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Effects of Age and Heart Failure on Human Cardiac Stem Cell Function

2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2011.03.036

1525-2191

Daniela Cesselli, Antonio Paolo Beltrami, Federica D’Aurizio, Patrizia Marcon, Natascha Bergamin, Barbara Toffoletto, Maura Pandolfi, Elisa Puppato, Laura Mariño, Sergio Signore, Ugolino Livi, Roberto Verardo, Silvano Piazza, Luigi Marchionni, Claudia Fiorini, Claudio Schneider, Toru Hosoda, Marcello Rota, Jan Kajstura, Piero Anversa, Carlo Alberto Beltrami, Annarosa Leri,

Muscle Physiology and Disorders

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21Cip1 and p16INK4a expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy. Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21Cip1 and p16INK4a expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy. The recognition that the human heart possesses a compartment of c-Kit-positive cardiac stem cells (CSCs) that can regenerate myocytes and coronary vessels offers the unique opportunity to reconstitute the damaged myocardium, restoring in part the physiological and anatomical characteristics of the normal heart. Human CSCs (hCSCs) can be isolated from small tissue samples and, after their expansion in vitro, can be delivered back to the same patient in an attempt to repair the failing heart.1Smith R.R. Barile L. Cho H.C. Leppo M.K. Hare J.M. Messina E. Giacomello A. Abraham M.R. Marbán E. Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens.Circulation. 2007; 115: 896-908Crossref PubMed Scopus (950) Google Scholar, 2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar, 3Beltrami A.P. Cesselli D. Bergamin N. Marcon P. Rigo S. Puppato E. D'Aurizio F. Verardo R. Piazza S. Pignatelli A. Poz A. Baccarani U. Damiani D. Fanin R. Mariuzzi L. Finato N. Masolini P. Burelli S. Belluzzi O. Schneider C. Beltrami C.A. Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).Blood. 2007; 110: 3438-3446Crossref PubMed Scopus (284) Google Scholar, 4Bearzi C. Leri A. Lo Monaco F. Rota M. Gonzalez A. Hosoda T. Pepe M. Qanud K. Ojaimi C. Bardelli S. D'Amario D. D'Alessandro D.A. Michler R.E. Dimmeler S. Zeiher A.M. Urbanek K. Hintze T.H. Kajstura J. Anversa P. Identification of a coronary vascular progenitor cell in the human heart.Proc Natl Acad Sci USA. 2009; 106: 15885-15890Crossref PubMed Scopus (171) Google Scholar, 5Itzhaki-Alfia A. Leor J. Raanani E. Sternik L. Spiegelstein D. Netser S. Holbova R. Pevsner-Fischer M. Lavee J. Barbash I.M. Patient characteristics and cell source determine the number of isolated human cardiac progenitor cells.Circulation. 2009; 120: 2559-2566Crossref PubMed Scopus (115) Google Scholar This discovery has laid the groundwork for the use of hCSCs in the treatment of the human disease. Preclinical studies have been completed, and two phase I clinical trials are in progress [NCT00474461 (last accessed December 22, 2010) and NCT00893360 (last accessed January 18, 2011) at http://www.clinical trials.gov]. Nonetheless, aging, cardiac hypertrophy, ischemic myocardial injury, and metabolic disorders, together with genetic and environmental factors, may dramatically affect the growth and differentiation behavior of resident hCSCs.6Chimenti C. Kajstura J. Torella D. Urbanek K. Heleniak H. Colussi C. Di Meglio F. Nadal-Ginard B. Frustaci A. Leri A. Maseri A. Anversa P. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure.Circ Res. 2003; 93: 604-613Crossref PubMed Scopus (336) Google Scholar, 7Urbanek K. Quaini F. Tasca G. Torella D. Castaldo C. Nadal-Ginard B. Leri A. Kajstura J. Quaini E. Anversa P. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy.Proc Natl Acad Sci USA. 2003; 100: 10440-10445Crossref PubMed Scopus (451) Google Scholar, 8Urbanek K. Torella D. Sheikh F. De Angelis A. Nurzynska D. Silvestri F. Beltrami C.A. Bussani R. Beltrami A.P. Quaini F. Bolli R. Leri A. Kajstura J. Anversa P. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure.Proc Natl Acad Sci USA. 2005; 102: 8692-8697Crossref PubMed Scopus (546) Google Scholar, 9Dimmeler S. Leri A. Aging and disease as modifiers of efficacy of cell therapy.Circ Res. 2008; 102: 1319-1330Crossref PubMed Scopus (311) Google Scholar This possibility raises two critical questions. First, is heart failure a stem cell disease characterized by severe depletion of the hCSC pool? Second, does a compartment of functionally competent hCSCs persist in the decompensated heart and do these cells have potential therapeutic implications? As is the case for other stem cells,10Sahin E. Depinho R.A. Linking functional decline of telomeres, mitochondria and stem cells during ageing.Nature. 2010; 464: 520-528Crossref PubMed Scopus (543) Google Scholar the life cycle of hCSCs is regulated by telomerase activity and telomere length.2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar Telomerase is a reverse transcriptase that extends the 3′ chromosomal ends by using its own RNA as a template.11Blackburn E.H. The end of the (DNA) line.Nat Struct Biol. 2000; 7: 847-850Crossref PubMed Scopus (133) Google Scholar Telomerase activity delays but cannot prevent telomere erosion, which is dictated by down-regulation of telomerase, oxidative stress, and loss of telomere-related proteins.12de Lange T. How telomeres solve the end-protection problem.Science. 2009; 326: 948-952Crossref PubMed Scopus (617) Google Scholar Shortening of telomeres beyond a critical length triggers cellular senescence, which corresponds to irreversible growth arrest in the G1 phase with loss of specialized functions, including stem cell proliferation, migration, and differentiation. Despite suggestive evidence in humans and in genetically manipulated mice13Lansdorp P.M. Lessons from mice without telomerase.J Cell Biol. 1997; 139: 309-312Crossref PubMed Scopus (34) Google Scholar, 14Xin Z.T. Beauchamp A.D. Calado R.T. Bradford J.W. Regal J.A. Shenoy A. Liang Y. Lansdorp P.M. Young N.S. Ly H. Functional characterization of natural telomerase mutations found in patients with hematologic disorders.Blood. 2007; 109: 524-532Crossref PubMed Scopus (82) Google Scholar, 15Alter B.P. Baerlocher G.M. Savage S.A. Chanock S.J. Weksler B.B. Willner J.P. Peters J.A. Giri N. Lansdorp P.M. Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita.Blood. 2007; 110: 1439-1447Crossref PubMed Scopus (266) Google Scholar that telomere shortening is a major variable of cellular senescence and organ aging, it remains to be demonstrated whether hCSCs with critically shortened or dysfunctional telomeres undergo replicative senescence and apoptosis, and whether loss of telomere integrity is one of the variables involved in the decline of stem cell function in the failing human heart. These issues were addressed in the present study, and the consequences of aging and ventricular decompensation on the clonogenicity, multipotency, and migratory properties of hCSCs were characterized, based on analysis of normal donor hearts and explanted hearts in end-stage heart failure. Moreover, the telomere-telomerase