Cardiovascular Research Advance Access published September 10, 2010
C-KIT+ CARDIAC PROGENITORS EXHIBIT MESENCHYMAL MARKERS AND PREFERENTIAL CARDIOVASCULAR COMMITMENT Elisa Gambini†,§, Giulio Pompilio†,‡,§, Andrea Biondi$, Francesco Alamanni‡,§, Maurizio C. Capogrossi#, Marco Agrifoglio‡,§, Maurizio Pesce†,*
†
Laboratorio di Biologia Vascolare e Medicina Rigenerativa, Centro Cardiologico Monzino-IRCCS
§
Dipartimento di Scienze Cardiovascolari, Università di Milano
‡
Dipartimento di Chirurgia Cardiovascolare, Centro Cardiologico Monzino-IRCCS
$
Centro Ricerca M. Tettamanti, Clinica Pediatrica Università degli Studi di Milano Bicocca
#
*
Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’ Immacolata, IDI-IRCCS
Corresponding Author: Maurizio Pesce, Ph.D., Laboratorio di Biologia Vascolare e Medicina
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E-mail:
[email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email:
[email protected]
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Rigenerativa, Centro Cardiologico Monzino, IRCCS, Via Parea, 4, 20138, Milan, Italy
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ABSTRACT
Aims - The heart contains c-kit+ progenitors that maintain cardiac homeostasis. Cardiac ckit+ cells are multipotent and give rise to myocardial, endothelial and smooth muscle cells, both in vitro and in vivo. C-kit+ cells have been deeply investigated for their stem cell activity, susceptibility to stress conditions and aging, as well as for their ability to repair the infarcted heart. Recently, expression of mesenchymal stem cell (MSCs) markers and MSC differentiation potency have been reported in CPCs. Based on these evidences, we hypothesised that c-kit+ cells may have phenotypic and functional features in common with cardiac MSCs.
Methods and Results – Culture of cells obtained from enzymatic dissociation of heart
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mesenchymal (MSC) markers. C-kit+ cells co-expressing MSC markers were identified in this population, were sorted by flow cytometry and were cultured in the presence or the absence of unselected cardiac cells from the same patient. Subsets of c-kit+ cells coexpressed MSCs markers also in vivo, as detected by immunofluorescence analysis of auricle tissue. Ex vivo expanded c-kit+ cells produced osteoblasts and adipocytes, although less preferentially than bone marrow derived MSCs, possessed vascular smooth muscle cells features, and were induced to differentiate into endothelial-like and cardiac-like cells.
Conclusions – In line with previous findings, our results indicate c-kit+ CPCs as primitive stem cells endowed with multilineage differentiation ability. They further suggest a possible relationship between these cells and a heart-specific MSC population with cardiovascular commitment potential.
Keywords: c-kit, cardiac progenitor, cardiac MSC, differentiation
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auricle fragments produced a fast growing fibroblast-like population expressing
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1. INTRODUCTION
After the seminal discovery that the adult heart is a self-renewing organ, undergoing continuous myocytes replacement during lifespan1-3, several candidate progenitor cells that sustain myocardium self-renewal have been found. These include: side population (SP) myocardial cells, identified on the basis of their ability to extrude drugs by activity of MDR-1 gene product4, progenitor cells characterized by expression of Sca-1/Sca-1-like antigens5, 6, cells derived from the so called “cardiospheres”
7, 8
, Isl-1+ cardioblasts9, 10, epicardial tissue stem cells11 and c-kit+ cardiac
progenitor cells (CPCs)12-14. While CPCs discovery has raised expectations for the devise of stem cell-based therapy of the heart, several issues still exist concerning an unambiguous definition of cardiac-derived stem cells phenotype.
