Patient profile modulates cardiac c-kit1 progenitor cellavailability and amplification potential

ELISA GAMBINI, MAURIZIO PESCE, LUCA PERSICO, BEATRICE BASSETTI, ANTONIO GAMBINI,FRANCESCO ALAMANNI, MARCO AGRIFOGLIO, MAURIZIO C. CAPOGROSSI,and GIULIO POMPILIO

MILAN, GENOA AND ROME, ITALY

From the Laboratorio di Biologia V

Centro Cardiologico Monzino-IRC

Scienze Cardiovascolari, Universi

Italy; Dipartimento di Chiru

Cardiologico Monzino-IRCCS,

Economia e Metodi Quantitativ

Universit�a degli Studi di Genova,

Patologia Vascolare, Istituto D

IDI-IRCCS, Rome, Italy.

Conflict of interests: None.

Human c-kit1 cardiac progenitor cells (CPCs) are multipotent and may be used forcardiac repair. The effect of cardiovascular risk factors andmedications onCPCs iso-lation efficiency, c-kit stemcellmarker expression, andex vivoproliferative potentialis unknown and was examined in the present work. Cells from human right atrialappendages (n 5 50) were expanded in culture; after �16 days (T0), it was estab-lished the percentage of CPCs and c-kit protein mean fluorescence intensity (MFI)by fluorescence activated cell sorting (FACS). Thereafter, CPCs were isolated byhigh throughput sorting; after culturing for 4 passages CPCs-derived cells were re-analyzed to assess c-kit1 cell percentage and enrichment vs T0. The association be-tween 19 demographic and clinical variables to CPCs number and MFI at T0, andCPCs enrichment at P4, was determined by multiple linear regression analysis withstepwise selection procedure. The results revealed that (1) at T0, the number of iso-lated CPCs directly correlated to b-blocker treatment; (2) at T0, c-kit protein expres-sion directly correlated to pulmonary hypertension (PH); (3) at P4, CPC’s enrichmentinversely correlated to smoke, atrial fibrillation (AF), a history of myocardial infarc-tion, whereas it directly correlated to PH and statins. Patient clinical profile andmed-ications differently modulate CPCs isolation and amplification potential ex vivo.These results may provide new insights for the understanding of cardiac homeostasisand suggest both limitations and possible enhancing strategies for the therapeuticuse of cardiac-resident progenitor cells. (Translational Research 2012;160:363–373)

Abbreviations:CPCs¼cardiacprogenitor cells;MFI¼mean fluorescence intensity; FACS¼ fluo-rescence activated cell sorting; EPCs ¼ endothelial progenitor cells; BMI ¼ body mass index;LV¼ left ventricular; Ab¼ antibody; OLS¼ ordinary least square; AIC¼Akaike’s information cri-terion; PH ¼ pulmonary hypertension; AF ¼ atrial fibrillation; PBS ¼ phosphate buffered saline;BSA ¼ bovine serum albumin; EDTA ¼ ethylenediaminetetraacetic acid

P rior in vitro and in vivo studies have shown thatthe heart is a self-renewing organ1-3 and that, atleast in mammals, cardiac homeostasis is largely

dependent upon a population of resident progenitor

ascolare e Medicina Rigenerativa,

CS, Milan, Italy; Dipartimento di

t�a degli Studi di Milano, Milan,

rgia Cardiovascolare, Centro

Milan, Italy; Dipartimento di

i (DI.E.M.), sezione Statistica,

Genoa, Italy; and Laboratorio di

ermopatico dell’ Immacolata,

cells.1,2 Different candidate cardiac progenitors havebeen identified in fetal and adult hearts, in variousmammalian species including mouse,3 rat,1 dog,4 andhuman.5-7 At present, the best-characterized cardiac

Elisa Gambini and Maurizio Pesce contributed equally to this work.

Submitted for publication December 13, 2011; revision submitted

May 2, 2012; accepted for publication May 31, 2012.

Reprint requests: Elisa Gambini, Centro Cardiologico Monzino, Lab-

oratory of Vascular Biology and Regenerative Medicine, Via Parea 4,

20138 Milan, Italy; e-mail: elisa.gambini@ccfm.it.

1931-5244/$ - see front matter

� 2012 Mosby, Inc. All rights reserved.

http://dx.doi.org/10.1016/j.trsl.2012.05.009

363

AT A GLANCE COMMENTARY

Gambini E, et al.

Background

Until the discovery of resident cardiac progenitor

cells (CPCs), the heart was considered terminally

differentiated. Today, CPCs are well characterized

to the extent that they are being tested in a phase I

clinical trial in patients with cardiomyopathy.

However, the effect of patient profile on CPC ex-

pression, isolation, and ex vivo proliferation is un-

known.

Translational Significance

It is well known that the features of human endo-

thelial progenitors correlate with cardiovascular

risk factors, patient characteristics, and pharmaco-

logic therapy. Understanding how CPCs are influ-

enced by patient profile is crucial to the

development of more effective cardiac cell thera-

pies.

