Patent Application: US-201213410358-A

Abstract:
modulation of the integrin - linked kinase signaling pathway is used to enhance the remodeling process relevant to a wide range of cardiac diseases . more specifically , a process to instigate beneficial human cardiac hypertrophy or for post myocardial infraction remodeling comprising illiciting an overexpression of ilk , is described . the ilk signaling pathway is also used as a means for cardiac stem cell proliferation and self - renewal .

Description:
all protocols were in accordance with institutional guidelines for animal care . all procedures and analyses were performed in a fashion blinded for genotype , and statistical comparisons were made between ilk transgenic mice and sex - matched littermate non - transgenic mice . a 1 . 8 kb ecori fragment comprising the full length ilk cdna was excised from a pbsk plasmid , and filled - in for blunt end ligation into a sali site downstream of the murine a - myosin heavy chain promoter . site directed mutagenesis ( quickchange kit , sratagene ) was performed to generate constitutively active ilk ( s343d ), and kinase - inactive ilk ( r211a ) mutants using the wild type a - mhc / ilk plasmid as template . dna sequencing confirmed the point mutations . pronuclear microinjection of the linearized a - mhc / ilk plasmids into 0 . 5 day fertilized embryos was performed at the core transgenic facility of the hospital for sick children research institute . transgene expression in c57bl / 6 founder and f1 progeny mice was confirmed by southern analysis and rt - pcr as described , using primers specific for the exogenous ilk transgene . the forward primer : 5 ′ gtccacattcttcaggattct3 ′, specific for exon 2 of - mhc promoter , and the ilk - specific reverse primer : ‘ acacaaggggaaatacc gt3 ’, were used for the reaction . these primers amplify a 1460 bp across the α - mhc - ilk fusion junction . f1 progeny derived from one of several independent founder lines were selected for detailed phenotypic analysis based on readily discernible increases in ilk expression ( fig4 ). all transgenic mouse procedures were performed in conformance with the policies for humane animal care governing the core transgenic facility of the hospital for sick children research institute and the animal research act of ontario . all surgical procedures were performed in accordance with institutional guidelines . mice were anesthetized in the supine position using ketamine - hcl ( 100 mg / kg ip ) and xylazine - hcl ( 10 mg / kg ip ), and maintained at 37 ° c . the right common carotid artery was isolated after midline neck incision and cannulated using a millar micro - tip pressure transducer ( 1 . 4f sensor , 2f catheter ; millar instruments , houston , tex .). heart rate ( beats per minute ), systolic and diastolic lv pressures ( mm hg ) were recorded , and peak positive and negative first derivatives ( maximum / minimum +/− dp / dt ; mmhg / second ) were obtained from lv pressure curves using origin 6 . 0 ( microcal software , inc ., northampton , mass .). serial two - dimensional echocardiography ( 2 - d echo ) was performed in male ilk transgenic and non - transgenic littermate mice at 10 - 12 weeks , at 5 , and 15 months of age . an ultrasound biomicroscope ( ubm ) ( vs40 , visualsonics inc ., toronto ) with transducer frequency of 30 mhz was used to make m - mode recordings of the lv . mice were lightly anesthetized with isoflurane in oxygen ( 1 . 5 %) by face mask , and warmed using a heated pad and heat lamp . heart rate and rectal temperature were monitored ( thm100 , indus instruments , houston , tex .) and heating adjusted to maintain rectal temperature between 36 and 38 ° c . once anesthetized , the mouse pericardial region was shaved and further cleaned with a chemical hair - remover to minimize ultrasound attenuation . with the guidance of the two - dimensional imaging of the ubm , m - mode recording of left ventricular wall motion was obtained from the longitudinal and short axis views of the lv at the level with the largest ventricular chamber dimension . anterior and posterior lv free wall thickness , and ventricular chamber dimensions were measured at end - systole and end - diastole ; the contractility indices , velocity of circumferential fiber shortening ( vcf ) and % fractional shortening , and lv ventricular mass , were calculated as described . determination of significant , genotype - specific differences in 2 - d echo and cardiac catheterization data relied on a paired t - test or anova in the case of serial measurements . cells were lysed in np40 buffer , supplemented with 1 mm sodium orthovanadate and 5 mm sodium fluoride as phosphatase inhibitors . equal amounts of protein from these cell lysates were immunoprecipitated with - ilk polyclonal antibody as previously described 10 , and immune complexes were incubated at 30 c for 30 min with myosin light chain ii regulatory subunit ( mlc20 ) ( 2 . 