axis and the presence of telomere dysfunction-induced foci (TIFs), in combination with markers of DNA damage response and replicative senescence, were determined, to obtain a common denominator for the processes that regulate the growth and death of hCSCs. Discarded atrial specimens, weighing 3 to 6 g, were collected over a period of 5 years from donor hearts at the time of transplantation and from explanted hearts of patients undergoing cardiac transplantation at the Cardiac Surgery Unit of the University Hospital of Udine in Italy. The clinical data are presented in Supplemental Tables S1 and S2 (available at http://ajp.amjpathol.org). Informed consent was obtained in accordance with the Declaration of Helsinki and with approval by the Independent Ethics Committee of the University of Udine. Histological analysis of samples from donor hearts did not show pathological changes. Samples were used for the isolation of c-Kit-positive hCSCs.2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar, 3Beltrami A.P. Cesselli D. Bergamin N. Marcon P. Rigo S. Puppato E. D'Aurizio F. Verardo R. Piazza S. Pignatelli A. Poz A. Baccarani U. Damiani D. Fanin R. Mariuzzi L. Finato N. Masolini P. Burelli S. Belluzzi O. Schneider C. Beltrami C.A. Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).Blood. 2007; 110: 3438-3446Crossref PubMed Scopus (284) Google Scholar, 4Bearzi C. Leri A. Lo Monaco F. Rota M. Gonzalez A. Hosoda T. Pepe M. Qanud K. Ojaimi C. Bardelli S. D'Amario D. D'Alessandro D.A. Michler R.E. Dimmeler S. Zeiher A.M. Urbanek K. Hintze T.H. Kajstura J. Anversa P. Identification of a coronary vascular progenitor cell in the human heart.Proc Natl Acad Sci USA. 2009; 106: 15885-15890Crossref PubMed Scopus (171) Google Scholar Fragments were also fixed in formalin and embedded in paraffin2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar, 3Beltrami A.P. Cesselli D. Bergamin N. Marcon P. Rigo S. Puppato E. D'Aurizio F. Verardo R. Piazza S. Pignatelli A. Poz A. Baccarani U. Damiani D. Fanin R. Mariuzzi L. Finato N. Masolini P. Burelli S. Belluzzi O. Schneider C. Beltrami C.A. Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).Blood. 2007; 110: 3438-3446Crossref PubMed Scopus (284) Google Scholar, 4Bearzi C. Leri A. Lo Monaco F. Rota M. Gonzalez A. Hosoda T. Pepe M. Qanud K. Ojaimi C. Bardelli S. D'Amario D. D'Alessandro D.A. Michler R.E. Dimmeler S. Zeiher A.M. Urbanek K. Hintze T.H. Kajstura J. Anversa P. Identification of a coronary vascular progenitor cell in the human heart.Proc Natl Acad Sci USA. 2009; 106: 15885-15890Crossref PubMed Scopus (171) Google Scholar for the identification of stem cell niches and for the quantitative analysis of lineage-negative hCSCs.8Urbanek K. Torella D. Sheikh F. De Angelis A. Nurzynska D. Silvestri F. Beltrami C.A. Bussani R. Beltrami A.P. Quaini F. Bolli R. Leri A. Kajstura J. Anversa P. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure.Proc Natl Acad Sci USA. 2005; 102: 8692-8697Crossref PubMed Scopus (546) Google Scholar, 16Urbanek K. Cesselli D. Rota M. Nascimbene A. De Angelis A. Hosoda T. Bearzi C. Boni A. Bolli R. Kajstura J. Anversa P. Leri A. Stem cell niches in the adult mouse heart.Proc Natl Acad Sci USA. 2006; 103: 9226-9231Crossref PubMed Scopus (376) Google Scholar, 17Hosoda T. D'Amario D. Cabral-Da-Silva M.C. Zheng H. Padin-Iruegas M.E. Ogorek B. Ferreira-Martins J. Yasuzawa-Amano S. Amano K. Ide-Iwata N. Cheng W. Rota M. Urbanek K. Kajstura J. Anversa P. Leri A. Clonality of mouse and human cardiomyogenesis in