Translational Research364 Gambini et al November 2012

progenitor cell type in the adult mammalian heart isrecognized by the expression of the transmembranetyrosine kinase receptor c-kit, CD117. It has beenfound7 that CPCs, harvested from different cardiacsources, are stored in cardiac niches, are clonogenic,multipotent, and differentiate into mature progeniesin vitro1,4 and in vivo,1,3,5 giving rise to myocardial,endothelial, and smooth muscle lineages.8,9

Although activation of CPCs follows myocardialinjury, the intrinsic ability of the myocardium to self-renew does not unfortunately compensate for themassive loss of cardiac myocytes observed in severepathologic conditions such as acute myocardial infarc-tion or heart failure.3,4,10,11 This is due to the inabilityof CPCs to produce enough myocytes to replace alllost myocardial cells and also to stem cell lossfollowing ischemia.3,4 Thus, over the last decade,direct stem/progenitor cell transplantation into thedamaged heart has emerged as an innovative therapyto replace lost myocardium. Currently, the potential ofCPCs to promote heart repair has been investigated ina phase 1 clinical trial (Stem Cell Infusion in Patientswith Ischemic cardiOmyopathy study), with promisinginitial results.12

Among key factors for the success of cell therapyin the treatment of ischemic heart disease, potencyof transplanted cells and availability of an adequatecell number are likely to be the most relevant.13 Un-fortunately, similarly to what has been found for bone

marrow-derived endothelial progenitor cells (EPCs),cardiovascular risk factors appear to limit residentCPCs ability to repair the injured heart in experimen-tal animal models.14 At the current time, it is stillunknown whether cardiovascular risk factors and car-diovascular drugs influence c-kit1 CPCs isolationfrom cardiac specimens and their expansion in cul-ture, thus, affecting their therapeutic potential. Thisissue was addressed in the present work. To thisaim, biologic features of c-kit1 CPCs have been cor-related to patient characteristics, known cardiovascu-lar risk factors, cardiac function, and pharmacologictherapy.

MATERIALS AND METHODS

Ethical statement. The collection of right atrial ap-pendages was performed after patient informed consent.The study was approved by the Local Ethical Commit-tee (approved on August 4, 2008; reference numberCCFM C9/607 and CCFM C10/607), and was per-formed according to Italian laws.

Patients and tissue samples. Specimen from rightatrial appendages (from 36.8 to 216.9 mg) werecollected from 50 Caucasian patients undergoingcardiac surgery; patient age ranged between 45 and80 years (67.66 1.2 years). In addition to the completemedical record, the following relevant informationwas obtained about each patient: age, sex, body massindex (BMI), cardiovascular risk factors, type ofcardiac procedure, and drug therapy at hospital admis-sion. Further, preoperative echocardiography datawere collected and included: left ventricular (LV)ejection fraction, LV dilatation and pulmonary pulmo-nary artery pressures.

Cell isolation and expansion. The procedure to obtainand expand c-kit1 CPCs from right atrial appendageshave been previously reported by our laboratory.7 Allatrial fragments were processed with the samemethodology. Fibrotic tissue was discarded andremaining myocardial tissue cut into 1 to 2 mm3

pieces, washed with phosphate buffered saline (PBS)(Lonza, Milan, Italy) and digested four times for30 min at 37�C with 3 mg/ml collagenase NB4 (ServaHeidelberg, Germany). After digestion, the solutionwas filtered using 70 um mesh nylon filters (BD-Biosciences, Milan, Italy) and the cells were seededinto a 10 cm uncoated Petri dish (Corning, Turin,Italy) containing Ham’s F12 medium (Lonza, Milan,Italy) supplemented with 10% fatal bovine serum(Thermo Scientific HyClone, Logan, UT), 2 mM L-glutathione (Sigma, Milan, Italy), 5 3 10-3U/mLhuman erythropoietin (Sigma, Milan, Italy), 10 ng/mLbFGF (Peprotech, London, UK), and antibiotics

Translational ResearchVolume 160, Number 5 Gambini et al 365

(Lonza, Milan, Italy). At 48 h after the beginning of theculture, the medium was completely changed to removedead non-adherent cells. When reaching 70%confluence, cells were split 1:10 and seeded into 10cm uncoated Petri dishes (Corning, Turin, Italy) untilreaching 70% confluence.

High troughput cell sorting and immunophenotypeanalysis by flow cytometry. At the end of the ‘‘primaryexpansion’’ period (ie, after reaching 70% confluence),the cells were stained with an anti-c-kit antibody (Ab),analyzed, and sorted by flow cytometry (T0).To do this, the cells were detached using a non-

enzymatic cell dissociation solution (Sigma) to avoidreduction of c-kit antigen immunoreactivity, and incu-bated in the dark in PBS containing 0.1% bovine serumalbumin (BSA) (Life Technologies, Paisley, UK) and 2mM ethylenediaminetetraacetic acid (EDTA) (LifeTechnologies, Paisley, UK) for 15 min in the presenceof APC-conjugated monoclonal antibodies directedagainst human c-kit receptor (anti-CD117, cloneYB5.B8; BD Biosciences, Milan, Italy), at 10 mg/mLconcentration. A FACSAria (Beckton-Dickinson,Milan, Italy) flow cytometer/cell sorter was used toidentify and separate c-kit1 from c-kit2 cells. Sortingsetup and appropriate gating was established eachtime using cells labeled with APC-conjugated isotypecontrol antibody at the same concentration. To mini-mize cell death and maximize recovery, cells weresorted at low pressure (20 PSI).Two different gating strategies were adopted to dis-

