current treatments at best only slow the progression of disease leading to heart failure and death ; once scar has developed , the injury to heart muscle is generally cumulative and irreversible . stem cell therapy represents a promising strategy to mitigate remodelling and regenerate the damaged myocardium . cardiospheres ( csps ) and cardiosphere - derived cells ( cdcs ) are natural mixtures of resident cardiac stem cells and supporting cell types . studies have shown that csps and cdcs are able to ameliorate cardiac dysfunction in acute and chronic ischaemic cardiomyopathies [ 110 ] . despite emerging pre - clinical and clinical evidence of the beneficial effects of cell - based therapy , previous clinical trials using bone marrow mononuclear cells   suggested a cell dose - dependent effect in myocardial function improvement and scar size reduction . however , dose dependence has generally been poorly characterized in animal models prior to clinical application . most studies in rodents have used astronomically high cell doses when extrapolated to large animal or human doses . for instance , in the scipio trial ( clinicaltrials.gov ; nct00474461 ) 1 million c - kit cardiac stem cells are administered to humans . interestingly , the same dose ( 1 million cells ) was employed in a pre - clinical rat model by the same group , despite the > 200-fold difference in recipient size . most other cell types have suffered from similar discrepancies in advancing from rodents to large animals and humans . this study was designed to examine the dose - dependent effects of intramyocardial injection of human csps in a mouse model of acute myocardial infarction ( mi ) . in addition , we sought to determine , on the basis of histological analysis , the mechanisms for this dose - dependent benefit . the hope is to systematically optimize dose in mice and then use these results to guide dosing in large animals and humans . human csps were derived as described [ 6 , 7 ] . briefly , human right ventricle biopsies from male donors were minced into small fragments . explants on dishes coated with fibronectin ( bd biosciences , bedford , ma , usa ) . within 12 weeks , stromal - like flat cells , and phase - bright round cells , emerged from the tissue fragments and became confluent . these csp - forming cells were harvested and then plated on poly - d - lysine ( bd biosciences)-coated plates for suspension culture . acute mi was created in adult male scid - beige mice ( 1012 weeks old ) , as described [ 1 , 7 ] , by ligating the left anterior descending artery . as the csp is a relatively heterogenous product and the numbers may vary from different batches , we define the dose by the number of starting csp - forming cells . the mice were then subjected to intramyocardial injections with a 30-gauge needle at four points in the infarct border zone , with one of the following five randomly assigned treatments : ( i ) 20 l pbs ( control group ) ; ( ii ) 1  10 cells formed csps ; ( iii ) 5  10 cells formed csps ; ( iv ) 1  10 cells formed csps ; ( v ) 5  10 cells formed csps . cardiac functions were assessed by echocardiography at baseline ( 4 hrs post - mi ) and 3 weeks afterwards [ 1 , 7 ] . a sub - population of animals from each group was sacrificed at 24 hrs and the hearts were excised for determining the numbers of engrafted cells by quantitative pcr ( utilizing the sry gene located on the y - chromosome of the injected male human cells ) . all other animals were killed at 3 weeks and hearts were excised for morphometry and immunohistochemisty . was determined using the two - tailed unpaired t - test and among groups by anova followed by bonferroni 's post - hoc test . when csp - forming cells were seeded on poly - d - lysine - coated plates , they readily aggregated into 3d clusters , within 35 days , to yield a suspension of csps ( fig . the phenotypes of csps have been well characterized in previous studies [ 14 , 15 ] . generally , csps consist of distinct layers with the expression of c - kit in the core and the mesenchymal marker cd105 in their periphery ( fig . qpcr quantitation of human cells in the mouse hearts revealed a 10% cell retention rate 24 hrs after injection into the injured hearts , regardless of the doses ( fig . . the cardiospheres are consistently positive for cd105 ( b ) and negative for cd45 ( c ) . ( d ) qpcr quantitation of cell retention rates in hearts injected with male human cardiospheres ( n = 5 mice per group ) . the most meaningful indicator of therapeutic benefit , in clinical practice , is the ability to produce functional preservation or improvement in the injured heart . echocardiography revealed that baseline left ventricular ejection fraction ( lvef ) after surgery did not differ among groups , indicating comparable degrees of initial injury ( fig . three weeks after treatment , lvefs in mice receiving the three highest doses ( 5  10 , 1  10 and 5  10 ) outperformed the saline group ( fig . no significant difference was seen between the saline and the 1  10 groups , although a positive trend was evident . to facilitate comparisons of treatment effects among groups , we calculated the change of lvefs over the 3-week time course . over the 3 weeks 2c ) , but not in the hearts that had received high doses of csps . this is in line with previous reports that csp transplantation preserves lvefs in post - mi hearts [ 6 , 7 ] . lvef slightly declined in the mice which received the 1  10 dose , but not as much as in the saline group ( not statistically different ) . coinciding with the dose - dependent functional benefit is the dose - dependent attenuation of ventricular remodelling . masson 's trichrome staining clearly distinguished scar tissue ( blue ) from normal myocardium ( pink ; fig . quantitative morphometry at 3 weeks showed severe lv chamber dilatation and infarct wall thinning in the saline - injected hearts ( fig . in contrast , high - dose csp - treated hearts exhibited attenuated lv remodelling and less abnormal heart morphology . magnification of the infarct region revealed the preservation and/or regeneration of normal myocardium tissue in the infarct . 3c ) in the infarct area from the hearts received the three high doses of csps . the measures from the low - dose group ( 1  10 cells ) were somewhat higher than that in the saline group , although not significantly so . cardiac function : left ventricular ejection fraction ( lvef ) measured by echocardiography at baseline ( post - myocardial infarction ; a ) and 3 weeks afterwards ( b ) in saline or various dosage groups ( n = 56 mice per group ) . * p < 0.05 when compared with the saline group or the 1  10 group . ( a ) representative masson 's trichrome - stained myocardial sections 3 weeks after treatment . ( b , c ) quantitative analysis and left ventricle morphometric parameters of the masson 's trichrome images ( n = 5 mice per group ) . * p < 0.05 when compared with the saline group or the 1  10 group . we have found that csps exert their functional benefits through both direct regeneration and indirect paracrine mechanisms , with the latter as the predominant factor . following this lead , we sought to investigate whether the dose - dependent functional benefit of csps also involves paracrine contributions , which involve various mechanisms such as promoting angiogenesis and cell cycle re - entry of mature cardiomyocytes and preserving myocardium after ischaemic injury . myocardial neovascularization assessed by vascular density at 3 weeks was dose dependently enhanced in mice receiving csp transplantation . hearts receiving high doses of csps exhibited higher vascular densities than those in the low - dose group ( 1  10 cells ) or the saline group ( fig . the vascular densities in the low - dose group were similar to that in the saline group . a similar dose - dependent enhancement was found in promoting cell cycle re - entry of cardiomyocytes . more ki67 cardiomyocytes were evident in hearts from the three high - dose groups ( fig . 5 ) , as compared with the low - dose or the saline group . again in addition to tissue regeneration , tissue preservation may be a salutary mechanism of cell therapy for acute mi . to quantify tissue preservation , apoptotic cells in the hearts were terminal deoxynucleotidyl transferase dutp nick end labeling ( tunel)-stained . fewer apoptotic cells were detected in the hearts from the three high - dose groups ( fig . it should be noted that , at 3 weeks , the numbers of tunel cells are a cumulative reflection of maladaptive lv remodelling rather than acute cell death due to ischaemia . ( a  c ) representative confocal images showing -smooth muscle actin - positive vasculature in the heart . ( d ) quantitation of -smooth muscle actin - positive vasculature in various groups ( n = 5 mice per group ) . * p ( b ) quantitation of ki67 cardiomyocytes in various groups ( n = 5 mice per group ) . * p < 0.05 when compared with the saline group or the 1  10 group . ( b ) quantitation of tunel cells in various groups ( n = 5 mice per group ) . * p < 0.05 when compared with the saline group or the 1  10 group . the last decade witnessed a burst of pre - clinical and clinical data indicating that the transplantation of stem cells may benefit diseased myocardium . despite mounting pre - clinical and clinical evidence of the positive effects of cell - based therapy , some salient questions include : ( i ) is there a minimally effective cell dose for therapeutic benefit ? ( ii ) when cell dose keeps escalating , does it reach a