Christelle Bielmann

Functional late outgrowth endothelial progenitors isolated from peripheral blood of burned patients

BACKGROUND Bioengineered skin substitutes are increasingly considered as a useful option for the treatment of full thickness burn injury. Their viability following grafting can be enhanced by seeding the skin substitute with late outgrowth endothelial progenitor cells (EPCs). However, it is not known whether autologous EPCs can be obtained from burned patients shortly after injury. METHODS Late outgrowth EPCs were isolated from peripheral blood sampled obtained from 10 burned patients (extent 19.6±10.3% TBSA) within the first 24h of hospital admission, and from 7 healthy subjects. Late outgrowth EPCs were phenotyped in vitro. RESULTS In comparison with similar cells obtained from healthy subjects, growing colonies from burned patients yielded a higher percentage of EPC clones (46 versus 17%, p=0.013). Furthermore, EPCs from burned patients secreted more vascular endothelial growth factor (VEGF) into the culture medium than did their counterparts from healthy subjects (85.8±56.2 versus 17.6±14pg/mg protein, p=0.018). When injected to athymic nude mice 6h after unilateral ligation of the femoral artery, EPCs from both groups of subjects greatly accelerated the reperfusion of the ischaemic hindlimb and increased the number of vascular smooth muscle cells. CONCLUSIONS The present study supports that, in patients with burns of moderate extension, it is feasible to obtain functional autologous late outgrowth EPCs from peripheral blood. These results constitute a strong incentive to pursue approaches based on using autotransplantation of these cells to improve the therapy of full thickness burns.

Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1.

Brain Natriuretic Peptide (BNP) injections in adult “healthy” or infarcted mice led to increased number of non-myocyte cells (NMCs) expressing the nuclear transcription factor Nkx2.5. The aim of this study was to identify the nature of the cells able to respond to BNP as well as the signaling pathway involved. BNP treatment of neonatal mouse NMCs stimulated Sca-1+ cell proliferation. The Sca-1+ cells were characterized as being a mixed cell population involving fibroblasts and multipotent precursor cells. Thus, BNP treatment led also to increased number of Sca-1+ cells expressing Nkx2.5, in Sca-1+ cell cultures in vitro and in vivo, in the hearts of neonatal and adult infarcted mice. Whereas BNP induced Sca-1+ cell proliferation via NPR-B receptor and protein kinase G activation, CNP stimulated Sca-1+ cell proliferation via NPR-B and a PKG-independent mechanism. We highlighted here a new role for the natriuretic peptide receptor B which was identified as a target able to modulate the proliferation of the Sca-1+ cells. The involvement of NPR-B signaling in heart regeneration has, however, to be further investigated.

0232: A new role of the brain natriuretic peptide in the heart: Modulation of cardiac precursor cell proliferation and differentiation

The actual role of the brain natriuretic peptide (BNP) in the heart remains elusive despite its reported protective effect in ischemic animal hearts. Because recently BNP was shown to control the proliferation and differentiation of murine embryonic stem cells, we asked in this study whether BNP could influence the proliferation and differentiation of cardiac progenitor cells (CPC) in vitro and in vivo . We first identified a c-kit + Sca-1 + cell population present in neonatal and adult hearts which expressed the NPR-A and NPR-B receptors. In vitro, these cells proliferated and in presence of BNP differentiated into CPCs (c-kit + Sca-1 + Nkx2.5 + ) and into mature cardiomyocytes. In parallel, BNP was injected to newborn and adult healthy mice (n=6 mice per group). In the hearts of both neonatal and adult mice, BNP injection increased the number of newly formed cardiomyocytes (neonatal: + 23%, p= 0.009 and adult: +68%, p= 0.005) and the number of CPCs (neonatal: + 142%, p= 0.002 and adult: +134%, p= 0.04). BrdU injection to neonatal BNP treated mice demonstrated that BNP stimulated CPC proliferation. In anticipation that BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction (n=6-7 mice per group). Higher numbers of Nkx2.5+ cells were detected in both the infarcted (+38%, p=0.03) and non infarcted areas (+69%, p=0.02) of BNP treated hearts one week after surgery. Finally, by isolating neonatal cardiac cells from the hearts of NPR-A or NPR-B deficient mice, we demonstrated that BNP modulates the fate of CPCs via NPRB binding and that long term BNP treatment is correlated in vitro and in vivo with decreased Protein Kinase G activity. Our results highlight a new key role for BNP in the control of CPC proliferation and/or differentiation. This new function of BNP should be evaluated in therapies aimed to induce cardiac cell regeneration and should reopen the debate about the therapeutic use of BNP for patients suffering from heart diseases.