criminate between cells expressing c-kit at high levels(c-kitsorted) and total cells expressing c-kit (c-kittotal).The more stringent criterion was used to maximize pu-rity of c-kit1 cells for subsequent culture experiment;the less stringent one was applied to recognize c-kit1

cells in the total population (c-kittotal). Statistical analy-sis followed (Fig S1). In a subset of experiments, at theend of the ‘‘primary expansion’’ phase, cells were co-stained with an anti-c-kit antibody (Ab) and 7-AADdye, and analysed for detection of living c-kit1 cells.Sorted c-kit1 cells (104/well) were sub-cultured into

transwell inserts (pore size 0.4 mm; Corning, Turin,Italy), onto un-selected cells from the same patient with-out physical contact. Under these conditions, c-kitsorted

CPCs were amplified for 4 passages after which cellswere detached from transwell inserts using the non en-zymatic method to preserve extracellular antigens andincubated in the dark in PBS containing 0.1% BSA(Life Technologies, Paisley, UK) and 2 mM EDTA(Life Technologies, Paisley, UK) for 15 min with thefollowing monoclonal antibodies or isotype-matchedcontrol: c-kit-APC (clone YB5.B8), CD34- FITC (clone581), CD45-PE (clone HI30), and CD14-FITC (cloneTUK4; MiltenyiBiotec, Bologna, Italy).

Immunofluorescence. Sorted c-kit1 cells were imme-diately fixed and stained with c-kit antibody (Ab) toassess the purity of the selected cell population. Afterpermeabilization with PBS containing 0.1% Triton-X100 for 10 min at room temperature (RT) andblocking with PBS containing 10% serum cells wereincubated with primary antibodies for c-kit (Dako,Milan, Italy). Alexa 488-conjugated secondaryantibodies (Life Technologies, Paisley, UK) wereused. Nuclear staining was performed incubating cellswith Hoechst 33342 (Sigma-Aldrich, Milan, Italy).Cells Images were acquired with Zeiss Axio ObserverZ1 microscope equipped with Apotome imagedeconvolution system and Axiovision software (CarlZeiss, G€ottingen, Germany).

Confocal analysis. Fragments from atrial appendageswere fixed in formaldehyde and processed for tissueinclusion and sectioning. Paraffin embedded sections(4–6 mm thick) were dewaxed in xylene and rehydratedin ascending alcohols. Following antigen retrieval withNa citrate, sections were incubated with primaryantibody at 4�C overnight. The following primary anti-bodies were used: anti-c-kit (Abcam) and anti-a-sarcomeric actin (Sigma-Aldrich). Primary antibodieswere amplified with secondary antibody at room temp-erature for 1 h. The following secondary antibodieswere used: Alexa 488 (Life Technologies, Paisley, UK)and DyLight 549-conjugated (Jackson Immuno-Research, West Grove, Pa). To reduce autofluorescencethe sections were stained with the lipid dye SudanBlack B (Sigma-Aldrich, Milan, Italy). Nuclear stainingwas performed incubating cells with Hoechst 33342(Sigma-Aldrich). Sections were observed using a ZeissLSM 710 confocal microscope (Carl Zeiss, G€ottingen,Germany).

Immunohistochemical analysis. Human heart sampleswere embedded in paraffin and processed. Sectionswere deparaffinized in xylene, hydrated, and rinsed inPBS. Antigen retrieval was performed microwaving inDiva Decloaker Solution (pH 7, Biocare Medical, Con-cord, Calif). Slides were washed in PBS and the endog-enous peroxidase activity was blocked in 3% hydrogenperoxide (H2O2) in H2O for 20 min. To minimize non-specific antibody binding, slides were preincubatedwith PBS 10% BSA overnight. Rabbit polyclonal c-kit(Abcam, Cambridge, UK) antibody was diluted with10%BSA and then incubated overnight at 4�C. Sectionswere incubated first with a biotinylated goat- anti-rabbitfor 45 min at 37�C and subsequently in Vectastain EliteABC kit (Vector Laboratories, Burlingame, CA) for 45min at RT. Finally, they were developed in DABsubstrate kit (Vector Laboratories) for 4 min,counterstained with hematoxylin to identify nuclei,dehydrated, and mounted in Eukitt (Bioptica, Milan,

Table I. Clinical characteristics of patients included

in the multivariable regression analysis

Patient characteristics

Age (mean 6 SE) 50 (67.6 6 1.2)Male sex, n (%) 43 (86)Body mass index, kg/m2, (mean 6 SE) 50 (27.4 6 0.7)Cardiovascular risk factors, n (%)

Smoking 32 (64)Hypertension 37 (74)Dyslepidemia 38 (76)Diabetes 17 (34)

CABG, n (%) 40 (80)VR, n (%) 6 (12)CABG 1 VR, n (%) 4 (8)Atrial fibrillation, n (%) 8 (16)Medications, n (%)