5 g / reaction ) and [ 32p ] atp ( 5 ci / reaction ). the reactions were stopped by addition of 4 × concentrated sds - page sample buffer . phosphorylated proteins were separated on 15 % sds - page gels . [ 32 ] mlc20 was visualized by autoradiography with x - omat film . measurement of activated rhoa was performed using a pull - down assay based on specific binding of rho - gtp to rho - binding domain ( rbd ) of the rho effector molecule , rhoketin43 . cdc42 and rac1 activation were measured using a pull - down assay , based on the ability of the p21 - binding domain of p21 associated kinase ( pak ) to affinity precipitate rac1 - gtp and cdc42 - gtp , as described . rbd expressed as a gst fusion protein bound to the active rho - gtp form of rho was isolated using glutathione affinity beads according the manufacturer &# 39 ; s protocol ( cytoskeleton ). the amount of activated rho was determined by western blot using a rho - specific antibody ( santa cruz ) and normalized as a ratio to the total amount of anti - rho antibody detected in a 1 / 20 fraction of clarified lysate . activated rac and cdc42 were measured by the same protocol using the p21 - binding domain of pak to affinity precipitate rac - gtp , which was quantitated using an anti - rac antibody ( cytoskeleton , inc .) or anti - cdc42 ( santa cruz ). blots were developed with supersignal west femto substrate ( pierce ) for the gst - pak / rbd pull - down assays . the hearts were weighed , paraffin - embedded , sectioned at 1 mm intervals , and stained with hematoxylin and eosin and sirius red using standard methods . micrographs were taken using both low magnification (× 2 . 5 ) and higher magnification (× 40 ) using fluorescent microscopy and genotype - specific cardiomyocyte areas determined based on digital measurements of & gt ; 500 cells per animal and 5 animals per genotype using image j software ( http :// rsb . info . nih . gov / ij /). scanning electron microscopy was performed on ventricular samples placed in 1 % universal fixative for several hours at 4 ° c . and post - fixed in oso4 , using the jsm 6700fe sem microscope . lv infarction was created in 6 month ilk tgs343d and littermate control mice by lad ligation as described . planimetric scar dimensions measured in six levels of hematoxylin and eosin - stained cross - sections of the lv at 7 days post - infarction . total and phospho - specific protein expression was measured in lysates derived from human fetal cardiomyocytes in culture and from transgenic and control mouse ventricular tissue as described previously . immunoblotting was performed with the following commercially available antibodies . polyclonal rabbit antibodies against ilk , p38mapk , p70s6k , p44 / 42 mapk ( erk1 / 2 ), and atf - 2 were purchased from cell signaling technologies . phospho - specific antibodies of pp 38mapk ( thr180 / tyr182 ), pp70s6k ( thr421 / scr424 ), ppkb ( ser473 ), pgsk3β ( ser9 ), pp 44 / 42 mapk ( thr202 / tyr204 ), and patf - 2 ( thr69 / 71 ) were purchased from cell signaling . mouse monoclonal antibodies recognizing pkb , gsk3β , and rhoa were purchased from transduction labs . rabbit polyclonal hemaglutinin ( ha ), and monoclonal cdc42 antibodies were obtained from santa cruz biotechnology . rabbit polyclonal rac1 antibody was purchased from cytoskeleton , inc . we generated a 13 - parvin ( parvb ) rabbit polyclonal serum and affinity - purified these antibodies over an immobilized gst - parvb column . mouse monoclonal gapdh was purchased from ambion , inc . proteins were visualized with an enhanced chemiluminescence ( ecl ) detection reagent ( amersham pharmacia biotech ) and quantified by densitometry . human fetal cardiomyocytes ( hfcm ) ( gestational age 15 - 20 weeks ) were obtained under an institutional review board - approved protocol and cultured to approximately 50 % confluency ( day 3 - 4 post - plating ) in preparation for adenovirally - mediated infection of ilk constructs , as previously described . replication - deficient serotype 5 adenovirus encoding either the human wild - type ilk gene ( ad - ilk wt ), kinase inactive ( ad - ilk r211a ) or empty virus constructs previously shown to modulate ilk expression and activity in l6 myoblasts , were used for infection of hfcm . hfcm were infected at 37 ° c . at multiplicity of infection of 2 . kp392 is a small molecule inhibitor of ilk which was used to probe the effects of ilk on the profile of rho family gtpase activation . human right ventricular samples were derived from two patients with congenital outflow tract obstruction undergoing surgical repair , and left ventricular myocardial samples from five patients with hypertrophic obstructive cardiomyopathy ( hocm ) presenting with discrete subaortic muscular obstruction . control human ventricular tissue was acquired from structurally normal hearts ( n = 5 ) which were not used for cardiac transplantation . all human tissue samples were snap - frozen in liquid nitrogen at the time of procurement . all human tissue was acquired following protocol review and approval by the appropriate research ethics board , and the protocols were conducted in accordance with the tri council policy statement for research involving humans . ilk protein levels are elevated in cases of human cardiac hypertrophy . in order to test for the participation