0006 : Proliferation of the cardiac precursor cells expressing the stem cell antigen-1 is controlled by activation of the natriuretic peptide receptors

A part of the cardioprotective role of the Brain Natriuretic Peptide (BNP) in mouse hearts is due to its effect on the cardiac precursor cell (CPC) proliferation and differentiation. Thus, in this study we identified the CPC subset able to respond to BNP as well as the signaling pathway involved. We demonstrated by immunohistochemistry and by flow cytometry analysis that the c-kit+ and the Sca-1+ cell subsets in neonatal and adult murine hearts express the NPR-A and NPR-B receptors and are thus able to be stimulated by BNP. In vitro, BNP only stimulated the proliferation of the Sca-1+ cells and not of the c-kit+ cells. Among Sca-1+ cells, BNP treatment led to increased number of Sca-1+ Nkx2.5+ cells, which were able to differentiate into cardiomyocytes. To determine by which receptor BNP acts on Sca-1+ cells to stimulate their proliferation, cells were isolated from neonatal hearts of mice deficient for the NPR-A (NPRA-KO) or NPR-B receptor. BNP stimulated the proliferation of the Sca-1+ NPR-A KO cells but not of the Sca-1+ cells lacking the NPR-B receptor, demonstrating that Sca-1+ cell proliferation is linked to NPR-B activation. This was confirmed by stimulating the Sca-1+ cells by the C-Natriuretic Peptide able also to activate the NPR-B receptor. BNP binding to NPR-B receptor led in Sca-1+ cells to Protein Kinase G activation and increased phosphorylation of phospholamban and p38. Reducing PKG activation inhibited BNP-induced-Sca-1+ cell proliferation, whereas reducing p38 phosphorylation increased Sca-1+ cell proliferation after BNP treatment. Phosphorylation of p38 was not mediated by BNP binding to NPR-B receptor but by its binding to NPR-A. In this work, we identified the Sca-1+ cells as being the targets of BNP in vitro and in vivo. BNP via NPR-B binding and PKG activation clearly stimulated the proliferation of the CPCs expressing Sca-1. Interestingly, is the dual role of the NPR-A and NPR-B receptors which control Sca-1+ cell proliferation. The author hereby declares no conflict of interest

0128 : Sca-1 positive cells, but not c-kit positive cells, differentiate into mature cardiomyocytes after brain natriuretic peptide treatment

The Brain Natriuretic Peptide (BNP) is a cardiac hormone, which promotes the recovery of cardiac function and the preservation of cardiac tissue in animal models of heart diseases. Its cardiac protective role in animals was attributed to fibrosis inhibition, as well as to reduction of cardiomyocyte apoptosis and hypertrophy. Recently, we demonstrated that BNP induces heart regeneration via the stimulation of cardiac precursor cell (CPC) proliferation and differentiation into mature cardiomyocytes. The aim of our study was to identify which CPC’s subset is able to respond to BNP stimulation. Cardiac precursor cells identified as being Sca-1 + Nkx2.5 + or c-kit + Nkx2.5 + cells, expressed in neonatal and adult hearts BNP’s receptors (NPR-A and NPR-B), showing their ability to be activated by BNP treatment. Cell sorting experiments based on the expression of Sca-1 or c-kit were performed on nonmyocyte cells isolated from neonatal wild-type hearts. Sca-1 + and c-kit + cells were cultured up to 3 weeks with or without BNP in differentiating medium. Sca-1 positive cells, which contained few c-kit + cells, responded clearly to BNP stimulation by upregulating mRNA levels of genes coding for Nkx2.5, Mlc-2v, c-kit, Sca-1, beta and alpha MHC. Furthermore, higher number of Troponin I + cells was detected in BNP treated cells compared to untreated cells, suggesting that Sca-1 + cells differentiated after BNP stimulation into mature cardiomyocytes. BNP treatment of c-kit + cells didn’t induce the upregulation of mRNA coding for cardiomyocyte specific genes. However, we determined that c-kit positive cells spontaneously differentiated into mature cardiomyocytes during the 3 weeks of cell culture without BNP stimulation. To determine which receptor is involved, Sca-1 + cells, isolated from neonatal hearts of NPR-A or NPR-B deficient mice, were treated with BNP. The effects of BNP on wild type and NPR-A KO cells did not differ substantially. However, Sca-1 + cells isolated from NPR-B deficient hearts couldn’t respond anymore to BNP stimulation. Thus, BNP specifically stimulates via NPR-B Sca-1 + cell differentiation into cardiomyocytes. c-kit + cells display clearly a cardiogenic potential which is BNP independent.