vivo.Proc Natl Acad Sci USA. 2009; 106: 17169-17174Crossref PubMed Scopus (114) Google Scholar Lineage-negative hCSCs were defined as cells positive for c-Kit and negative for CD45, to exclude the bone marrow origin and the mast cell phenotype, and negative for GATA4 and α-sarcomeric actin (α-SA), to exclude commitment to cardiac lineages. The localization of OCT3/4 and NANOG was determined in CD45-negative, c-Kit-positive hCSCs. Magnitude of sampling is presented in Supplemental Table S3 (available at http://ajp.amjpathol.org); antibodies are listed in Table 1.Table 1Antibodies for FACS, Immunolabeling, and Western BlottingAntigenAntibodySourceFluorochromePseudocolorFACS c-kitAPC-conjugated mouse mAbBDAPC c-kitPE-conjugated mouse mAbBDPE CD34FITC-conjugated mouse mAbBDFITC CD44FITC-conjugated mouse mAbBDFITC CD45FITC-conjugated mouse mAbBDFITC CD45Pacific Blue-conjugated mouse mAbDakoPacific Blue CD49aPE- conjugated mouse mAbBDPE CD73PE-conjugated mouse mAbBDPE CD90PE-conjugated mouse mAbBDPE CD133PE-conjugated mouse mAbMiltenyi BiotecPE CD146PE-conjugated mouse mAbBDPE CD105PE-conjugated mouse mAbSerotecPE ABCG2PE-conjugated mouse mAbBDPE KDRPE-conjugated mouse mAbSerotecPEImmunolabeling c-kitGoat pAbR&DA488, A555,Green, red c-kitRabbit pAbDakoA488, A555Green, red Oct3/4Rabbit pAbAbcamA488Yellow, red Oct3/4Goat pAbSanta CruzA555Yellow, red Oct3/4Mouse mAbSanta CruzCy5Yellow, red NanogMouse mAbAbcamA555, Cy5Red, white NanogRabbit pAbAbcamA488Magenta Nkx2.5Goat pAbSanta CruzCy5White GATA-6Rabbit pAbSanta CruzCy5Yellow Ets1Mouse mAbSanta CruzCy5Magenta vWFRabbit pAbSigma-AldrichA488Yellow α-SMAMouse mAbDakoA488Magenta α-CAMouse mAbSigmaTRITCRed α-SAMouse mAbSigma-AldrichTRITC, Cy5Red, white connexin 43Rabbit pAbSanta CruzA488Green p16INK4AMouse mAbNovocastraA488Green p21Mouse mAbCalbiochemA555Yellow phospho-p53ser15Mouse mAbCell SignalingA555Red γH2AXMouse mAbUpstateA488Green 53BP1Rabbit pAbCell SignalingA555RedWestern blotting phospho-p53ser15Mouse mAbCell Signaling RanMouse mAbSigma-AldrichA488 and A555, Alexa Fluor 488 and 555; APC, allophycocyanin; Cy5, cyanine 5; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; pAb, polyclonal antibody; PE, phycoerythrin; TRITC, tetramethylrhodamine isothiocyanate; vWf, von Willebrand factor; α-CA, α-cardiac actinin; α-SA, α-sarcomeric actin; α-SMA, α-smooth muscle actin. Open table in a new tab A488 and A555, Alexa Fluor 488 and 555; APC, allophycocyanin; Cy5, cyanine 5; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; pAb, polyclonal antibody; PE, phycoerythrin; TRITC, tetramethylrhodamine isothiocyanate; vWf, von Willebrand factor; α-CA, α-cardiac actinin; α-SA, α-sarcomeric actin; α-SMA, α-smooth muscle actin. Spectral analysis was performed with a TCS-SP2 confocal microscope (Leica, Wetzlar, Germany) using the lambda acquisition mode; myocardial sections were labeled by c-Kit, OCT3/4, and NANOG conjugated with Alexa Fluor 555, Cy5, and Alexa Fluor 555, respectively. Myocytes were stained with α-SA (Cy5) and nuclei with DAPI. The emission signal for Alexa Fluor 555 was excited at 543 nm with an argon laser, and the fluorescence intensity was recorded, generating a lambda stack ranging from 558 to 798 nm at 4.9-nm intervals. The emission signal for Cy5 was excited at 633 nm with a helium/neon laser, and the fluorescence intensity was recorded, generating a lambda stack ranging from 653 to 798 nm at 6-nm intervals. Lens and corresponding numerical aperture were 63× and 1.4, respectively. Sampling consisted of 30 c-Kit-positive, OCT3/4-positive, and