of ilk in hypertrophic heart disease in vivo , we examined ilk expression in human ventricular tissue samples from patients with and without clinically evident hypertrophy . ventricular samples were acquired from two patients in the first year of life with ventricular hypertrophy secondary to congenital outflow tract obstruction ; control ventricular tissue was derived from structurally normal 19 week human fetal hearts ( n = 2 ), and examined in parallel for levels of ilk expression . ventricular tissue from these hearts exhibited a 5 - 6 fold increases in ilk protein levels over control levels ( fig1 a ). we then investigated whether ilk protein expression was elevated in hypertrophy caused by left ventricular outflow tract obstruction ( lvot ), since clinical hypertrophic heart disease more commonly affects the lv . surgical specimens were acquired from the lvot in adult patients ( n = 4 ) with hypertrophic obstructive cardiomyopathy ( hocm ) exhibiting resting lvot gradients & gt ; 50 mmhg control ventricular tissue was obtained from structurally normal hearts ( n = 5 ) at the time of multi - organ transplantation procurement . myocardial samples from hocm patients exhibited a ˜ 2 fold increase in ilk protein levels relative to control hearts ( fig1 b ). thus , the cases of clinical hypertrophy all demonstrate elevation of ilk protein , suggesting this is a critical molecular response to increased cardiac loading and the development of hypertrophy . ilk has been shown to activate rho family gtpases , which have also been causally implicated in experimental hypertrophy . we therefore assayed the ventricular tissues directly for activation of rhoa , cdc42 and rac1 gtpases , using specific affinity binding assays that distinguish the gdp - bound ( inactive ) and gtp - bound ( active ) states of each . strikingly , there was a ˜ 2 - fold and 10 - fold increase in rac1 gtp loading in the hypertrophic ventricular samples from patients with acquired and congenital and outflow tract obstruction , respectively ( fig2 ab ). cdc42 activation of ˜ 2 - fold was also evident in both acquired and congenital hypertrophic lesions . conversely , the levels of gtp - bound rhoa were unchanged between the control and hypertrophied ventricles . these results indicate selective activation of rac1 , and to a lesser extent , cdc42 , coincident with increased ilk protein levels , in human ventricular hypertrophy induced in both left and right ventricles by obstructive hemodynamic loading . as the pro - hypertrophic kinases , akt / pkb , gsk313 , and erk1 / 2 , are known targets of ilk , we ascertained whether these proteins were also elevated in the cases of human hypertrophy . western blotting for total protein indicated equivalent levels of gsk313 and erk1 / 2 in the hypertrophied hearts , and an increase in pkb ( fig3 ). we tested the hypertrophic hearts for concordant increases in the phosphorylation state of known kinase targets of ilk that have also been implicated in the promotion of cardiac hypertrophy . surprisingly , the phosphorylation state of the classical hypertrophic signaling targets , akt / pkb and gsk3b , was not increased above control levels in any of the samples from the human hypertrophic ventricles ( fig3 ab ), despite the increased ilk protein levels in these samples . this result suggests that a putative ilk - rac1 hypertrophic pathway is separable from ilk signaling through pkb / akt and gsk3b . erk1 / 2 p38mapk8 , and p70s6k , are kinases downstream of ilk which have also been implicated in promotion of experimental cardiac hypertrophy in vivo . in contrast to akt / pkb and gsk313 , erk1 / 2 and p70s6k were strongly phosphorylated in ventricular lysates in the setting of lvot obstruction ( fig3 c ), indicative of an activation profile of ilk kinase targets induced during human hypertrophy which appears to exhibit a degree of selectivity . cardiac - specific expression of activated ilk in transgenic mice induces hypertrophy . the selective elevation of ilk levels in clinical cases of cardiac hypertrophy prompted us to ask whether increased ilk expression is causative of cardiac hypertrophy . to directly test hypertrophic responses to ilk in vivo , we derived independent lines of transgenic mice harboring different ilk transgenes , expressed under control of the cardiac specific - mhc promoter . as discussed above , ilk is a multifunctional protein24 , thus our strategy was to generate lines expressing ilk variants that would allow us to differentiate kinase - dependent and - independent ilk functions in the heart . toward this end , lines expressing : 1 ) constitutively activated , ilk s343d , 2 ) wildtype , ilk tgwt , and 3 ) kinase - inactive ilk , ilk r211a , were derived . southern blot analyses of genomic dna identified mice carrying the ilk s343d transgene ( fig4 a ), and rt - pcr analysis indicated cardiac - specific expression of ilk s343 ° ( fig4 b ). densitometric analysis of western blots indicated that transgenic ilk s343d protein levels were approximately 3 - fold higher in transgenic animals , relative to non - transgenic littermates ( fig4 c ), and comparable to the increased levels seen in the clinical hypertrophic samples . importantly , immune complex kinase assays confirmed that ilk activity in transgenic heart tissue measured in the ilk s343d genotype was elevated relative to non - transgenic controls , in parallel with ilk protein levels ( fig4 d ). similar analyses confirmed generation of ilk wt and ilk r211a transgenic lines ( not shown ). hearts from ilk s343d tg mice exhibited concentric hypertrophy , evidenced by gross enlargement and increased heart weight : body weight ratio ( fig5 a ; supplementary table 1 ), and echocardiographic measurements showing significant lv wall thickening , compared to ntg mice ( supplementary table 2 ). we observed an approximately 29 % increase ( p & lt ; 0 . 