Carolyn A. Carr

Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron labeling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine‐magnetic resonance imaging (cine‐MRI) and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterized by flow cytometry, transduced with lentiviral vectors expressing green fluorescent protein (GFP), and labeled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation, and rat hearts were imaged at 1, 4, 10, and 16 weeks postinfarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whereas the signal void volume did not decrease significantly in severely infarcted hearts. High‐resolution three‐dimensional magnetic resonance (MR) microscopy identified hypointense regions at the same position as in vivo. Donor cells containing iron particles and expressing GFP were identified in MR‐targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labeled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron‐labeled donor cells can be used for their isolation from host tissue to enable further characterization.

Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: an in vivo cine-MRI study

Basic and clinical studies have shown that bone marrow cell therapy can improve cardiac function following infarction. In experimental animals, reported stem cell-mediated changes range from no measurable improvement to the complete restoration of function. In the clinic, however, the average improvement in left ventricular ejection fraction is around 2% to 3%. A possible explanation for the discrepancy between basic and clinical results is that few basic studies have used the magnetic resonance (MR) imaging (MRI) methods that were used in clinical trials for measuring cardiac function. Consequently, we employed cine-MR to determine the effect of bone marrow stromal cells (BMSCs) on cardiac function in rats. Cultured rat BMSCs were characterized using flow cytometry and labeled with iron oxide particles and a fluorescent marker to allow in vivo cell tracking and ex vivo cell identification, respectively. Neither label affected in vitro cell proliferation or differentiation. Rat hearts were infarcted, and BMSCs or control media were injected into the infarct periphery (n = 34) or infused systemically (n = 30). MRI was used to measure cardiac morphology and function and to determine cell distribution for 10 wk after infarction and cell therapy. In vivo MRI, histology, and cell reisolation confirmed successful BMSC delivery and retention within the myocardium throughout the experiment. However, no significant improvement in any measure of cardiac function was observed at any time. We conclude that cultured BMSCs are not the optimal cell population to treat the infarcted heart.

Cine-MRI versus two-dimensional echocardiography to measure in vivo left ventricular function in rat heart

Two‐dimensional echocardiography is the most commonly used non‐invasive method for measuring in vivo cardiac function in experimental animals. In humans, measurements of cardiac function made using cine‐MRI compare favourably with those made using echocardiography. However, no rigorous comparison has been made in small animals. Here, standard short‐axis two‐dimensional (2D) echocardiography (2D‐echo) and cine‐MRI measurements were made in the same rats, both control and after chronic myocardial infarction. Correlations between the two techniques were found for end diastolic area, stroke area and ejection fraction, but cine‐MRI measurements of ejection fraction were 12u2009±u20096% higher than those made using 2D‐echo, because of the 1.8‐fold higher temporal resolution of the MRI technique (4.6u2009ms vs 8.3u2009ms). Repeated measurements on the same group of rats over several days showed that the cine‐MRI technique was more reproducible than 2D‐echo, in that 2D‐echo would require five times more animals to find a statistically significant difference. In summary, caution should be exercised when comparing functional results acquired using short‐axis 2D‐echo vs cine‐MRI. The accuracy of cine‐MRI allows identification of alterations in heart function that may be missed when using 2D‐echo. Copyright

Novel MRI method to detect altered left ventricular ejection and filling patterns in rodent models of disease.

The aim of this study was to determine whether high‐temporal‐resolution (HTR) cardiac cine‐MRI could be used to identify subtle alterations in contractility and diastolic function in rodent models of disease. Following standard 45‐min in vivo MRI measurements of left ventricular (LV) volumes, a single mid‐ventricular slice was selected for 3‐min HTR imaging. Cavity volume was measured every 2.4 ms, yielding approximately 60 images through the cardiac cycle. From these images, peak ejection and filling rates were calculated and two separate filling phases (comparable with the early (E) and late (A) phases of a Doppler echocardiogram) were identified during diastole. Repeated HTR imaging of the same animals on sequential days indicated reproducibility of E′/A′ ratios of 11%. In chronically infarcted rat hearts, HTR imaging revealed lower peak ejection rates (PERs), peak early filling rates (E′) and E′/A′ ratios, and higher peak late filling rates (A′) than in sham‐operated rats. Diabetic db/db mouse hearts had the same function as controls when using standard cine‐MRI, yet HTR imaging identified significantly lower PERs, early filling rates and E′/A′ ratios in diabetic mouse hearts. In conclusion, the HTR MRI technique revealed changes in function that were below the limits of detection of standard cine‐MRI. Magn Reson Med 60:582–587, 2008.

Human cardiosphere-derived cells from patients with chronic ischaemic heart disease can be routinely expanded from atrial but not epicardial ventricular biopsies.

To investigate the effects of age and disease on endogenous cardiac progenitor cells, we obtained right atrial and left ventricular epicardial biopsies from patients (nu2009=u200922) with chronic ischaemic heart disease and measured doubling time and surface marker expression in explant- and cardiosphere-derived cells (EDCs, CDCs). EDCs could be expanded from all atrial biopsy samples, but sufficient cells for cardiosphere culture were obtained from only 8 of 22 ventricular biopsies. EDCs from both atrium and ventricle contained a higher proportion of c-kit+ cells than CDCs, which contained few such cells. There was wide variation in expression of CD90 (atrial CDCs 5–92xa0% CD90+; ventricular CDCs 11–89xa0% CD90+), with atrial CDCs cultured from diabetic patients (nu2009=u20094) containing 1.6-fold more CD90+ cells than those from non-diabetic patients (nu2009=u200918). No effect of age or other co-morbidities was detected. Thus, CDCs from atrial biopsies may vary in their therapeutic potential.