Statins 25 (51)b-blockers 28 (56)Ca11 antagonists 14 (29)Angiotensin II antagonists 11 (23)ACE Inhibitors 20 (40)

History of acute myocardial infarction 14 (28)Echocardiographic data,

Impaired LV function*, n(%) 11 (22)Left ventricular dilation†, n(%) 15 (30)Pulmonary hypertension‡, n(%) 8 (17)

Abbreviations: ACE, angiotensin-converting enzyme;CABG, coro-nary artery bypass grafting; LV, left ventricular; VR, valve replace-ment.*Impaired LV function defined as LV ejection function #50%.†Left ventricular dilation defined as indexed end-systolic volume$25 ml.‡Pulmonary hypertension definedas calculated systolic pulmonaryartery pressure $40 mm Hg.

Translational Research366 Gambini et al November 2012

Italy). Sections were observed using a Zeiss Axioskoplight microscopy.

Statistical methods. To investigate the relationship be-tween the c-kit1 cell populations at T0 and P4 with the19 selected demographic and clinical characteristicvariables (Table I), a stepwise regression analysiswith backward selection of variables was performed.Variables entered into the analysis were selected tofully depict the clinical, cardiologic, and pharmacologicprofile of donor patients.According to the regression analysis theory, the ordi-

nary least square (OLS) method leads to parameter esti-mations, which guarantee a pre-assigned probability oferror. From the hypothesis testing point of view, themultiple regression analysis output contains an overalltest of significance based on F statistic that tests thenull hypothesis that all the parameters are equal tozero and k tests of significance (where k is the numberof parameter of the model) based on t statistic, whichtests the null hypothesis that a certain parameter is equalto zero. If the P value (the probability value associatedwith the F statistic value and with the t statistics values)is lower than the assigned probability of error ɑ, the null

hypothesis is rejected and the parameters of the modelare statistically significant.The P value associated with F statistic leads to the re-

jection of the null hypothesis and to the acceptation ofthe entire model. For each variable has been providedthe coefficient estimate (the sign 1 indicates a directlinear correlation whereas the sign 00 indicates an inverseone), the standard error value, the t statistic value, andthe P value. Since the number of variables involved inthe regression analysis was high, to reduce model com-plexity and to identify the most important predictors,a stepwise selection method with backward eliminationwas adopted.In the backward stepwise model selection procedure,

variables are sequentially removed from a completemodel (all regression variables included). In any roundof backward stepwise iterations, the regression variablesare each removed from the starting model, and a mea-sure of the goodness of fit of the estimated statisticalmodel is calculated to find which variable has to be de-leted. As a measure of goodness of fit, we adopt theAkaike’s information criterion (AIC), which not onlyrewards goodness of fit but also includes a penaltythat is an increasing function of the number of estimatedparameters. The preferred model is the one with thelowest AIC value. The AIC methodology attempts tofind the model that best explains the data with a mini-mum of free parameters. The statistical power hasbeen checked for each run of the stepwise analysis.To investigate a possible influence of specimen size

on number of cells obtained after the primary amplifica-tion phase, a Spearman correlation analysis was adop-ted. Finally, a Kruskal–Wallis one-way analysis ofvariance was run to detect differences in fold enrich-ment in patients with vs without a previous myocardialinfarction. All calculations were performed in R Soft-ware environment (www.r-project.org). R is one of themost powerful and flexible statistical software pack-ages. It is an open-source program developed throughthe contribution of all the statistician academiccommunity.

RESULTS

Cells from the right atrial appendage of each patientwere studied according to the experimental protocoldepicted in Figure 1. Briefly, after surgical biopsy,auricle fragments were either dissociated for cell cultureor fixed for immunofluorescence. Culture was per-formed in 2 consecutive amplification rounds calledprimary expansion and T0-P4 amplification, whichallowed to expand c-kit expressing cells before highthroughput sorting, and assess the kinetics of CPCsgrowth to perform statistical correlations with patientcharacteristics.

Fig 1. Experimental flow chart and time line. C-kit1 cells were obtained from the atrial appendage of each patient

and used for histology and immunohistochemistry/immunofluorescence staining or for cell culture. Cell culture

was performed according to a 2-step program involving a primary amplification step, followed by high throughput

sorting of CPCs and secondary expansion of these cells in a transwell co-culture system.7 Data obtained during the

primary and the secondary amplification steps (T0 c-kittotal cell percentage and c-kit staining MFI; T0-P4 c-kittotal

cell percentage) were used for multiple linear regression analysis. (Color version of figure is available online.)

