Background.
Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate
in vivo
.
Aim.
To develop a reporter gene that permits
in vivo
stem cell labeling. We examined the sodium-iodide symporter (NIS), a protein that is not expressed in the heart, but promotes cellular uptake of
99m
Tc or
124
I, thus permitting cell tracking by SPECT or PET imaging, respectively.
Methods.
The human NIS gene (
h
NIS) was expressed in rat cardiac derived stem cells (rCDCs) using lentivirus driven by the CAG or CMV promoter. NIS function in transduced cells was confirmed by
in vitro
99m
Tc uptake. Eleven rats were injected with 1 or 2 million rCDCs intramyocardially immediately after LAD ligation; 6 with CMV-NIS and 5 with CAG-NIS cells. Dual isotope SPECT imaging was performed on a small animal SPECT/CT system, using
99m
Tc for cell detection and
201
Tl for myocardial delineation, 24 hrs after cell injection. PET was performed on a small animal PET scanner using
124
I for cell tracking and
13
NH
3
for myocardial delineation, 48hrs after cell injection. Contrast Ratio (CR) was defined as [(signal in the cells)-(signal in blood pool)]/signal in blood pool. High resolution
ex vivo
SPECT scans of explanted hearts (n=3) were obtained to confirm that
in vivo
signal was derived from the cell injection site. The presence of
h
NIS mRNA was confirmed in injected hearts after animal sacrifice (n=2), by real-time RT-PCR.
Results.
NIS expression in rCDCs did not affect cell viability/proliferation (p=0.718, ctr vs NIS). In vitro
99m
Tc uptake was 6.0±0.9% vs 0.07±0.05, without and with perchlorate (specific NIS blocker), respectively. NIS-transduced rCDCs were easily visualized as spots of
99m
Tc or
124
I uptake within a perfusion deficit in the SPECT and PET images. CR was considerably higher when cells were transduced by the CMV-NIS virus in comparison to the CAG-NIS virus (70±40% vs 28±29%, p=0.085). Ex vivo small animal SPECT imaging confirmed that
in vivo
99m
Tc signals were localized to the injection sites. PCR confirmed the presence of
h
NIS mRNA in injected hearts.
Conclusion.
NIS expression allows non invasive
in vivo
stem cell tracking in the myocardium, using both SPECT and PET. This reporter gene has great potential for translation in future clinical applications.
Cardiosphere-derived cells (CDCs) were grown from rat hearts and percutaneous endomyocardial adult human biopsy specimens. Rat CDCs plated as single cells formed clones with a doubling time of 42.2 ± 0.7 hours (n = 9). Clones from rat CDCs divided steadily for 27 days before proliferation spontaneously slowed and morphological changes occurred in most cells. After 56 days, rat clonal populations contained a small fraction of c-Kit
+
cells as determined by flow cytometry, and large subsets of cells expressing cardiac troponin I, α-smooth muscle actin, and von Willebrand factor as determined by immunofluorescence, indicative of their multipotentiality
in vitro
. To assess therapeutic potential, acute myocardial infarcts (MIs) were created in immunodeficient mice and actively proliferating polyclonal human CDCs were injected into the border zone. Echocardiographic left ventricular function, histological examination, and immunofluorescence served as endpoints. CDC-injected animals showed no significant deterioration in ejection fraction (EF) from 2 days (EF = 45.2 ± 4.8%) to 6 weeks post-MI (EF = 40.2 ± 4.5%, n = 7, p = NS), in contrast to fibroblast-injected control animals (EF = 42.8 ± 4.3% at 2 days vs 27.3 ± 4.0% at 6 weeks, p < 0.01). At the 6 week endpoint, the CDC group had thicker infarct walls as measured histologically compared to the fibroblast group (0.26 ± 0.03mm vs 0.12 ± 0.01mm, n = 5, p < 0.01). CDC engraftment was determined by immunofluorescence using a human-specific antibody. CDCs stably engrafted for up to 6 weeks and could be found distributed primarily throughout the infarct (57 ± 3% of engrafted CDCs, n = 5 animals), as well as the border zone (30 ± 5%) and viable tissue (13 ± 3%). After 6 weeks, CDCs within the infarct had formed small myocytes with little cytoplasmic cardiac troponin I, while CDCs within the viable myocardium had formed large myocytes with well-defined sarcomeric organization. We conclude that CDCs are clonogenic and spontaneously multipotent
in vitro
and capable of preserving heart function in a mouse infarct model. Functional preservation is presumably due in part to maintenance of infarct wall thickness, likely secondary to stable CDC engraftment within the infarct, as well as the formation of morphologically mature myocytes throughout the non-infarcted tissue.
Allogeneic cardiosphere-derived cells (CDCs) have proven safe and effective in a small animal model of myocardial infarction (MI), and have been shown to act primarily via paracrine mechanisms to stimulate endogenous regeneration. The present translational study tested allogeneic CDCs in a large animal model (mini-pigs).
To establish a robust allogeneic model, all pigs were swine leukocyte antigen (SLA) typed by PCR. A male donor and female recipients with full SLA I, II mismatch were used. Pigs underwent balloon occlusion of the LAD for 2.5 hours, followed by reperfusion. Two weeks later, 12.5 million CDCs (n=8) or vehicle (n=6) were infused. Animals were sacrificed 2 weeks or 2 months post-infusion to assess the cellular (histology using a clinical rejection scale) and humoral (donor-specific antibodies, complement-dependent cytotoxicity) immune responses, as well as cardiac function (left ventriculography, hemodynamics, morphometry). Numerous in-life assessments for safety were performed and CDC engraftment was assessed by FISH for Y chromosome.
All immunological assays indicated an undetectable response to CDCs. Cardiac enzymes and systemic inflammation showed no differences between groups. There were no systemic histological findings related to CDCs. CDCs did not permanently engraft, with <0.1% persisting 2 weeks post-infusion and none evident 2 months post. Despite evanescent engraftment, functional benefits were seen following infusion of CDCs. Ejection fraction (EF) and infarct size (IS) were significantly improved in CDC-treated animals compared to vehicle-treated 2 weeks post-infusion (EF: 48.6±2.1% vs 38.2±2.6%, p<0.05; IS: 9±2 vs 14±2%, p<0.05). At 2 months, EF still trended higher in CDC-treated animals (42±5 vs 32±10%, p=0.09), and IS still trended lower (14±4 vs 16±7%, p=0.60). Pressure-volume relationships revealed a trend for enhanced contractility in CDC-treated animals (Emax: 3.2±2.2 vs 1.0±1.1 mmHg/mL, p=0.26). The magnitude of the functional benefits was similar to that seen in a prior pig study using autologous CDCs.
Overall, results of the present study demonstrate that allogeneic CDCs are largely equivalent to autologous in terms of efficacy, and elicit no detectable immunological response or safety concern.
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