001 ) in cardiomyocyte area in ilk s343d tg animals , as assessed in laminin - stained sections of lv ( fig5 b ), which is sufficient to account for the observed cardiac enlargement in ilk s343d tg mice , suggesting ilk activity regulates cardiomyocyte size , rather than proliferation . there was no conspicuous increase in collagen deposition in the ilk s343d tg hearts , as assessed histologically using masson &# 39 ; s trichrome ( fig5 b ) or picrosirius red staining . the ilk s343d tg mice appeared healthy , with no evidence of peripheral edema or cardiac failure , as there were no ilk - induced differences in absolute or body weight - indexed lung and liver weights ( supplementary table 1 ). these data indicate that expression of activated ilk in the heart induces hypertrophy without the development of cardiac failure . to further characterize ilk s343d - induced hypertrophy , m - mode echocardiography was performed at 3 , 5 and 15 months of age in male ilk tg s343d and ntg mice . at all time points , ilk s343d tg mice exhibited significant increases in lv mass as well as lv free wall dimensions at end - systole and end - diastole ( fig5 c , supplementary table 2 ). cardiac function , however , was preserved as assessed by measures of lv wall shortening fraction and the velocity of lv circumferential fiber shortening ( vcf ). invasive hemodynamic measurements performed at 3 months revealed no significant differences in measures of contractility ( dp / dtmax ), lusitrophy ( dp / dtmin ), afterload or heart rate in ilk tgs343d mice relative to ntg controls ( supplementary table 3 ), indicating that ilk - induced hypertrophy does not alter cardiac function . thus , based on the observed lack of cardiac failure and normal hemodynamic function , the cardiac phenotype associated with ilk s343d expression is indicative of a compensated form of hypertrophy . induction of cardiac hypertrophy is dependent on the activity of ilk . our results , showing hypertrophic induction by the activated ilk allele , as well as activity - dependent induction of mapk , erk1 / 2 , and p70s6k phosphorylation , suggested that ilk - induced hypertrophy is dependent on ilk activity . in order to test this idea directly , we compared the hypertrophic status of hearts from transgenic mice expressing ilk wt , with hearts from ilk r211a transgenic mice . ilk wt hearts exhibited a hypertrophic phenotype which closely mimicked that of the ilk343d mutant , as evident by the significant ( p & lt ; 0 . 001 ) increase in hw : bw ( supplementary table 4 ) and lv mass measured by echocardiography ( p & lt ; 0 . 001 ) in comparison to ntg littermate controls ( supplementary table 5 ). additionally , transgenic mice with cardiac - restricted expression of the kinase - inactive ilk construct ( ilk r211a ) did not develop cardiac hypertrophy , as assessed by echocardiography at 4 months of age ( supplementary table 6 ). the finding that cardiac over - expression of kinase - deficient ilk did not exhibit evidence of cardiac dysfunction suggests that the structural role of ilk is sufficient for maintenance of baseline ventricular function , whereas kinase activity is required for hypertrophic remodeling . the g - protein activation profile correlated with the cardiac phenotypic findings , featuring selective activation of rac1 and cdc42 in the ilk wt ( fig6 ab ) and ilk s343d tg ( supplementary fig1 ) genotypes , both of which develop hypertrophy , in comparison to the kinase - inactive ilk r211a , which exhibits a cardiac phenotype indistinguishable from control . heart , lung , liver weights of ilk wt and ilk r211a transgenic mice we found that expression of either wild type ( fig6 cd ) or constitutively active ( supplementary fig2 ) ilk , but not ilk r211a , increased phosphorylation of both erk1 / 2 , and p38mapk , indicating that activation of these kinases was dependent on ilk catalytic activity . whereas increased expression of ilk was confirmed in both the ilk wt and ilk r211a genotypes ( fig6 e ), phosphorylation - dependent activation of ilk targets , p70s6k , erk1 / 2 , p38mapk , and the p38 - dependent transcription factor , atf2 , was only evident in the wild - type over - expressing ventricles ( fig6 cd ). western blotting confirmed roughly equal expression levels from ilk wt and ilk r211a transgenes ( fig6 e ), suggesting these differences were due