Proceedings of the meeting - Department of Physics - University of ...

O15MRI tracking to establish the relationship between myocardial injury and stem cell homing.Carolyn Carr 1 , Daniel Stuckey 1 , Louise Tatton 2 , Damian Tyler 1 , Juergen Schneider 1 , Sian Harding 3 , Kieran Clarke 11 Department of Physiology, Anatomy and Genetics, University of Oxford; 2 National Blood Service, John Radcliffe Hospital, Oxford;3 National Heart and Lung Institute, Imperial College, LondonIntroductionStem cells offer a promising approach to the treatment of myocardialinfarction and prevention of heart failure. Clinical trials using mononuclearcells, bone marrow-derived stromal cells (BMSCs) and skeletalmyoblasts are completed or underway 1 . In these trials, cells havebeen administered either by direct injection into the myocardium or byinfusion into the coronary system. In order to optimise cell-basedtherapy, the fate of the administered stem cells should be known,particularly where they are administered by infusion. Homing of stemcells to the infarcted myocardium has been shown by histologicalmethods to be related to the extent of myocardial injury 2 . Previouslywe have used iron oxide-labelling of BMSCs to non-invasively trackdonor cells injected into the infarcted myocardium by MRI and toisolate the donor cells from the grafted hearts using the magneticproperties of the injected BMSCs 3 . Here we infused labelled cells intothe vasculature to investigate the relation between myocardial damageand cell homing.AEF 65%CBEF 45% 3000 BMSCsDMethodsRat BMSCs were isolated from the tibia and fibia of male Wistar ratsand adherent cells were cultured to passage two. Prior toadministration, cells were incubated with 0.9 µm iron oxide particles(Bangs Laboratories Inc) for 24 hours and Vybrant DiI cell trackerdye for 1 hour. Myocardial infarction was induced in female rats byligation of the coronary artery. One or three days after coronary arteryligation, 5 x 10 6 labelled BMSCs were infused via the tail vein. Heartfunction was determined using cine MRI at 2, 7 and 28 days afterinfarct surgery. Animals were sacrificed at 4 weeks and hearts takenfor ex vivo MR microscopy or cell isolation.Cine MR acquisition: cardiac cine-MRI was performed using an11.7 T MR system with a Bruker console and a 60 mm birdcage coilas described previously 4 . A stack of contiguous 1.5 mm true shortaxis ECG and respiration-gated cine images (FOV 51.2 mm 2 , matrixsize 256 x 256, TE/TR 1.43/4.6 ms, 17.5° pulse, 25-35 frames percardiac cycle) were acquired to cover the entire left ventricle.MR microscopy: hearts were excised, fixed in paraformaldehyde, andembedded in 1% agarose doped with Gadolinium DTPA for highresolution MRI (fast gradient echo, FOV, 20mm 3 ; matrix size,256×256×512; TR/TE, 1.8/30 ms).Cell re-isolation: hearts were excised and digested with collagenase.Total digest was visualised by microscopy. In some cases, ironoxide/DiI + cells were separated from digested heart tissue using amagnet.ResultsMRI: Infarct surgery resulted in hearts with a range of ejection fractions(EF; 35-75%) and scar sizes (2-35%). In some hearts, a cleararea of signal void could be seen in the in vivo cine MRI throughoutthe infarct scar (Figure A, B). This area of signal void was confirmedby ex vivo MR microscopy (Figure C). Hearts with low levels ofdamage did not contain these areas of signal void (Figure D).Cell re-isolation: Fluorescent DiI labelled cells could clearly be seenin the tissue digest. Hearts with a more severe infarct were found tocontain a greater number of BMSCs (Figure B). DiI+ BMSCs couldalso be seen in sections of undigested scar tissue. Low numbers ofcells, undetected by MRI, were observed in hearts with smaller infarcts(Figure D). On rare occasions, contracting DiI+ cardiomyocyteswere observed. Contraction analysis showed that these were of adultphenotype and therefore unlikely to have resulted from differentiationof the BMSCs. DiI is a cytoplasmic stain and thus can be transferredbetween cells during scavenging or cell fusion 5 .DiscussionIron oxide labelling can be used to track BMSCs successfully whenthe cells are injected directly into the myocardium 3 since this results ina large number of labelled cells located within a small area of tissue.Infusion of BMSCs into the vasculature is associated with high loss ofcells as BMSCs become trapped in other organs, such as the lungs,EF 67% 1200 BMSCsIn vivo cine MRI 4 weeks post infarct surgery (A, B, D) and ex vivoMR microscopy (C) of the heart shown in A. The number of DiI+BMSCs detected after cell isolation from hearts B and D is shown.liver and spleen 6 . Nevertheless, cells infused into the vasculature havebeen detected in the infarcted heart using histology by us and others 2 .In hearts with a moderate infarct, regions of signal void could bedetected through the scar by cine MRI (Figure A) and MR microscopy(Figure C). Visualisation of the iron oxide labelled BMSCs in severelyinfarcted hearts can be more challenging as the scar is very thin andsignal along the endocardial edge of the scar may be diminished dueto the presence of compact collagen fibres and fibroblasts. In heartswith low levels of damage (Figure D), signal voids were not observed.Isolation of cells from heart tissue by total heart digest enabled thenumber of BMSCs that had homed to the heart and remained there forfour weeks to be established. Previously 3 we have shown that retentionof BMSCs injected directly into the myocardium correlates withthe degree of damage to the heart. Where cells are infused, the largernumber of BMSCs in more infarcted hearts may be due to increasedhoming to the heart or to improved retention of cells by the damagedmyocardium. The low numbers of DiI-labelled cardiomyocytes observedand their adult phenotype indicates that myocyte formationfrom BMSCs did not occur in this study.ConclusionIron oxide labelling can be used to track BMSCs administered to theinfarcted heart by intravenous infusion and the magnetic labellingfacilitates separation of grafted cells from those of the hostmyocardium after digestion of the heart. However, due to the lownumbers of BMSCs remaining in the mildly infarcted heart and thethin scar of the severely infarcted heart, this technique is less robustwhen intravenous infusion is used rather than direct myocardialinjection. A labelling technique that gives positive contrast would bemore valuable for low cell numbers in scar tissue.References1) Cleland et al, 2006, Eur J Heart Fail, 8, 105-1102) Ciulla et al, 2004, Transfusion, 44, 239-443) Stuckey et al, 2006, Stem Cells, in press4) Tyler et al, 2006, J Cardiovas Magn Reson, 8, 327-335) Garbade et al, 2005, Eur J Cardiothorac Surg, 28, 685-6916) Aicher et al, 2003, Circulation, 107, 2134-9This project was funded by the British Heart Foundation