Medium A is composed of EGM-2 medium (Lonza Clonetics, Inc)46, medium B is composed of MEM Alpha (Invitrogen Corp

Medium A is composed of EGM-2 medium (Lonza Clonetics, Inc)46, medium B is composed of MEM Alpha (Invitrogen Corp.), 10% FBS (Invitrogen Corp.), 100?U/ml penicillin G, 100 microg/ml streptomycin (Invitrogen Corp.), 1?M dexamethasone (Sigma-Aldrich), 50?g/ml ascorbic acid (Sigma-Aldrich), and 10?mM -glycerophosphate (Sigma-Aldrich)22. limiting factor in the field of heart regeneration. Although more and more results demonstrate the involvement of paracrine signals, their identification as well as their origin are not yet known. Interestingly, in senescent hearts, the proliferation of c-kit+ cells can be re-activated by the stem cell factor15. Bone Morphogenetic Protein (BMP) gradient in the heart seems also to modulate the differentiation of the c-kit+ cells of cardiac neural crest origin12. Using R26R-confetti mice, it was shown that Sca-1+ cells contribute more to cardiomyocyte renewal in physiological (i.e. during physiological growth and ageing) than in pathophysiological (i.e. after ischemia or pressure overload) conditions4. Thus, the relative non-activation of the Sca-1+ CPCs in the ischemic hearts could be due either to the presence of an inactivating factor or to the absence of a stimulating factor. Identifying the factors able to stimulate CPC proliferation and differentiation will be essential for further development of therapeutically strategies aimed to stimulate heart regeneration even in elderly patients suffering from cardiac vascular diseases. Recently, we identified a factor able to increase the number of newly formed cardiomyocytes in mouse hearts during physiological growth and after myocardial infarction (MI)16. The Brain Natriuretic Peptide (BNP) is a cardiac hormone secreted through a constitutive mechanism by ventricular cardiomyocytes, fibroblasts, endothelial cells and even by infiltrating neutrophils, T-cells and macrophages after MI17. Interestingly, BNP is also secreted by immature cells, such as embryonic stem cells18, satellite cells19 or CPCs20. BNP binds to two guanylyl cyclase receptors, denoted NPR-A and NPR-B, which leads to the generation of intracellular cGMP21. The accumulation of cGMP in the cytoplasm activates protein kinase G (PKG) and the phosphodiesterases 2, 3 or 521. We recently demonstrated that BNP injections into neonatal and adult healthy or infarcted mice led to reduced heart dilation associated at the cellular level to increased number of CycLuc1 Nkx2.5+ actinin? cells and newly formed cardiomyocytes16. BNP clearly stimulated the proliferation of the Nkx2.5+ non myocyte cells (NMCs) and their differentiation into cardiomyocytes. Thus, in this report we determined the nature of the cell subset (i.e. from c-kit or Sca-1 origin) responding to BNP stimulation among NMCs and we identified the signaling pathway involved. Results BNP increases the number of Sca-1+ cells To determine whether T BNP treatment modified the number of c-kit+ or/and Sca-1+ cells, flow cytometry analysis using antibodies against c-kit or Sca-1 proteins were performed on NMCs isolated from neonatal mouse hearts and cultured with or without BNP for up to 11 days (i.e. until reaching confluence). BNP treatment didnt statistically modify the total number of cells (?27%, p?=?0.14 at 4 days and +12%, p?=?0.12 at 11 days) (Fig. 1A) but increased the percentages of Sca-1 positive cells after 4 (+18%, p?=?0.03) and 11 days (+95%, p?=?0.0001) (Fig. 1B). The percentages of c-kit+ cells remained similar between BNP treated and untreated cells (Fig. 1B). As a consequence, the total number of Sca-1+ cells was increased after 11 days of treatment (+89% compared to untreated cells, p?=?0.0001) and the number of c-kit+ cells remained unchanged (Fig. 1C). Accordingly, mRNA levels coding for Sca-1 was increased in BNP treated cells compared to the untreated ones (Supplemental Fig. 1A). Open in a separate window Figure 1 BNP stimulates Sca-1+ cell proliferation.(A) Non myocyte cells (NMCs) were isolated from neonatal hearts of C57BL/6 mice, cultured 4 and 11 days with or without BNP (untreated cells) and counted. (B) Percentages of c-kit+ and Sca-1+ cells obtained by flow cytometry analysis on BNP treated or untreated NMCs. (C) Number of cells expressing the c-kit or the Sca-1 protein in NMCs treated or not with BNP for 4 and 11 days calculated with the total number of cells and the percentages of the c-kit+ and Sca-1+ cells. (ACC) n?