Translational ResearchVolume 160, Number 5 Gambini et al 367

CPCs localization, isolation, amplification andanalysis. Confocal microscopy and immunohistochemi-cal analysis of human auricolae fragments showed thepresence of isolated or clustered c-kit1 cells in sub-epicardial or intra-myocardial zones (Fig 2, A and B).Therefore, in agreement with prior studies,3,7 it wasconfirmed that this tissue is a CPCs source.Using a protocol already established by us to culture

c-kit1 CPCs,7 monodispersed cellular suspensions, re-sulting from right atrial tissue digestion, were platedfor 2 rounds of expansion. This phase before sortingc-kit1 cells lasted 16.46 0.6 days (n5 50). To excludethat size of atrium specimens influenced number of cellsobtained during the primary expansion phase, a correla-tion analysis between total cell number and atrial tissueweight was performed. As no correlation was found (seeFig S2), we can infer that growth capacity of atrialtissue-derived cells was not affected by specimen size.Upon reaching 70% confluence, primary amplified

cells were stained with an anti-c-kit antibody and ana-lyzed by flow cytometry. This allowed determine thepercentage of cells expressing c-kit (c-kittotal popula-tion) and that of cells expressing the marker at highlevels suitable for high throughput sorting (c-kitsorted

population) (see Fig 3, A). This further allowed calcu-late the c-kit mean fluorescence intensity (MFI). MFIreflects the number of epitopes per cell that can binda given antibody and, therefore, provides an indirect es-timate of the epitope expression level at the cell sur-face15 (Fig S1). Since c-kit is the major antigendefining the most undifferentiated cardiac progeni-tors,16 we considered the MFI value as indirect indica-tion of cell stemness. As expected, the MFI valuerelative to anti-c-kit antibody staining was significantlyhigher compared with control cells stained with

matched isotype antibodies (Fig 3, C). C-kittotal andc-kitsorted cells represented 23.6% 6 2.5% and 1.5% 60.1% (mean 6 SE; n 5 50) of cells in the primary un-selected cell population, respectively. The purity ofc-kitsorted cells was checked by re-analyzing them im-mediately after sorting (Fig 3, D). C-kitsorted cellswere finally co-cultured into transwells onto parentalunselected cells for 4 passages. As already shown byus7, this system maximizes the growth of c-kit1 cellswhile partially prevents c-kit downregulation observedin the absence of supporting cells, as a result of celldifferentiation. Under these conditions, the time spanto reach P4 with 70% confluence was 26 6 1.48 days(Fig 1). At P4, co-cultured cells were again analyzedfor c-kit expression to assess c-kit1 cell fold enrich-ment, a parameter correlated to the ability of these cellsto grow. C-kit1 cell fold enrichment was calculatedby dividing the number of c-kit1 at P4 by the numberof sorted c-kit1 at T0. Finally to exclude the presenceof contaminating hematopoietic-derived cells at P4,the expression of different hematopoietic lineagemarkers, such as CD45, CD14 and CD34, was investi-gated (Fig S3).

Patient characteristics correlating to c-kittotal cellspercentage and MFI at T0. A multiple linear regressionanalysis with stepwise selection procedure was appliedto determine the association between 19 demographicand clinical variables to c-kittotal cellular population in50 patients and c-kit MFI in 40 patients at T0 (seeFig S1). The stepwise variables selection was basedon AIC (Akaike’s Information Criterion) and F-testfor significance of regression and validation of thetwo resulting models (p-values,0.02). Using thisstatistical modelling, the correlation between theconsidered biological variables at T0 (c-kittotal cells

Fig 2. Localization of c-kit1 cells in the human auricle tissue. A, Confocal microscopy images showing c-kit

expressing cells (green fluorescence) localized in epicardial tissue (ep) (upper left panel) and between myocytes

(red fluorescence; cm) in the myocardium (upper center panel). The figure also shows high power views of a di-

viding c-kit expressing cell (upper right panel) and a cluster of c-kit1/a-Sarc actin1 cells (lower panels) in the

atrial tissue. B, Light microscopy images of c-kit-expressing cells in the epicardium (ep). High power view in

the lower panel shows the presence of these cells (arrows) in the epicardial sheet shown in the area enclosed in

the upper, low magnification, panel. Counterstaining was performed by hematoxylin/eosin. C, Low (left panel)

and high power (right panel) views of c-kit expression in CPCs as detected by immunofluorescence after high

throughput sorting. Cells in the right panel are enclosed in the dashed rectangle in the left panel. (Color version

of figure is available online.)

Translational Research368 Gambini et al November 2012

percentage and c-kit MFI) and patient characteristicswas performed (Tables IIa and IIb). The sample sizewas consistent for estimating the model, witha statistical power of 0.7357 and 0.8199 for c-kittotal

percentage and MFI, respectively. Moreover,according to P value associated to the F statistic, theentire model reached statistical significance (P value 50.01615 and 0.01918 for c-kittotal percentage and MFIanalyses, respectively). Concerning the influence ofpatient variables on c-kittotal cells percentage, theanalysis revealed a positive and significant correlationwith b-blocker intake (P 5 0.0161) (see Table IIa).With the aim to further clarify the influence ofb-blockers on cell viability, a flow cytometry studywas then used to assess the percentage of livingc-kittotal cells at T0. To this aim, staining of primary

expanded cells with 7-AAD was performed inconjunction with c-kit staining. As shown in Figure 4,we found that the percentage of c-kittotal/7-AAD2

viable cells was significantly higher in patientsassuming b-blockers.As for c-kit protein expression level (MFI, Table IIb),

the applied model identified a positive significant corre-lation with pulmonary hypertension (P 5 0.0343). Al-though nonsignificant, an association with MFI wasalso found for b-blocker intake (positive) and for smok-ing habit (negative).