NANOG-positive cells. Additionally, 10 cells negative for these markers and present in the same samples were used as control, to discriminate background autofluorescence from specific labeling. Each determination was restricted to the region of interest within each hCSC. For each region of interest, a graph plotting mean pixel intensity and the emission wavelength of the lambda stack was generated. This protocol has repeatedly been used in our laboratory.17Hosoda T. D'Amario D. Cabral-Da-Silva M.C. Zheng H. Padin-Iruegas M.E. Ogorek B. Ferreira-Martins J. Yasuzawa-Amano S. Amano K. Ide-Iwata N. Cheng W. Rota M. Urbanek K. Kajstura J. Anversa P. Leri A. Clonality of mouse and human cardiomyogenesis in vivo.Proc Natl Acad Sci USA. 2009; 106: 17169-17174Crossref PubMed Scopus (114) Google Scholar, 18D'Alessandro D.A. Kajstura J. Hosoda T. Gatti A. Bello R. Mosna F. Bardelli S. Zheng H. D'Amario D. Padin-Iruegas M.E. Carvalho A.B. Rota M. Zembala M.O. Stern D. Rimoldi O. Urbanek K. Michler R.E. Leri A. Anversa P. Progenitor cells from the explanted heart generate immunocompatible myocardium within the transplanted donor heart.Circ Res. 2009; 105: 1128-1140Crossref PubMed Scopus (23) Google Scholar, 19Boni A. Urbanek K. Nascimbene A. Hosoda T. Zheng H. Delucchi F. Amano K. Gonzalez A. Vitale S. Ojaimi C. Rizzi R. Bolli R. Yutzey K.E. Rota M. Kajstura J. Anversa P. Leri A. Notch1 regulates the fate of cardiac progenitor cells.Proc Natl Acad Sci USA. 2008; 105: 15529-15534Crossref PubMed Scopus (165) Google Scholar, 20Kajstura J. Urbanek K. Perl S. Hosoda T. Zheng H. Ogórek B. Ferreira-Martins J. Goichberg P. Rondon-Clavo C. Sanada F. D'Amario D. Rota M. Del Monte F. Orlic D. Tisdale J. Leri A. Anversa P. Cardiomyogenesis in the adult human heart.Circ Res. 2010; 107: 305-315Crossref PubMed Scopus (252) Google Scholar In all cases, atrial samples were used for the collection of hCSCs; this approach was followed to obtain a direct comparison between control and failing hearts. Two protocols were used for the isolation of hCSCs: enzymatic dissociation of the samples with collagenase and primary explant technique. These protocols were developed in the original documentation of hCSCs2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar and were retained here for continuity and comparison. As previously,2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar these two methodologies yielded comparable results up to 20 to 25 population doublings; efficiency and viability of hCSCs were superimposable. Collagenase treatment was not found to affect these variables. The phenotype of the small cell population was defined by flow-cytometry and immunolabeling.2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar, 4Bearzi C. Leri A. Lo Monaco F. Rota M. Gonzalez A. Hosoda T. Pepe M. Qanud K. Ojaimi C. Bardelli S. D'Amario D. D'Alessandro D.A. Michler R.E. Dimmeler S. Zeiher A.M. Urbanek K. Hintze T.H. Kajstura J. Anversa P. Identification of a coronary vascular progenitor cell in the human heart.Proc Natl Acad Sci USA. 2009; 106: 15885-15890Crossref PubMed Scopus (171) Google Scholar Single-cell suspensions were labeled with directly conjugated antibodies (Table 1) specific for hematopoietic and endothelial cell (EC) lineages. Conjugated isotype antibodies were used as negative control. To exclude the presence of erythrocytes, the samples of freshly isolated cells were exposed to BD fluorescence-activated cell sorting (FACS) lysing solution; the flow cytometric analysis was