0130 : Is the stem cell antigen 1 involved in the brain natriuretic peptide effect on cardiac precursor cells?

Cardiac cellular therapy can be intended by stimulation of “endogenous” cardiac precusor cells (CPCs), identified by several markers such as the Stem Cell Antigen 1 (Sca-1) or the stem cell factor c-kit. CPCs are able to proliferate and differentiate into beating cardiomyocytes in vivo and in vitro after brain natriuretic peptide (BNP) treatment. BNP is a cardiac hormone, whose role in the heart remains to be clarified. Interestingly, BNP treatments, in vivo and in vitro , induced an upregulation of mRNA coding for Sca-1 and a 2 fold increase of the number of CPCs. In previous work, we showed that Sca-1 KO mice had two times less CPCs, and two times less BNP mRNA expression compared to Wild Type mice. The aim of this project is to determine whether the effect of BNP on CPCs is linked to Sca-1 expression. Sca-1 KO mice developed a dilated cardiomyopathy with ageing. To determine whether reduced amount of BNP is responsible for the development of this cardiac disease, neonatal Sca-1 KO mice were treated with BNP for 2 weeks. Mice were sacrificed 13 weeks later, and cardiac parameters were measured by echocardiography. The thickness of the left ventricular posterior wall and the ejection fraction were increased in BNP-treated Sca-1 deficient mice (+15% and + 28%, respectively) compared to untreated Sca-1 KO mice. To explore the cellular mechanisms, in vitro experiments were performed. Non Myocytes cells (NMCs), containing CPCs, have been isolated from the heart of Sca-1 KO mice and cultured with or without BNP in media favouring either cell proliferation or differentiation into cardiomyocytes. BNP is not able to induce Sca-1KO cell proliferation, whereas CPC differentiation into cardiomyocytes is maintained even in absence of Sca-1 expression. In conclusion, BNP modulates the cardiomyopathy developed by Sca-1 KO mice. BNP effect on CPC proliferation is linked to Sca-1 expression, but its effect on differentiation is Sca-1 independent.

0129 : Adult human mononuclear clones isolated from peripheral blood can differentiate into immature cardiomyocytes

Breakthroughs in stem cell biology and demonstrations of the heart’s endogenous regenerative capacities have incited an intense race towards cardiac regeneration, i.e. the replacement of lost myocardium after myocardial infarction. In preclinical trials, cell transplantation therapies, using adult human multipotent stem cells (e. g, hematopoietic stem cells) into infarcted myocardium has shown enhanced cardiogenesis in animal models. Nevertheless, results in clinical trials remain unsatisfactory and determining a suitable cell population that is easily harvested and improves cardiac repair is challenging. In previous research, our lab isolated human peripheral blood mononuclear clones (PBMCs) bearing cardiac mesodermal markers, e.g. c-kit, Islet-1 or Flk-1. Potentially, these cells can differentiate within the cardiac lineage to mature cardiomyocytes and partMyosin heavy chainicipate in heart repair. We sought to establish an in vitro cardioinstructive differentiation protocol to derive cardiomyocytes from our PBMC population. For this, we screened three differentiation protocols, i.e. Keller, Smith and He protocols. To follow cell differentiation RT-qPCR and immunostainings were performed for relevant genes, e.g. Nkx2.5, GATA4, Troponin T (cTNT), (β-Myosin heavy chain ((β-MHC) and (-actinin. Results for mRNA expression showed that at least 75% of PBMCs can derive to a cardiac precursor population (Nkx2.5+/GATA4+) following Keller and Smith protocol. In Smith protocol, 75% of PBMCs differentiated to immature cardiomyocytes (cTNT+) of which 50% expressed both cTNT and (- MHC when co-cultured with neonatal mice ventricular myocytes. Immunofluoresence assay showed that PBMCs in both Keller and Smith protocol are Nkx2.5+/(α-actinin+ demonstrating differentiation at the protein level. Next we evaluated the safety, survival and integration of injected PBMCs in neonatal Gt(ROSA)26-Tomato mice tissues in vivo. By staining PBMC with membrane marker PKH2GL, we followed cell homing within the animal tissues visualized by fluorescent microscopy on tissue sections. In conclusion, we demonstrated that human PBMCs can differentiate, under certain conditions, into cardiac precursor cells or immature cardiomyocytes in vitro. These results make them an interesting and promising cell source for stem cell therapies in cardiac repair.