Patient characteristics correlating to c-kit1 cells ex vivoamplification potency. The association between patientcharacteristics and c-kit1 cells ex vivo amplification po-tency was analysed in a subgroup of the initial popula-tion (33/50). The application of this model revealed

Fig 3. Flow cytometry identification and high throughput sorting of c-kit expressing cells. A, Two different

gating criteria were adopted to recognize c-kit expressing cells by flow cytometry. A first gate was established

to sort cells with a high level of c-kit expression (c-kitsorted, left dot plots), while a second gate was used to

identify the total population of cells expressing c-kit antigen (c-kittotal right dot plots). These gates were estab-

lished based on staining using an isotype control antibody conjugated with the same fluorochrome (APC; center

panels), to assess background staining of the cells of interest. B, Quantification of c-kitsorted and c-kittotal cell

percentage obtained using gating strategy described in panel A. (n 5 50). C, Panel shows the average log10

MFI of the staining of primary amplified cells using isotype and c-kit antibodies. As expected, statistical anal-

ysis by paired Student t-test (* indicates P, 0.0001; n5 42) showed that the log10MFI was significantly higher

in cells stained with the c-kit specific antibody. D, C-kit1 cells sorting strategy. The appropriate gating to dis-

criminate between c-kit expressing and non-expressing cells was established each time using APC-conjugated

isotype antibody (upper dot plots). To avoid false positive events during the sorting procedure, sorting gate was

set to include less than 0.1% events resulting from unspecific binding (middle panels). Using this criterion, only

cells expressing c-kit antigen at high level (c-kitsorted cells) were separated and cultured. C-kitsorted cells purity

was checked by analyzing sorted cells immediately after separation (lower dot plots). (Color version of figure is

available online.)

Translational ResearchVolume 160, Number 5 Gambini et al 369

characteristics positively or negatively correlated withfold enrichment of human c-kit1 cells. Again, thep-value associated to F statistics (P5 0.05464) showedthat the entire model reached statistical significance andthe sample size was adequate for estimating the model(statistical power 5 0.9131). Table IIC shows thesignificance of the identified positive and negativecorrelations. Smoke, AF and history of myocardialinfarction (MI) were significantly associated to animpairment of c-kit1 CPCs proliferation. In particular,the negative influence of a previous MI has been

further investigated by analysis of variance bycomparing the fold enrichment of c-kit expressing cellsin patients with vs without previous MI. A significantimpairment in growth capacity was confirmed in cellsobtained from patients with MI (see Fig 5).A positive correlation with c-kit1 cell amplification

potential was found for statins intake and for PH, inagreement with data obtained for cells at T0. As fortreatment with b-blockers, Ca11 antagonist and angio-tensin II antagonists, the correlations were negative,although at a non-significant level.

Table IIa. Regression analysis output on T0 dataset

(dependent variable: c-kit1 cell; stepwise selection

method: backward)

Coefficients Estimate SEr t value Pr(. jtj)

(Intercept) 17.314 3.623 4.779 1.70e-05 ***b-blockers 12.072 4.841 2.494 0.0161 *

Signif. codes: 0 ‘***’ 0.01 ‘*’Residual standard error: 16.99 on 48 degrees of freedomMultiple R-squared: 0.1147, Adjusted R-squared: 0.09624F-statistic: 6.218 on 1 and 48 DF, P value: 0.01615

Table IIb. Regression analysis output on T0 dataset

(dependent variable: MFI; stepwise selection

method: backward)

Coefficients Estimate SE t value Pr(. jtj)

(Intercept) 193.23 24.02 8.045 1.47e-09 ***Smoke 241.02 24.13 21.700 0.0977b-blockers 44.76 23.05 1.942 0.0600Pulmonary

Hypertension66.92 30.42 2.200 0.0343 *

Signif. codes: 0 ‘***’ 0.01 ‘*’

Residual standard error: 71.27 on 36 degrees of freedomMultiple R-squared: 0.2383, Adjusted R-squared: 0.1748F-statistic: 3.753 on 3 and 36 DF, P value: 0.01918

Translational Research370 Gambini et al November 2012

DISCUSSION

The main finding of the present study is that multiplefactors independently influence availability and proteinexpression of c-kit1 cells that can be isolated froma right atrial specimen and the ability of these cells toproliferate in vitro. Our findings correlate with theobservation that in humans, number and function of cir-culating endothelial progenitors correlate withcardiovascular risk factors, patient characteristics, andpharmacologic therapy.17

CPCs for cardiac repair. A phase I FDA-approvedclinical trial using autologous c-kit1 human cardiacstem cells in patients with post-infarct cardiomyopathyis still in progress and preliminary results are nowavailable.12 C-kit1 CPCs are harvested from rightappendage biopsies of patients undergoing coronaryartery bypass surgery and severe left ventriculardysfunction and reintroduced after ex vivo expansioninto the patient 3 to 4 months later via cardiaccatheterization. Preliminary results in patients are veryencouraging, suggesting that intracoronary infusion ofautologous CSCs is effective in improving LV systolicfunction and reducing infarct size.12 Therefore, theinitial population of CPCs obtained from humanspecimen and the capacity to culture them to obtain anexpanded population of cells in a limited amount of

time are emerging as crucial steps for the clinicaltransferability of CPCs-based cell therapy.