performed with a BD FACScan system (BD Biosciences, San Jose, CA) or a CyAn system (Beckman Coulter, Fullerton, CA). For immunolabeling, hCSCs were fixed in 4% paraformaldehyde for 15 minutes at room temperature and the expression of c-Kit, OCT3/4, NANOG, and SOX2 was evaluated.2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar, 4Bearzi C. Leri A. Lo Monaco F. Rota M. Gonzalez A. Hosoda T. Pepe M. Qanud K. Ojaimi C. Bardelli S. D'Amario D. D'Alessandro D.A. Michler R.E. Dimmeler S. Zeiher A.M. Urbanek K. Hintze T.H. Kajstura J. Anversa P. Identification of a coronary vascular progenitor cell in the human heart.Proc Natl Acad Sci USA. 2009; 106: 15885-15890Crossref PubMed Scopus (171) Google Scholar, 17Hosoda T. D'Amario D. Cabral-Da-Silva M.C. Zheng H. Padin-Iruegas M.E. Ogorek B. Ferreira-Martins J. Yasuzawa-Amano S. Amano K. Ide-Iwata N. Cheng W. Rota M. Urbanek K. Kajstura J. Anversa P. Leri A. Clonality of mouse and human cardiomyogenesis in vivo.Proc Natl Acad Sci USA. 2009; 106: 17169-17174Crossref PubMed Scopus (114) Google Scholar For clonal analysis, a FACSAria cell sorter (BD-Biosciences) was used to seed single c-Kit-positive hCSCs into 96-well Terasaki plates.2Bearzi C. Rota M. Hosoda T. Tillmanns J. Nascimbene A. De Angelis A. Yasuzawa-Amano S. Trofimova I. Siggins R.W. Lecapitaine N. Cascapera S. Beltrami A.P. D'Alessandro D.A. Zias E. Quaini F. Urbanek K. Michler R.E. Bolli R. Kajstura J. Leri A. Anversa P. Human cardiac stem cells.Proc Natl Acad Sci USA. 2007; 104: 14068-14073Crossref PubMed Scopus (840) Google Scholar, 4Bearzi C. Leri A. Lo Monaco F. Rota M. Gonzalez A. Hosoda T. Pepe M. Qanud K. Ojaimi C. Bardelli S. D'Amario D. D'Alessandro D.A. Michler R.E. Dimmeler S. Zeiher A.M. Urbanek K. Hintze T.H. Kajstura J. Anversa P. Identification of a coronary vascular progenitor cell in the human heart.Proc Natl Acad Sci USA. 2009; 106: 15885-15890Crossref PubMed Scopus (171) Google Scholar Total RNA was extracted from nonconfluent cultures of hCSCs at passage 3 using TRIzol reagent (Invitrogen, Carlsbad, CA). After treatment with DNase I (Ambion; Applied Biosystems, Austin, TX), first-strand cDNA synthesis was performed with 1 μg total RNA using random hexanucleotides and M-MLV reverse transcriptase (Invitrogen). PCR amplification was performed in a final volume of 50 μL, using 80 to 150 ng cDNA, 10 mmol/L Tris-HCl pH 9.0, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 25 pmol of each primer, and 2 U TaqI polymerase (Amersham; GE Healthcare, Little Chalfont, UK). The PCR conditions were as follows: 94°C for 2 minutes; 30 to 45 cycles at 94°C for 60 seconds, 60°C for 60 seconds, and 72°C for 90 seconds. The optimal conditions and the number of cycles were determined to allow amplification of samples within the linear phase of the PCR. The reaction products were analyzed on 1.8% agarose gels.19Boni A. Urbanek K. Nascimbene A. Hosoda T. Zheng H. Delucchi F. Amano K. Gonzalez A. Vitale S. Ojaimi C. Rizzi R. Bolli R. Yutzey K.E. Rota M. Kajstura J. Anversa P. Leri A. Notch1 regulates the fate of cardiac progenitor cells.Proc Natl Acad Sci USA. 2008; 105: 15529-15534Crossref PubMed Scopus (165) Google Scholar Primers are listed in Table 2.Table 2PCR Primers and Product LengthGeneForward primerReverse primerAmplicon size (bp)KIT⁎The protein product for the KIT gene is known also as c-kit.5′-CGTGGAAAAGAGAAAACAGTCA-3′5′-CACCGTGATGCCAGCTATTA-3′70POU5F1†The protein product for POU5F1 is known also as OCT3, OCT4, or OCT3/4.5′-GAGGATCACCCTGGGATATAC-3′5′-CGATACTGGTTCGCTTTCTCTTT-3′295NANOG5′-AATACCTCAGCCTCCAGCAGATG-3′5′-CTGCGTCACACCATTGCTATTCT-3′149SOX25′-GTTACCTCTTCCTCCCACTCCA-3′5′-CACCCCTCCCATTTCCCTCGTT-3′260KLF45′-GGGAGAAGACACTGCGTCA-3′5′-GGAAGCACTGGGGGAAGT-3′88GAPDH5′-CGACCACTTTGTCAAGCTCA-3′5′-AGGGGTCTACATGGCAACTG-3′228HPRT5′-CGTGATTAGTGATGATGAACCAG-3′5′-CGAGCAAGACGTTCAGTCCT-3′129 The protein product for the KIT gene is known also as c-kit.