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mononuclear cellular fraction isolated by density gradient centrifugation of crude bone marrow (BM), followed by culture under adhesive conditions. Compared to other stem cell types, MSCs cannot be uniquely distinguished by expression of tissue-specific markers. In fact, MSCs exist in the stromal compartment of virtually every tissue and are characterized by expression of unspecific antigen sets15. Recently, minimal consensus criteria for MSCs identification have been established; these are plastic adherence and formation of the so called Colony Forming Units-Fibroblast (CFUF) colonies, osteogenic and adipogenic differentiation ability, CD29 (Integrin-β1), CD44 (H-CAM), CD90 (Thy-1), CD105 (Endoglin) expression, and absence of hematopoietic (stem) cell markers such as CD34, CD45 and CD13316. Preclinical studies have shown that BM-derived MSCs have a potential to repair the infarcted heart by promoting cardioprotection and neo-vascularization through a potent paracrine effect also by direct differentiating into cardiac myocytes
20-22
17-19
, but
. The advantage of MSCs over CPCs is the
relative easiness and reproducibility of techniques to obtain and expand them in culture; a potential disadvantage of these cells compared to CPCs may arise from their commitment to differentiate into osteogenic cells, therefore raising the risk for unwanted bone formation in the
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Mesenchymal stem cells (MSCs) have been first derived from the adherent component of the
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heart23-25. Despite this, the feasibility of BM-derived MSCs use for myocardial repair in patients has been demonstrated in a recent safety clinical study26 where no side effects of this kind were observed. The existence of a heart-specific MSCs has not yet been formally demonstrated. On the other hand, results with cardiosphere-derived cells have suggested the existence of these cells based, e.g., on expression of CD90 and CD105 markers7, 27, 28; furthermore, CPCs isolated from the human heart have been shown to differentiate into adipocytes and osteocytes in vitro29. These results led us to hypothesize that c-kit+ cells may have phenotypic and functional features in common with cardiac-specific MSCs. In the present study, we therefore investigated expression of MSCs markers in c-kit+ cells in the human heart tissue and devised a protocol to culture cardiac cellular populations and isolate c-kit+ cells by high throughput flow cytometry. Using this system,
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finally investigated in c-kit+ CPCs before and after ex vivo amplification steps.
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expression of cardiovascular and mesenchymal markers as well as multilineage differentiation were
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2. MATERIALS AND METHODS
2.1 Ethical statement The collection of auricle fragments fragments and bone marrow specimens was performed after patients’ formal approval of an informed consent. The study was approved by Local Ethical Committee and review Board (approved on Aug 4, 2008; reference no CCFM C9/607 and CCFM C10/607), and was performed according to Italian national laws. Experimentation conformed with the principles outlined in the Declaration of Helsinki.
2.2 Cell isolation, culture and differentiation The procedure to obtain primary and secondary cultures of human CPCs from auricolae fragments
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supplementary material. To assess mesenchymal and cardiovascular in vitro commitment, CPCs were cultured under conditions known to induce adipogenic, osteogenic, endothelial and cardiac differentiation. The methods used to perform these experiments are detailed in the online supplementary material.
2.3 Flow Cytometry A FACSAria (Beckton-Dickinson) flow cytometer/cell sorter was used to identify and separate c-kit+ from c-kit- cells. Cell sorting set up and parameters are described in the online supplementary material. Analysis of mesenchymal, endothelial, HLA and hematopoietic markers was performed by multicolour analysis flow cytometry. Antibody panels and staining conditions are described in the online supplementary material.
2.4 q-RT-PCR Total RNA was purified from primary cells, sorted c-kit+ cells, transwell c-kit+-derived cells at P4 before and after culturing into pro-adipogenic, pro-osteogenic or cardiac inducing conditions. After
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is adapted from an already published method13. They are described in details in the online
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reverse transcription, primers specific for cardiac, stem cell, differentiated mesenchymal cells and vascular cells were used to assess cell phenotype of human CPCs by real time PCR. Experimental procedures, conditions and a full list of primers used in these experiments are described in the online supplementary material.
2.5 Immunofluorescence, confocal analysis Transwell c-kit+-derived cells at P4 were fixed or sub-cultured for 1-3 weeks into cardiac differentiation media; the cells were fixed using 4% paraformaldheyde (PFA) in PBS. After blocking with PBS containing 5% serum and 0.3% Triton-X100 for an overnight at 4°C; cells were incubated with primary antibodies for alpha-Smooth Muscle Actin (α-SMA) or cardiac markers. Auricle fragments tissue biopsies were fixed with 4% PFA and further processed for tissue
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supplementary material.
2.6 Cytokine, chemokine and growth factor detection Bio-Plex assay (Bio-Rad Laboratories, Italy), a bead-based multiplex immunoassay, was used to quantify cytokines, chemokines and growth factors released in the culture supernatant conditioned for 24 hrs by similar amounts of primary cells at T0 and supporting stromal cells at P4. Supernatants were collected after 24h of conditioning and analysed by Bio-plex assay.
2.7 Statistical analysis Comparison of results was performed by using paired or unpaired Student’s t-test using GraphPad statistical software. A P