=?8 and 16 different experiments after 4 and 11 days of culture, respectively. (D) Representative histogram of NMC sorting for Sca-1 expression. The numbers represent the percentage.Gene expression data were normalized to the expression levels of 18S (CT values). identification as well as their origin are not yet known. Interestingly, in senescent hearts, the proliferation of c-kit+ cells can be re-activated by the stem cell factor15. Bone Morphogenetic Protein (BMP) gradient in the heart seems also to modulate the differentiation of the c-kit+ cells of cardiac neural crest origin12. Using R26R-confetti mice, it was shown that Sca-1+ cells contribute more to cardiomyocyte renewal in physiological (i.e. during physiological growth and ageing) than in pathophysiological CycLuc1 (i.e. after ischemia or pressure overload) conditions4. Thus, the relative non-activation of the Sca-1+ CPCs in the ischemic hearts could be due either to the presence of an inactivating factor or to the absence of a stimulating factor. Identifying the factors able to stimulate CPC proliferation and differentiation will be essential for further development of therapeutically strategies CycLuc1 aimed to stimulate heart regeneration even in elderly patients suffering from cardiac vascular diseases. Recently, we identified a factor able to increase the number of newly formed cardiomyocytes in mouse hearts during physiological growth and after myocardial infarction (MI)16. The Brain Natriuretic Peptide (BNP) is a cardiac hormone secreted through a constitutive mechanism by ventricular cardiomyocytes, fibroblasts, endothelial cells and even by infiltrating neutrophils, T-cells and macrophages after MI17. Interestingly, BNP is also secreted by immature cells, such as embryonic stem cells18, satellite cells19 or CPCs20. BNP binds to two guanylyl cyclase receptors, denoted NPR-A and NPR-B, which CycLuc1 leads to the generation of intracellular cGMP21. The build up of cGMP in the cytoplasm activates protein kinase G (PKG) and the phosphodiesterases 2, 3 or 521. We recently shown that BNP injections into neonatal and adult healthy or infarcted mice led to reduced heart dilation associated in the cellular level to improved quantity of Nkx2.5+ actinin? cells and newly formed cardiomyocytes16. BNP clearly stimulated the CycLuc1 proliferation of the Nkx2.5+ non myocyte cells (NMCs) and their differentiation into cardiomyocytes. Therefore, in this statement we determined the nature of the cell subset (i.e. from c-kit or Sca-1 source) responding to BNP activation among NMCs and we recognized the signaling pathway involved. Results BNP increases the quantity of Sca-1+ cells To determine whether BNP treatment altered the number of c-kit+ or/and Sca-1+ cells, circulation cytometry analysis using antibodies against c-kit or Sca-1 proteins were performed on NMCs isolated from neonatal mouse hearts and cultured with or without BNP for up to 11 days (i.e. until reaching confluence). BNP treatment didnt statistically improve the total quantity of cells (?27%, p?=?0.14 at 4 days and +12%, p?=?0.12 at 11 days) (Fig. 1A) but increased the percentages of Sca-1 positive cells after 4 (+18%, p?=?0.03) and 11 days (+95%, p?=?0.0001) (Fig. 1B). The percentages of c-kit+ cells remained related between BNP treated and untreated cells (Fig. 1B). As a consequence, the total quantity of Sca-1+ cells was improved after 11 days of treatment (+89% compared to untreated cells, p?=?0.0001) and the number of c-kit+ cells remained unchanged (Fig. 1C). Accordingly, mRNA levels coding for Sca-1 was improved in BNP treated cells compared to the untreated ones (Supplemental Fig. 1A). Open in a separate window Number 1 BNP stimulates Sca-1+ cell proliferation.(A) Non myocyte cells (NMCs) were isolated from neonatal hearts of C57BL/6 mice, cultured 4 and 11 days with or without BNP (untreated cells) and counted. (B) Percentages of c-kit+ and Sca-1+ cells acquired by circulation cytometry analysis on BNP treated or untreated NMCs. (C) Quantity of cells expressing the c-kit or the Sca-1 protein in NMCs treated or not with BNP for 4 and 11 days calculated with the total quantity of cells and the percentages of the c-kit+ and Sca-1+ cells. (ACC) n?=?8 and 16 different experiments after 4 and 11 days of tradition, respectively. (D) Representative histogram of NMC sorting for Sca-1 manifestation. The figures represent the percentage of the cells compared to the total number of sorted NMCs. n?=?18C43 different experiments. (E) Quantity of sorted.