First-pass perfusion CMR two days after infarction predicts severity of functional impairment six weeks later in the rat heart

BackgroundIn humans, dynamic contrast CMR of the first pass of a bolus infusion of Gadolinium-based contrast agent has become a standard technique to identify under-perfused regions of the heart and can accurately demonstrate the severity of myocardial infarction. Despite the clinical importance of this method, it has rarely been applied in small animal models of cardiac disease. In order to identify perfusion delays in the infarcted rat heart, here we present a method in which a T1 weighted MR image has been acquired during each cardiac cycle.Methods and resultsIn isolated perfused rat hearts, contrast agent infusion gave uniform signal enhancement throughout the myocardium. Occlusion of the left anterior descending coronary artery significantly reduced the rate of signal enhancement in anterior regions of the heart, demonstrating that the first-pass method was sensitive to perfusion deficits. In vivo measurements of myocardial morphology, function, perfusion and viability were made at 2 and 8 days after infarction. Morphology and function were further assessed using cine-MRI at 42 days. The perfusion delay was larger in rat hearts that went on to develop greater functional impairment, demonstrating that first-pass CMR can be used as an early indicator of infarct severity. First-pass CMR at 2 and 8 days following infarction better predicted outcome than cardiac ejection fraction, end diastolic volume or end systolic volume.ConclusionFirst-pass CMR provides a predictive measure of the severity of myocardial impairment caused by infarction in a rodent model of heart failure.

The effect of 4′-substituents on the kinetics of ligand substitution in 2,2′:6′,2″-terpyridine platinum(II) complexes

The rates of hydrolysis of five (4′-substituted-2,2′:6′,2″-terpyridine) platinum(II) (n-propylamine) complexes exhibit a high sensitivity to the electronic properties of the terpyridine ligand; analysis using the Hammett relationship gives a ρ value of +2.34.

Ongoing Dual-Angle Measurements for the Correction of Partial Saturation in 31P MR Spectroscopy

Use of a repetition time similar to, or shorter than, metabolite T1s is common in NMR spectroscopy of biological samples to improve the signal‐to‐noise ratio. Conventionally, the partial saturation that results from this is corrected using saturation factors. However, this can lead to erroneous results in the presence of chemical exchange or nonconstant T1s. We describe an alternative approach to correction for saturation, based on ongoing dual‐angle T1 measurement. Using 31P magnetic resonance spectroscopy of the perfused rat heart undergoing ischemia‐reperfusion, we demonstrate that signal alternations in the data acquired by the dual‐angle approach are eliminated by the ongoing dual‐angle T1 measurement correction scheme, meaning that metabolite concentration and T1 measurement can be made throughout the course of the ischemia‐reperfusion protocol. Simulations, based on parameters pertinent to the perfused rat heart, demonstrate that accurate saturation correction is possible with this method except at times of rapid concentration change. Additionally, compared to the conventional saturation factor correction method, the ongoing dual‐angle T1 measurement correction scheme results in improved accuracy in determining the [phosphocreatine] recovery time constant. Thus, the ongoing dual‐angle T1 measurements procedure permits accurate monitoring of metabolite concentrations even in the setting of chemical exchange and T1 changes and allows more accurate analysis of bioenergetic status. Magn Reson Med, 2010.

EFFICIENT CARDIAC DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS, THAT ENGRAFT AND PROTECT AGAINST ISCHAEMIC DAMAGE IN THE INFARCTED RAT HEART

Induced pluripotent stem (iPS) cells provide a promising source of cardiac progenitor cells for administration to the infarcted heart. Here, we describe a protocol for efficient, serum free, directed differentiation of human iPS cells, as a monolayer, that yields a mixed population in which cardiomyocytes, endothelium and smooth muscle cells constitute over 50% of the differentiated population, with no additional selection strategies utilised. GFP positive cardiac progenitors from human iPS cells (2×106) were administered by direct injection into the myocardium of athymic nude rats following 50u2005minu2005of ischaemia. Cardiac function was measured using MRI at 2u2005days, 2, 6 and 10u2005weeks. At 10u2005weeksu2005the hearts were removed for histology. By 6u2005weeks, control infarcted hearts (n=5) had significantly larger end systolic volumes and lower ejection fractions than sham operated hearts (n=3) whereas hearts treated with iPS-derived cardiac differentiated cells (n=4) maintained cardiac function; such that by 10u2005weeksu2005the end systolic volumes and ejection fractions were not significantly different from those of the sham operated animals (ejection fraction at 10u2005weeks: sham 77±5%, infarct 45±9% (p<0.05 vs sham), iPS 62±4%). Histological analysis showed that the human iPS-derived cardiac progenitor cells engrafted, differentiated into cardiomyocytes and smooth muscle, and persisted for at least 10u2005weeksu2005post-infarct. Thus efficient directed differentiation of human iPS cells towards the cardiac lineage generates cells that can prevent loss of heart function after ischaemic heart damage.