C-kit1 isolation, amplification and correlation withclinical parameters. C-kit1 cells are cardiac progenitorsdistributed in various districts in the human heart.3 Fortherapeutic purpose, the right atrium is an appealingsource of CPCs, because it easily accessible. Evidencefrom animal models3 and humans7,18 suggest that theright appendage is one of the preferential locations ofc-kit cardiac progenitor cell niches. Two recent studieshave attempted to associate ex vivo cultured CPCsproperties with patient characteristics. Itzhaki-Alfiaand coworkers18 have correlated, by multivariateanalysis, patient profile, with c-kit cells percentage incultures, derived from the mean of cell passages 1, 2,and 3. However, a limitation of this approach is that c-kit cells percentage progressively decreases withculture passages.7 Furthermore, only 6 variables wereentered in the stepwise regression analysis (5 clinical),which are likely not to be fully representative ofpatient profile. Aghila Raniand colleagues19 haveassociated clinical parameters and c-kit cell countmigrating from explanted atrial tissue samples.However, the authors did not fully characterize c-kit1

cardiosphere-forming population for its cardiac origin(co-expression of CD341 antigen) nor investigate,using a multivariate analysis, the correlation betweencell count and clinical variables. More recently,D’Amario et al13 isolated CPCs from the right side ofleft ventricular septum or apex of patients withadvanced heart failure. They showed the highefficiency of CPCs isolation process, although noinferences have been advanced about the correlationbetween cell quality and patient profile.We have previously described a new amplification

system tailored to expand CPCs ex vivo, where themain specificities consisted in a pre-expansion phase,a sorting phase using high throughput flow cytometry,and a transwell culture phase onto autologous unse-lected cells.7 Using this strategy, by means of the com-putational model we have adopted, we were able tocorrelate both initial and final c-kit cell populations(percentage and c-kit expression in primary culturesand fold enrichment after expansion) with a panel ofvariables representative of patient demographics, clini-cal and myocardial status, and pharmacologic therapy.

Association between patient characteristics and CPCsfeatures. The only variable found to be independentlyand positively associated with CPCs percentage at T0was b-blocker intake, which was also related, withoutreaching a statistical significance, with CPC protein ex-pression and growth capacity. Moreover, a higher viabil-ity rate at T0 has been found in c-kit cells obtained frompatients taking b-blocker therapy. These findings are

Fig 4. b-blockers improve CPCs survival during primary amplification period. Primary expanded cells from

auricle specimens of patients taking or not taking b-blockers were assessed for vitality. Cells were stained with

7-AAD (a dye staining dead cells) and anti-CD117 antibody, and analyzed by flow cytometry. A, Upper panels

show multicolor gating of dead (red) and alive (green) cells in the physical dimensions. Plots in the center

show the 7-AAD staining of the same cells. The dashed bar corresponds to the limit of fluorescence that was es-

tablished (based on negative staining, not shown) to discriminate dead from alive cells. Lower panels show c-kit

expression in live cells. Again, dashed lines indicate the limit in c-kit fluorescence that was established (based on

isotype antibody staining, not shown) to discriminate between c-kit expressing and c-kit non expressing cells.

B, Quantification of viable 7-AAD2/c-kit1 cells obtained from patients with and without b-blocker therapy.

* indicates P 5 0.0356 (unpaired t-test; n 5 3). (Color version of figure is available online.)

Translational ResearchVolume 160, Number 5 Gambini et al 371

new and deserve further investigation. Recent resultsfrom murine models indicate beneficial effects ofb-blockers on EPCs number and function, throughnitric-oxide-mediated and antioxidative mechanisms.20-22

Moreover, the positive action of b-blockers on heart rateand function may also represent an explanation for theincreased availability and viability of CPCs. Irrespectiveof the underlying mechanism, these data may open newinsights on the mode of action of this class of drugs onthe diseased myocardium.Pulmonary hypertension correlated with both c-kit

cells protein expression and expansion capacity. Ithas been previously described that PH induces prolifer-ation of resident smooth muscle cells.23 Similarly, underour experimental conditions, CPCs from patients withPH exhibit enhanced proliferation in vitro, which maybe linked to an activation of the cardiac progenitorcell pool consequent to pressure overload in the rightatrium.