benefit plateau or even induce detrimental effects ? in this study , we explored these questions by using human csp in a mouse model of acute mi . the 24-hr cell retention rate was independent of cell dose : 10% of injected cells engrafted in the myocardium , regardless how many cells were injected ( fig . this indicates that the myocardial capacity to host transplanted cells is not saturated , at least at the dose of 5  10 cells . previous studies from our laboratory and other groups indicate that cardiac contraction and venous drainage account for a great amount of initial cell loss [ 2 , 9 ] . a large fraction of cells are simply  washed out immediately after injection . therefore , a straightforward way to increase total cell engraftment is to increase the delivered dose . the three highest doses ( 5  10 , 1  10 and 5  10 cells ) of csps exerted superior functional benefit ( i.e. higher values of lvef ) than the saline group or the low - dose ( 1  10 ) group . there was a trend for the low - dose group to outperform the saline group , but the differences are not statistically significant . moreover , higher resolution is needed to elucidate whether there are dose - dependent patterns within the three highest doses . a similar dose 3 ) . better attenuation of ventricular remodelling was evident in the hearts at the high doses but not in the low - dose group . this indicates that the functional benefit from the high - dose groups was at least partially because of prevention of ventricular dilation and wall thinning . we sought to dissect the mechanisms underlying the dose - dependent functional benefit and heart morphology preservation brought about by csp injections . our previous studies indicated that cell engraftment at 3 weeks is extremely low ( i.e. < 1% of total injected cells could be detected at 3 weeks ) . thus , although the high - dose groups could have relatively more engrafted cells at 3 weeks , the absolute numbers of engrafted cells are small and seem insufficient to explain the striking functional benefit . to that end , we focused on examining the indirect paracrine effects of various csp doses . these effects include promotion of angiogenesis , cardiomyocyte cell cycle re - entry and inhibition of apoptosis . factories , secreting various cytokines and growth factors ( e.g. vascular endothelial growth factor , insulin - like growth factor , hepatocyte growth factor , stromal cell - derived factor-1 ) . it has been shown that these factors can promote angiogenesis , tissue protection and cardiomyocyte proliferation . taking these facts together , our data suggest that the dose - dependent functional benefit from csps may largely come from paracrine effects . our study demonstrated dose - dependent functional benefit of csp transplantation in mice with acute mi . given that the mouse body weight is 2025 g and average human weight is 70 kg , the corresponding doses in humans , relative to those used here in mice , would be 30 million , 150 million , 300 million and 1.5 billion cells . based on our results , we would extrapolate 150 m as an efficacious dose in humans and one where the benefit has nearly reached saturation . taking together these considerations and manufacturing feasibility , we anticipate using 150 m csp cell - equivalent dosage in large animals and humans . nevertheless , future studies with large animals are needed to confirm that extrapolations from rodents are useful for defining a plausible dose range for clinical trials .
despite mounting pre - clinical and clinical evidence of the beneficial effects of cell - based therapy , optimal cell dosing and delivery approaches have not been identified . 
 cardiospheres are self - assembling three - dimensional ( 3d ) microtissues formed by cardiac stem cells and supporting cell types . the ability of cardiospheres to augment cardiac function has been demonstrated in animal models of ischemic cardiomyopathy . in this study 
 , we studied the dose dependence of the benefits of human cardiospheres , delivered via intramyocardial injection , upon cardiac function and ventricular remodelling in scid mice with acute myocardial infarction . 
 four doses of cardiospheres were used : 1  104 , 5  104 , 1  105 and 5  105 ( expressed as number of plated cardiosphere - forming cells ) . 
 acute ( 24 hr ) cell retention rates in all groups were similar . 
 functional assessment and quantitative heart morphometry indicated benefit from higher cell doses ( 5  104 ) in terms of ejection fraction , infarct size and capillary density . 
 histological analysis indicated that the dose - dependent benefit was primarily because of indirect effects of transplanted cells . 
 the results provide scalable data on cardiosphere dosing for intramyocardial injection .