to ilk catalytic activity . acute ilk - dependent rac1 activation in isolated human cardiomyocytes . in order to evaluate the effect of acute ilk up - regulation on gtpase activation , we infected human fetal cardiomyocytes with adenoviruses expressing ilk ( ad - ilk ), or an empty virus control . infection with ad - ilk stimulated an ˜ 3 - fold increase in levels of gtp - bound rac1 and an ˜ 7 - fold increase in gtp - bound cdc42 , 24 hours post - infection ( fig7 ). these stimulations were blocked by treatment of the ad - ilk infected cells with the small molecule ilk inhibitor , kp - 392 , suggesting that ilk kinase activity is required for activation of these small gtpases . infection of the cardiomyocytes with empty adenovirus , carrying no ilk sequences , had no effect on the activation state of rac1 , cdc42 , or rhoa . these results indicate that , as in the transgenic mouse hearts and during human hypertrophy caused by mechanical loading , acute up - regulation of ilk in isolated cardiomyocytes directly activates rac1 and cdc42 . genetic ilk over - expression enhances post - infarction remodeling . in order to test for potential cardioprotective effects of ilk , we analyzed lv infarct size in aged 6 month ilk tgs343d and littermate control mice at 7 days post - lad ligation , based on planimetric scar dimensions measured in six levels of cross - sections of the lv ( fig8 ). the ilk tgs343d genotype exhibited a significantly greater lv mass ( p = 0 . 01 ), a trend towards reduction in absolute lv scar area ( p = 0 . 106 ), and a reduction in scar area indexed to lv mass ( p = 0 . 047 ) ( fig8 b ). thus , cardiac ilk activation resulted in a post - infarction remodeling phenotype featuring a reactive increase in lv mass . recent studies have challenged the traditional thinking that the adult mammalian heart lacks inherent regenerative capacity . cardiac stem cells ( cscs ) derived from bone marrow or niches within the heart have been identified and shown to participate in the regeneration of myocardium in vivo xii , xiii , xiv . tissue - resident cardiac progenitor cells expressing various stem cell markers such as sea - 1 , mdr - 1 , and c - kit , exhibit the hallmarks of adult stem cells : self - renewal , clonogenicity , and multi - lineage differentiation . however , the population of progenitor cells in the heart is very low , and the inability to expand this population of cells in vitro or in vivo represents a major barrier to therapeutic stem cell applications . integrin - linked kinase ( ilk ) is a multi - functional protein kinase , which coordinates signal transduction by integrins and growth factor receptors , and serves as a nodal regulator of protein kinase cascades important to cell proliferation , differentiation and apoptosis xv , xvi . ilk functions as the effector of phosphoinositide - 3 ′- oh kinase ( pi3k ) signaling following distinct signal inputs from integrins and growth factor receptor tyrosine kinases xvii , xviii . ilk also inhibits glycogen synthase kinase - 3β ( gsk - 3β ) xvi , xix , which leads to the nuclear accumulation of β - catenin , which , in turn , leads to the activation of wnt target genes implicated in the maintenance and symmetric replication of embryonic stem cells , as well as their more tissue - and lineage - restricted progeny . the canonical wnt / β - catenin signaling pathway has been shown to be important in both embryonic and adult stem cell maintenance and self - renewal in hematopoetic , gastrointestinal and neural tissue xx , xxi , xxii , xxiii , xxiv , xxv , although this pathway has not been studied in cscs . the demonstrable utility of integrin - linked kinase ( ilk ) to promote cardiac stem cell proliferation and self renewal is herein set forth . while it was known that integrin - linked kinase ( ilk ) is a multi - functional protein kinase , which coordinates signal transduction by integrins and growth factor receptors , and activates wnt target genes implicated in the maintenance and symmetric replication of embryonic stem cells , the effect of ilk on cardiac stem cells has been heretofore unknown . recent evidence suggests that the adult heart contains stem cells , which are capable of self - renewal as well as tissue - specific , multi - lineage differentiation . however , their inherent capacity for self - renewal is limiting to cell replacement applications . we herein demonstrate that a cardiac stem cell population is susceptible to amplification through ilk gain - of - function . primary cultures derived from human fetal cardiac tissue ( 19 - 22 weeks gestation ) were grown in serum - free media supplemented with growth factors and evaluated for the appearance of cells with the properties of stem cells , including self - renewal and the capacity to differentiate into definitive cardiac myocytes . the effect of ilk was ascertained using adenoviral over - expression of ilk cdna constructs conveying either gain - or loss - of - function . cultures infected with wild type ilk yielded a significant ( p = 0 . 