Interestingly, AF correlated with a depression of c-kitexpansion capacity. Although new, this observation par-allels with the notion that AF causes an impairment ofcardiomyocyte function and structural changes in atrialmyocardium, leading to a progressive fibrosis,24 inwhich a depressed progenitor compartment may playa role.Notably, patients with a history of myocardial infarc-

tion showed a limitation in fold enrichment of c-kit cells,suggesting that in these patients, CPCs growth is im-paired. Anversa and coworkers have previously reportedthat in chronic ischemic myocardium myocyte replace-ment by the progenitor pool is markedly less than myo-cyte death in the long term.11 Although our source wasthe atrium and not the left ventricle, our findings maycouple Anversa’s inferences on the loss of myocardiumin patients with chronic ischemic heart disease.No information is presently available, to our know-

ledge, on interactions between CPC and pharmacologic

Table IIc. Regression analysis output on P4 dataset

(dependent variable: c-kit1 cell fold enrichment;

stepwise selection method: backward)

Coefficients Estimate SE t value Pr(. jtj)

(Intercept) 49.94 13.66 3.657 0.00125 **History of acute

myocardial infarction228.16 11.89 22.369 0.02622 *

Atrial fibrillation 256.77 22.97 22.471 0.02095 *Smoke 226.87 11.21 22.396 0.02472 *b-blockers 219.48 11.22 21.737 0.09521.Statins 32.66 11.54 2.831 0.00923 **Ca11 antagonist 214.6 10.97 21.331 0.19562.Angiotensin II antagonist 230.89 17.08 21.809 0.08299.Pulmonary hypertension 61.88 24.57 2.519 0.01886 *

Signif. codes: 0.001 ‘**’ 0.01 ‘*’Residual standard error: 27.07 on 24 degrees of freedom

Multiple R-squared: 0.4341, Adjusted R-squared: 0.2455F-statistic: 2.302 on 8 and 24 DF, P value: 0.05464

Fig 5. A history of myocardial infarction impairs CPCs ability to

grow. The graph shows a higher ability of CPCs obtained from patients

without a history of MI to be enriched in culture compared to patients

having a pre-existing MI, as assessed by Kruskal–Wallis one-way

analysis of variance. * indicates P 5 0.0462 by Kruskal–Willis test;

no MI: n 5 23; MI: n 5 11

Translational Research372 Gambini et al November 2012

therapy in humans. As for b-blockers, it was veryintriguing to find that treatment with HMG-CoAinhibitors (statins) was profoundly correlated with a bet-ter ability of c-kit1 cells to proliferate in culture. Vast lit-erature has reported the effects of statins to rescue riskfactor-associated EPCs functional failure via enhance-ment of pro-survival signaling dependent on activationof the PI3K/AKT signaling pathway.25-28 Interestingly,in mice, AKT-dependent pro-survival signaling pro-motes myocardial repair through suppression of myo-cyte apoptosis,29 stimulates growth factor-dependentmigratory activity, and CPC proliferation.3,30 Notably,increases of mitotic myocytes and of the growth phaseof the cell cycle were observed.20 Moreover, it hasbeen previously shown that statins functionally amelio-rate swine hibernating myocardium.31 Taken together,this evidence suggests that statin assumption may notcritically modify the absolute number of CPCs available,but likely stimulate CPCs clonogenic potential. Hence,pharmacologic upregulation of the PI3K/AKT signalingappears to be an appealing modality to obtain enoughprogenitors to repair the ischemic heart. Our data maybe then open to new insights on the ability of statins todirectly influence myocardial homeostasis.Finally, cigarette smoking inversely correlated with

fold enrichment at P4. This suggests an influence ofsmoking habit on CPCs growth capacity. This findinghas not been previously described for c-kit cells; how-ever, although the mechanisms are still unknown, thereis a consistent body of evidence that smoking has a ma-jor effect in reducing the number and function of circu-lating endothelial progenitor cells.32

Study limitations. It has to be mentioned that except forsmoking, none of the other classic cardiovascular riskfactors (ie, aging, hypertension, dyslipidemia, diabetes),

well-known to reduce bio-availability33 or function33 ofhuman endothelial progenitor cells, have emergedas independent variables in our statistical model.Although this finding is in agreement with the onlyother report that has systematically correlated patientcharacteristics with CPCs number using a comparablestatistical model,31 we cannot exclude that our findingsmay be at least partially due to a selection bias of ourpopulation. In particular, with respect to aging, onepossible limitation of our study was that patientsenrolled in the present work had a relatively youngmean age (67.5 years) with a narrow SD (1.2 years).This selection bias may have masked the effects ofaging on CPCs.

CONCLUSIONS

In conclusion, the main finding of this work was thata number of variables associated with patient demo-graphic, cardiologic status, and medications have a dif-ferent impact on resident CPCs number and function.Although we do not have a mechanistic explanationfor the correlations observed, our findings may haveimplications for either the understanding of myocardialhomeostasis or the clinical transferability of autolo-gous cardiac cell therapy. One potential limitation aris-ing from this work is that some patients (ie, smokersand subjects with previous myocardial infarction orwith AF) may be ‘‘bad-self-CPC-donors,’’ and theircells may require ex vivo treatment with ‘‘enhancingstrategies’’34 to restore their therapeutic potential. Onthe other hand, our data suggest the existence ofsome risk factors is potentially modifiable and,

Translational ResearchVolume 160, Number 5 Gambini et al 373

importantly, that some drugs such as b-blockers andstatins may influence the number and expansion capac-ity of CPCs.

The authors acknowledge Laura Facchinetti BSc for her precious

technical collaboration.

Supplementary Data

Supplementary data associated with this article canbe found, in the online version, at doi: 10.1016/j.trsl.2012.05.009.

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