† The protein product for POU5F1 is known also as OCT3, OCT4, or OCT3/4. Open table in a new tab Expression of c-Kit (KIT) was measured by quantitative real-time PCR, performed using a LightCycler 480 real-time PCR system (Roche Applied Sciences, Indianapolis, IN). Primers were designed from available human sequences using primer analysis software Primer3 version 0.4.0 (available at http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000) (Table 2). A dual-color assay with Hydrolysis Probe 2 and HPRT as internal control was used for normalization (Universal Probe Library, Roche). LightCycler 480 Basic software (Roche) used the second-derivative maximum method to identify the crossing point (Cp). Glioma cell line A172 was used as positive control. The expression of pluripotency genes and cardiac cytoplasmic proteins was determined by immunolabeling (Table 1). Image acquisition was performed with a Leica TCS-SP2 confocal laser microscope, using a 63× oil immersion objective (numerical aperture, 1.40) or a 40× oil immersion objective (numerical aperture, 1.25). Migration analysis was performed using a Cultrex 24-well cell migration assay (Trevigen, Gaithersburg, MD). Approximately 200,000 cells were seeded per well. Growth medium containing 10% fetal bovine serum was used as chemoattractant. The tumorigenic potential of hCSCs was established by in vitro and in vivo protocols. In vitro, the soft agar assay was used; 1 × 105 hCSCs were seeded in a 0.5% soft agar solution placed on top of a 1% hard agar. The number of colonies grown within the semisolid medium was measured 28 days later. For the in vivo analysis, 1 × 106 hCSCs were injected subcutaneously in NOD/Scid mice (HARLAN Italy S.R.L., San Pietro al Natisone, Italy). SKOV-3 ovarian cancer cells were used as positive control (Sigma-Aldrich, St. Louis, MO). Animals were kept in pathogen-free conditions and tumor growth was evaluated monthly for a period of 6 months, or until SKOV-3 ovarian cancer cells generated a tumor 1 cm in diameter. hCSCs were loaded with 10 μmol/L Fluo-3 AM dye (Invitrogen) and were placed on the stage of a two-photon microscope: a BX51WI Olympus microscope (Olympus, Tokyo, Japan) coupled with a Bio-Rad Radiance 2100MP system (Bio-Rad Laboratories, Hercules, CA). Cells were bathed with Tyrode's solution containing (in mmol/L) NaCl 140, KCl 5.4, MgCl2 1, HEPES 5, glucose 5.5, and CaCl2 2.0 (pH 7.4, adjusted with NaOH). The Fluo-3 was excited at 900 to 960 nm with a Tsunami mode-locked Ti:sapphire femtosecond laser (Spectra-Physics; Newport Corporation, Irvine, CA), and the emission signal was collected at 535 nm. Series of images were acquired at 10-second intervals for a period of 33 minutes. Changes of intracellular Ca2+ in individual hCSCs were determined by measuring the fluorescent signal of Fluo-3. In each cell, the oscillations in fluorescence with time were graphically visualized using ImageJ (NIH, Bethesda, MD) and Microsoft Office Excel 2003 software. These traces were used to assess the number, amplitude, and duration of Ca2+ oscillations in hCSCs. Fluo-3 signals were expressed as normalized fluorescence (F/F0). Th