001 ), ˜ 5 - fold increase in both the absolute number and the frequency of c - kit pos , myosin neg cells , which reached ˜ one cell in 250 . cardiospheres ( cs ), comprised on morphologically homogeneous , anchorage - independent cells , were reproducibly present at day 7 - 10 , and formed derivative cs in multiple passages . ilk infection of primary cardiac cell cultures resulted in a greater number of primary spheres at each cell density tested , compared with untreated and virus controls ( p = 0 . 001 ). secondary spheres transferred to differentiation medium consisting of imdm with 10 % fbs and 5 - aza - deoxycytodine ( 10 um ) generated cells exhibiting biochemical evidence of differentiation into cardiomyocytes , smooth muscle cells and endothelial cells . this study demonstrates that self - renewing cardiospheres generated from human fetal cardiac cells are comprised of cells exhibiting the properties of stem cells , including the capacity for self - renewal and multilineage differentiation . ilk - transformed stem cells are shown to be equally susceptible to cardiac differentiation , even while exhibiting an increased capacity for proliferation and cs formation . our results suggest that ilk promotes stem cell amplification and can be applied therapeutically to overcome a major limitation in the field of cardiac regenerative medicine . here we show that the overexpression of ilk in human fetal cardiac tissue in vitro increases the population of cardiac stem cells , which exhibit self - renewal and multi - lineage differentiation . our results suggest that gain - of - function of a gene which promotes stem cell amplification can be applied therapeutically to overcome a major limitation in the field of regenerative medicine . human fetal hearts were harvested during elective pregnancy termination at the gestational ages of 19 to 22 weeks , in accordance with the guidelines of the institutional human research ethics board and after obtaining maternal consent . the hearts were minced and washed using phosphate buffered saline ( pbs ). cell isolation was accomplished using 0 . 2 % trypsin and 1 . 0 mg / ml type ii collagenase in a 0 . 02 % glucose pbs solution at 37 ° c . after dissection , cells were incubated on pre - coated plastic culture dishes ( starstedt ) for 2 hours at 37 ° c . to remove fibroblasts , with imdm ( gibco ) containing penicillin and streptomycin and supplemented with 10 % fetal bovine serum ( gibco ). after incubation , the supernatant was removed and added to pre - coated culture dishes ( starstedt ) and placed in a 5 % co2 incubator at 37 ° c . cells were cultured to 60 - 70 % confluency in preparation for adenovirally - mediated infection of ilk constructs incorporating green fluorescent protein ( gfp ), as previously described xxvi . replication - deficient serotype 5 adenovirus encoding either the human wild - type ilk gene ( ad . ilk - gfp ) or empty virus constructs ( ad . c ) previously shown to modulate ilk expression and activity in l6 myoblasts xxvii , were used for the infection of cells . cells were infected at 37 ° c . at multiplicity of infection of 1 . 5 in imdm medium with 10 % fetal bovine serum for 24 h . effective gene transfer was confirmed by more than 80 % of gfp positivity . western blot analysis was performed to confirm that the transduction of ad . ilk in cardiac cell cultures . the cells were washed with pbs and harvested by scraping in lysis buffer . after measurement of protein expression , analyses were performed with polyclonal anti - ilk antibody ( cell signaling ). proteins were visualized with an enhanced chemiluminescence ( ecl ) detection reagent ( amersham pharmacia biotech ) and quantified by densitometry . cells were fixed using methanol at − 20 ° c . for 20 minutes . cells were then reacted with c - kit antibody ( diluted 1 : 20 ; assay design inc . ), human monoclonal anti - cd34 ( cymbus biotechnology ), human monoclonal anti - α - smooth muscle actin ( 1 : 100 ; santa cruz ), human polyclonal anti - von willebrand factor ( 1 : 200 ), myosin monoclonal antibody ( mf20 diluted 1 : 10 ), or monoclonal anti - α actin in ( 1 : 200 ) from sigma . nuclei were stained with dapi . all slides were analyzed at 20 × magnification using a lcica fluorescent microscope with a coupled camera . all analysis was done using openlab 4 . 0 . 2 software . more than ten fields were randomly chosen and photographed , and the total cell number (˜ 5000 / dish ) was counted manually in a fashion blinded to viral status . cell viability of cells was confirmed with trypan blue staining prior to plating at densities from 10 cells / μl to 1 cell / μl in 24 - well plates . the culture medium was composed of dmem / f - 12 ( 1 : 1 ) including hepes buffer ( 5 mm ), glucose ( 0 . 