFP+,*P<0.05 vs R-GFP). o,p, Tamoxifen treatment between 8 – 12 weeks of age with MI injury occurring 3 days after tamoxifen cessation. p, Quantitation of eGFP+ cardiomyocytes from histological images taken from 2 hearts of these mice. eGFP+ cardiomyocytes were quantified as a percentage of the total cardiomyocyte fraction in >50 histological sections across the entire heart. *P<0.05 vs R-GFP. All error bars represent s.e.m.
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Figure 3: Inducible Cre expression from the Kit locus shows limited adult cardiomyocyte formationa, Genetic cross between Kit+/MCM and R-GFP reporter mice to lineage trace c-kit expressing cells when tamoxifen is present. b, Schematic showing tamoxifen treatment between day 1 and 6 months of age (panels c–g). c, d, Representative FACS plots with c-kit antibody (APC) vs eGFP from bone marrow of Kit+/MCM × R-GFP mice without (c) or with tamoxifen (d). e, FACS quantification of eGFP+ cells from bone marrow of these mice (n=2 mice for R-GFP and n=4 for Kit+/MCM × R-GFP). *p<0.05 vs R-GFP. f, g, Representative heart sections from Kit+MCM × R-GFP mice showing c-kit+ lineage cells in green and cardiomyocytes in red (desmin antibody). White arrow indicates eGFP+ adult cardiomyocyte. h,i,j, Tamoxifen treatment of Kit+/MCM × R-GFP mice between 6 – 12 weeks of age followed by disassociation of cells from the hearts of these mice in h (white arrow shows rare cardiomyocyte) that is quantified in j (127,284 cardiomyocytes across 2 hearts, 7 were eGFP+,*P<0.05 vs R-GFP). k,l,m,n, Tamoxifen treatment of Kit+MCM × R-GFP mice between 8 and 14 weeks of age with myocardial infarction (MI) on week 10. l, Immunohistological heart section for desmin (red) and eGFP (green) with nuclei in blue (arrow shows a cardiomyocyte from the c-kit+ lineage). m, n, Disassociated cardiomyocytes show rare but definitive myocyte labeling (white arrow), which was quantified in n (225,760 cardiomyocytes from 2 MI hearts, 37 were eGFP+,*P<0.05 vs R-GFP). o,p, Tamoxifen treatment between 8 – 12 weeks of age with MI injury occurring 3 days after tamoxifen cessation. p, Quantitation of eGFP+ cardiomyocytes from histological images taken from 2 hearts of these mice. eGFP+ cardiomyocytes were quantified as a percentage of the total cardiomyocyte fraction in >50 histological sections across the entire heart. *P<0.05 vs R-GFP. All error bars represent s.e.m.
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