In vivo imaging of cell fate decisions in cardiac cell therapy using cardioCEST MRI.
Ashley Pumphrey1, Zhengshi Yang2, Shaojing Ye2, Ahmed Abdel-Latif2, and Moriel Vandsburger3

1CVRC, University of Kentucky, Lexington, KY, United States, 2University of Kentucky, Lexington, KY, United States, 3Physiology, University of Kentucky, Lexington, KY, United States


We developed a cardiac specific chemical exchange saturation transfer pulse sequence and applied it to the tracking of cell survival/proliferation or rejection in murine models of cardiac cell therapy.


Research into cell therapies for cardiac regeneration is stymied by an inability to track in vivo cell survival, proliferation, and differentiation. MRI tracking of iron oxide nanoparticle labeled cells following cardiac implantation has been explored, however concerns of tissue retention of particles after cell death and difficulty differentiating between labeled cells and tissue necrosis remain major limitations. Chemical exchange saturation transfer (CEST) - MRI has been used to image cells labeled with paraCEST contrast agents1 or cells expressing CEST active reporter genes2 in stationary organs and tumors but not the heart. We developed a cardiorespiratory gated CEST-encoded steady state cine pulse sequence (cardioCEST), and demonstrated the ability to selectively visualize CEST contrast from Eu-HPDO3A labeled cells following intra-myocardial implantation3. Here we examined whether cardioCEST imaging could differentiate cell survival/proliferation from rejection in a longitudinal mouse model of cardiac cell therapy.


Pulse Sequence Design: CEST encoding used a 2s train of Gaussian saturation pulses (720°, B1average = 14μT, bandwidth = 200Hz, duration = 8.8ms, number of pulses = 196, offsets = ±15ppm) prior to a constant repetition time cine gradient echo readout that was cardiorespiratory gated (TR/TE= 7.1/3.1ms, flip angle = 10°). After each saturation 4 averages of one phase-encode step were acquired for each cardiac phase, and dummy pulses maintained steady state magnetization during respiratory motion. Additional parameters were FOV=2.56x2.56cm, Matrix = 256x128, and slice thickness = 1mm. All imaging was performed on a 7T Bruker Clinscan (Bruker, Germany) scanner using a cylindrical volume coil for excitation and a 4-channel phased-array surface coil for reception. Cell and Animal Experiments: C2C12 cells derived from C3H mice were labeled with Eu-HPDO3A via hypotonic swelling4 that yielded 22,355.67 ng(Eu)/mg cell mass as determined by ICPMS. Implantation of ~106 cells into the left ventricular wall was performed using a surgical pop-out technique5. In order to model the survival/proliferation vs. the rejection of implanted cells, labeled C2C12 cells were implanted into syngeneic C3H mice (survival, n=6) or C57B6 mice (rejection, n=7). Separately, saline-labeled C2C12 cells implanted into different C3H (n = 6) or C57B6 (n = 9) mice served as controls. Pairs of cardioCEST images were acquired in 2-3 mid-ventricular slices per mouse at 1, 10 and 20 days following cell implantation. Afterwards hearts were excised and stained using hematoxylin and eosin staining. Registration of cardioCEST image pairs was performed to account for slight movement between acquisitions. Maps of CEST contrast were generated by matrix calculation of MTRasym = (S-15ppm – S15ppm)/S-15ppm*100. Regions of interest in mice receiving Eu-HPDO3A labeled cells were defined in the area of cell implantation (cell graft), and a control area in the septum. In mice receiving saline labeled cells, one region of interest was defined in the area of cell injection (saline graft).


MTRasym values were similar at the sites of implanted Eu-HPDO3A labeled cells in C3H and C57B6 mice (Figures 1, 2) at 1 day after injection. In C3H mice (proliferation model, Figure 1), MTRasym remained higher for 20 days in areas of implanted labeled cells compared to septal regions and saline labeled controls (Figure 2), but decreased compared to day 1. Elevated MTRasym in C3H mice at day 20 was localized to areas of C2C12 cell proliferation as identified by histology (Figure 1). In C57B6 mice (rejection model), MTRasym in the area of implanted cells decreased significantly within 10 days of injection. By 20 days post injection, MTRasym values were similar in areas of implanted cells, control regions in the septum, and in control mice receiving saline labeled cells (Figures 1, 2). Also, MTRasym was significantly (p < .01) lower in the cell graft in C57B6 mice compared to C3H mice at 10 and 20 days after injection. Finally, graft size in C3H mice was similar when calculated from cardioCEST images as compared to histology (Figure 3).


Longitudinal cardioCEST imaging enabled in vivo monitoring of cell survival/proliferation or rejection in a mouse model of cardiac cell therapy. Following rejection of labeled cells, Eu-HPDO3A is likely cleared from the myocardium similar to other lanthanide-HPDO3A complexes, leading to elimination of heightened MTRasym values by 20 days post implantation. In proliferating cells the dilution of label with cell division led to a reduction in MTRasym values by 20 days post implantation that remained elevated compared to non-labeled and rejected cells. Further investigation is needed to understand the association between MTRasym values and cell density.


Imaging of cell fate decisions using cardioCEST MRI could support the design of novel methods to improve cell survival and differentiation in cardioregenerative therapies.


No acknowledgement found.


(1) Ferrauto et al. MRM. 2013; 69(6): 1703-11. (2) Gilad et al. Nat. Biotech. 2007; 25: 217-19. (3) Pumphrey et al. NMR in Biomedicine. In Press. 2015. (4) Di Gregorio et al. CMMI. 2013; 8(6): 475-86. (5) Gao et al. Circ. Res. 2010; 107: 1445-53.


Figure 1. MTRasym maps acquired in C3H mice (proliferation) and C57B6 mice (rejection), reveal elevated MTRasym in areas of Eu-HPDO3A labeled cells at 1 day post-implantation. CEST contrast is preserved in proliferating cells but eliminated following cell rejection as confirmed by H&E staining.

Figure 2. MTRasym­ in proliferating Eu-HPDO3A labeled cells remains higher than septal tissue and proliferating saline labeled grafts for 20 days. MTRasym in Eu-HPDO3A labeled cells undergoing rejection decreases rapidly to levels of control tissue and saline labeled grafts. (Bars = P<0.05, *P<0.05 vs. Day 1).

Figure 3. (A) Graft size assessed as percentage of LV mass in the imaged slice was similar when calculated from histological data and from cardioCEST maps using an MTRasym threshold of 15%. (B) Bland-Altman analysis confirms similar assessment of graft size by both methods.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)