6 %), sodium bicarbonate ( 3 mm ), and glutamine ( 2 mm ), insulin ( 25 μg / ml ), transferrin ( 100 μg / ml ), progesterone ( 20 nm ), putrescine ( 60 μm ), sodium selenite ( 30 nm ), human recombinant egf ( 20 ng / ml ), and bfgf ( 20 ng / ml ). the number of primary spheres generated in each well was assessed 14 days after plating . primary spheres were dissociated into single cells consisting of ˜ 200 - 500 cells , which were placed in 96 - well plates . the number of secondary spheres was assessed 14 days after replating dissociated cells . secondary spheres were transferred to differentiation medium , which was composed of imdm containing 10 % fbs and 10 um 5 - aza - 2 ′- deoxycytodine ( 5azad ). cells migrating out from the spheres were analyzed by immunocytochemistry on day 14 . cells were fixed and characterized by staining with the following markers : α - cardiac actinin antibody ( diluted 1 : 100 , sigma ), von willebrand factor antibody ( diluted 1 : 200 dako ), or α - smooth muscle actin antibody ( diluted 1 : 100 , santa cruz ). to determine whether the overexpression of ilk increases the stem cell number in the human heart , fetal hearts of gestational ages 19 - 22 weeks were acquired during elective pregnancy termination , and the hearts were enzymatically dissociated into single cell suspension . the cells were incubated on pre - coated plastic culture dishes for 2 hours at 37 ° c . to remove fibroblasts , which were shown to be devoid of c - kit pos cells . at 2 - 3 days after isolation and at 60 - 70 % confluency , cells were infected with replication defective adenovirus containing wild type ( ad . ilk ), or virus control ( ad . c ). effective gene transfer was confirmed by more than 80 % gfp positivity ( fig1 a ) and by ˜ 3 - fold increase in ilk protein expression ( fig1 b ) in cell cultures . c - kit pos cells imaged by fluorescence microscopy were invariably negative for the cardiac myosin markers α - cardiac actinin ( fig1 c ), mf20 and the hematopoetic stem cell marker cd34 . cultures infected with wild type ilk yielded a significant ( p = 0 . 001 ), ˜ 5 - fold increase in both the absolute number and the frequency of c - kit pos cells , which reached ˜ one cell in 250 ( fig1 d ). to determine if primary human fetal cardiac cells generate cardiospheres in vitro , cells were infected with ad . ilk or control virus and plated in serum - free medium supplemented with 20 ng / ml each of egf and bfgf at clonal density of a single cell per well in 24 - well plates . primary cardiospheres ( cs ), comprised on morphologically homogeneous cells , were reproducibly present at day 7 - 10 ( fig2 , upper panel ). cs were noted to be uniformly free - floating , presumably reflecting anchorage - independence , in distinction to cardiac myocytes which became rapidly adherent to the culture plate surface . cells from dissociated primary cs were plated at a density corresponding to one sphere (˜ 200 - 400 cells )/ well . secondary cs , which were morphologically indistinguishable from primary cs , were evident in ˜ 60 % of wells at day 14 ( fig2 , lower panel ). cs were shown to be comprised of cells expressing the c - kit pos surface receptor ( fig3 ). occasional cells at the periphery of the spheres stained for the cardiac marker α - actinin ( arrowhead ). ilk infection of primary cardiac cell cultures resulted in a greater number of primary spheres at each cell density tested , compared with untreated and virus controls ( fig4 a ). among cs generated from ilk - infected cultures , ˜ 80 % stained homogeneously for ilk - gfp ; ˜ 20 % exhibited no evidence of gfp staining ; and no spheres were observed which were mosaic for gfp , suggesting origin from a single cell rather than cellular aggregation . the frequency of sphere - initiating cells , as measured by the ratio of sphere number : total cell number , was significantly greater in ilk - overexpressing cultures ( fig4 b ). the frequency of secondary or tertiary spheres generated from primary spheres comprised of ad . ilk , ad . c or uninfected cells was highly similar (˜ 60 % of wells ), indicating that while ilk gain - of - function increases the formation of primary spheres , it does not alter their inherent capacity for subsequent self - renewal . cardiac stem cells are multipotent and have the capacity to differentiate into smooth muscle , cardiac and endothelial cells xxx , xxxi , xxxii . secondary spheres were transferred to differentiation medium consisting of imdm with 10 % fbs and the methyltransferase inhibitor , 5azad ( 10 um ). within 4 - 5 days spheres became attached to the plate and individual cells migrated from spheres , which exhibited biochemical evidence of differentiation into cardiomyocytes , smooth muscle cells and endothelial cells ( fig5 ). the profile of differentiated cells among ilk - over expressing and control cells was highly similar ( fig5 , lower left panel ), indicating that ilk - induced clonal proliferation of cardiac stem cells does not impair their capacity for multilineage differentiation . these experiments show that primary cultures derived from human fetal cardiac tissue grown in non - serum , growth factor - supplemented media form macroscopic cardiopsheres , analogous to neurospheres containing multipotent neural stem cells xxvii , xxix . cardiospheres ( cs ) have been previously characterized as lineage - negative ( lin neg ) c - kit pos , morphologically homogeneous cells devoid of cardiac markers such as sarcomeric structures , having the capacity for self - renewal , as well as differentiation into functional cardiac myocytes , and to participate in the regeneration of functional myocardium in vivo xxx , xxxi , xxxii . cells comprising cs did not express the hematopoetic stem cell marker cd34 , suggesting that cs were derived from a cardiac resident , rather than from a bone marrow - mobilized , cell population xxxiii . the evolutionarily conserved canonical wnt pathway has been implicated in both human and mouse es cell self - renewal competence xxii . the wnt / β - catenin signaling pathway is required for maintaining proliferation of neuronal progenitors xxiii , and for haematopoietic stein cell homeostasis xxv . ilk negatively modulates of gsk - 3β activity and promotes nuclear activation of β - catenin xxiii , xxxiv , xxxv , xxxvi , xxxvii , and is a candidate kinase activator of wnt pathway signaling xvi . ilk over - expression or constitutive activation promotes cell cycle transit through a signaling pathway comprising the wnt components gsk - 3β and β - catenin , leading to increased expression of cyclin d1 xxxviii , and providing a molecular basis for the inherent proliferation ( self - renewal ) property of stem cells . moreover , ilk promotes anchorage - independent survival , which appears to be a generic and poorly understood feature of stem cells , including c - kit - containing cs isolated from adult rat xxxix and human hearts xl . these experiments validate our initial theory that human fetal cardiac tissue would be enriched for stem cells , which are important during cardiogenesis xli . since it has been reported that cardiac c - kit positive cells can grow and differentiate into the various cardiac lineages , including cardiomyocytes , smooth muscle and endothelial cells xiv , c - kit antibody was used as a marker for cardiac stem cells . the proto - oncogene c - kit encodes a transmembrane tyrosine kinase receptor , and the ligand for c - kit has been identified to be stem cell factor ( scf ) xlii . we took advantage of the tendency of cardiac cells to form macroscopic cs when grown on non - adhesive substrata in the presence of growth factor supplementation . using the capacity to form cs as a readout for stem cell frequency , we tested whether adenoviral ilk overexpression would cause proliferation of cs - forming cells with self - renewal , clonogenic , and multi - differentiation properties . we have thus demonstrated that self - renewing cardiospheres generated from human fetal cardiac cells are comprised of cells exhibiting the properties of stem cells , including the capacity for self - renewal and multilineage differentiation . this result has been also reported in cardiac cells isolated from adult rodent xxx , murine xxxii , and human atrial biopsies xxxi . overexpression of ilk resulted in an ˜ 10 - fold increase in the frequency of sphere - initiating cells . importantly , ilk - transformed stem cells are shown to be equally susceptible to cardiac differentiation , even while exhibiting an increased capacity for proliferation and cs formation . ilk is positioned to transduce distinct signal inputs from integrins and growth factor receptor tyrosine kinases xliii , xliv , is an activator of the wnt pathway xlv , and promotes anchorage - independent cellular proliferation xvi , xviii , thus providing a putative molecular basis for the observed amplification effect on the cardiac stem cell population . an ilk - dependent increase in cardiac stem cell frequency is consistent with the finding that vascular endothelial growth factor ( vegf ) has been shown to positively regulate hematopoetic stem cell survival xlvi , since ilk positively regulates vegf expression through an hypoxia - inducible factor - 1α - dependent pathway xv . the fact that ilk effect was evident even under conditions of growth factor supplementation supports the rationale of exploiting upregulation the ilk signaling pathway as a novel strategy to promote therapeutically useful expansion of a target stem cell population . all patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains . all patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference . it is to be understood that while a certain form of the invention is illustrated , it is not to be limited to the specific form or arrangement herein described and shown . it will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification and any drawings / figures included herein . one skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned , as well as those inherent therein . the embodiments , methods , procedures and techniques described herein are presently representative of the preferred embodiments , are intended to be exemplary and are not intended as limitations on the scope . changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims . although the invention has been described in connection with specific preferred embodiments , it should be understood that the invention as claimed should not be unduly limited to such specific embodiments . indeed , various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims . i nelson w j , nusse r . convergence of wnt , beta - 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