Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors.

UNLABELLED: Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 µg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. SIGNIFICANCE: The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its utility in future cardiac stem cell therapy trials.

In-vivo comparison of the acute retention of stem cell derivatives and fibroblasts after intramyocardial transplantation in the mouse model.

PURPOSE: Various strategies have been applied to increase the engraftment of an intramyocardial cell transplant (Tx) to treat ischemic myocardium. Thereby, co-transplanted fibroblasts (FB) improve the long-term survival of stem cell derivatives (SCD) in a murine model of myocardial infarction. For therapeutic use, the time frame in which FB exert putative supportive effects needs to be identified. Therefore, we tracked the biodistribution and retention of SCD and FB in vivo using highly sensitive positron emission tomography (PET) imaging. METHODS: Murine [(18)F]-fluorodeoxyglucose (FDG) labeled SCD and FB were transplanted after left anterior descending artery (LAD) ligation into the border zone of the ischemic area in female C57BL/6 mice. Cardiac retention and biodistribution during the initial 2 h after injection were measured via PET imaging. RESULTS: Massive initial cell loss occurred independently of the cell type. Thereby, FB were retained slightly, yet significantly better than SCD until 60 min post-injection (7.5 ± 1.7 vs. 5.2 ± 0.7% ID at 25 min and 7.0 ± 1.5 vs. 4.8 ± 0.8% ID at 60 min). Thereafter, a fraction of ∼ 5% that withstood the massive initial washout remained at the site of injection independently of the applied cell type (120 min, SCD vs. FB P = 0.64). Most of the lost cells were detected in the lungs (∼ 30 % ID). CONCLUSIONS: We were able to quantitatively define the retention and biodistribution of different cell types via PET imaging in a mouse model after intramyocardial Tx. The utmost accuracy was achieved through this cell- and organ-specific approach by correcting PET data for cellular FDG efflux. Thereby, we observed a massive initial cell loss of ∼ 95%, causing low rates of long-term engraftment for both SCD and FB. We conclude that FB are not privileged compared to SCD regarding their acute retention kinetics, and therefore exert their beneficial effects at a later time point.

Positron emission tomography based in-vivo imaging of early phase stem cell retention after intramyocardial delivery in the mouse model.

PURPOSE: To establish PET as a tool for in-vivo quantification and monitoring of intramyocardially transplanted stem cells after labelling with FDG in mice with induced myocardial infarction. METHODS: After inducing myocardial infarction in C57BL/6 mice, murine embryonic stem cells were labelled with FDG and transplanted into the border zone of the infarction. Dynamic PET scans were acquired from 25 to 120 min after transplantation, followed by a scan with 20 MBq FDG administered intravenously for anatomical landmarking. All images were reconstructed using the OSEM 3D and MAP reconstruction algorithms. FDG data were corrected for cellular tracer efflux and used as marker for cellular retention. FACS analysis of transplanted cells expressing enhanced green fluorescent protein was performed to validate the PET data. RESULTS: We observed a rapid loss of cells from the site of transplantation, followed by stable retention over 120 min. Amounts of retention were 5.3 ± 1.1 % at 25 min, 5.0 ± 0.9 % at 60 min and 5.7 ± 1.2 % at 120 min. FACS analysis showed a high correlation without significant differences between the groups (P > 0.05). FDG labelling did not have any adverse effects on cell proliferation or differentiation. CONCLUSION: Up-to-date imaging is a powerful method for tracking and quantifying intramyocardially transplanted stem cells in vivo in the mouse model. This revealed a massive cell loss within minutes, and thereafter a relatively stable amount of about 5 % remaining cells was observed. Our method may become crucial for further optimization of cardiac cell therapy in the widely used mouse model of infarction.

In vivo imaging of embryonic stem cell therapy.

Embryonic stem cells (ESCs) have the most pluripotent potential of any stem cell. These cells, isolated from the inner cell mass of the blastocyst, are "pluripotent," meaning that they can give rise to all cell types within the developing embryo. As a result, ESCs have been regarded as a leading candidate source for novel regenerative medicine therapies and have been used to derive diverse cell populations, including myocardial and endothelial cells. However, before they can be safely applied clinically, it is important to understand the in vivo behavior of ESCs and their derivatives. In vivo analysis of ESC-derived cells remains critically important to define how these cells may function in novel regenerative medicine therapies. In this review, we describe several available imaging modalities for assessing cell engraftment and discuss their strengths and limitations. We also analyze the applications of these modalities in assessing the utility of ESCs in regenerative medicine therapies.

Non-invasive imaging of human embryonic stem cells.

Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography (PET), and multimodality approaches. Although the focus of this review article is primarily on hESCs, the labeling/tracking strategies described here can be readily applied to other (stem) cell types as well. Non-invasive imaging can provide convenient means to monitor hESC survival, proliferation, function, as well as overgrowth (such as teratoma formation), which could not be readily investigated previously. The requirement for hESC tracking techniques depends on the clinical scenario and each imaging technique will have its own niche in preclinical/clinical research. Continued evolvement of non-invasive imaging techniques will undoubtedly contribute to significant advances in understanding stem cell biology and mechanisms of action.

Death and proliferation time course of stem cells transplanted in the myocardium.

PURPOSE: Noninvasive positron emission tomography (PET) imaging of reporter gene is combined with quantitative real-time polymerase reverse transcription (RT-PCR) method to study the time course of death and proliferation of stem cells transplanted in the myocardium. METHODS: Male murine embryonic stem cells (ESCs) were stably transfected with a mutant version of herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter gene; 5 x 10(6) such cells were injected into the myocardium of female athymic rats. While the transplanted cells was monitored by in vivo 9-(4-[F-18]fluoro-3-hydroxymethylbutyl)guanine ([F-18]FHBG) PET imaging of the heart, their absolute number was estimated by RT-PCR from hearts harvested at 3-5 h, 24 h, days 4, 7, and 14 after transplantation. RESULTS: (1) Forty percent of injected cells were retained in the heart while majority of injected cells were lost within a few hours after injection. Cell death was peaked at 24 h when 18% of donor cells retained in the heart were dead. (2) The substantial cell loss was reversed by significant proliferation of ESCs. This led to the recovery of cell number to 3.4 million (70% of injected dose) at day 4 and first visual observation of in vivo [F-18] signal in the heart. (3) A robust correlation (R (2) = 0.9) between percent of injected dose per gram of tissue derived from in vivo PET signal and the number of donor cells estimated by RT-PCR was revealed. CONCLUSIONS: The time course of transplanted stem cells surviving in the heart reveals a process of substantial cell loss within 24 h of injection and subsequent recovery of cell number through proliferation. Such proliferation can be noninvasively monitored by reporter gene imaging.

Noninvasive de novo imaging of human embryonic stem cell-derived teratoma formation.

Teratoma formation can be a serious drawback after the therapeutic transplantation of human embryonic stem (hES) cells. Therefore, noninvasive imaging of teratomas could be a valuable tool for monitoring patients undergoing hES cell treatment. Here, we investigated the angiogenic process within teratomas derived from hES cells and now report the first example of using (64)Cu-labeled RGD tetramer ((64)Cu-DOTA-RGD4) for positron emission tomography imaging of teratoma formation by targeting alpha(v)beta(3) integrin. H9 hES cells (2 x 10(6)), stably expressing firefly luciferase, and enhanced green fluorescence protein (Fluc-eGFP) were injected into adult nude mice (n=12) s.c. Eight weeks after transplantation, these hES cell grafts evolved into teratomas as confirmed by longitudinal bioluminescence imaging. Under micropositron emission tomography imaging, 2-deoxy-2-[(18)F]fluoro-D-glucose and 3'-deoxy-3'-[(18)F]-fluorothymidine both failed to detect hES cell-derived teratomas (0.8+/-0.5 versus 1.1+/-0.4 %ID/g, respectively; P=not significant versus background signals). By contrast, (64)Cu-DOTA-RGD4 revealed specific and prominent uptake in vascularized teratoma and significantly lower uptake in control tumors (human ovarian carcinoma 2008 cell line), which had low integrin expression (10.1+/-3.4 versus 1.4+/-1.2 %ID/g; P<0.01). Immunofluorescence staining of CD31 and beta(3) integrin also supported our in vivo imaging results (P<0.05). Moreover, we found that the cells dissociated from teratomas showed higher alpha(v)beta(3) integrin expression than the 2008 cells. In conclusion, by targeting alpha(v)beta(3) integrin, we successfully showed the ability of (64)Cu-DOTA-RGD4 to noninvasively visualize teratoma formation in vivo for the first time.

Embryonic stem cell grafting in normal and infarcted myocardium: serial assessment with MR imaging and PET dual detection.

PURPOSE: To use magnetic resonance (MR) imaging and positron emission tomography (PET) dual detection of cardiac-grafted embryonic stem cells (ESCs) to examine (a) survival and proliferation of ESCs in normal and infarcted myocardium, (b) host macrophage versus grafted ESC contribution to serial MR imaging signal over time, and (c) cardiac function associated with the formation of grafts and whether improvement in cardiac function is related to cardiac differentiation of ESCs. MATERIALS AND METHODS: All animal procedures were approved by the institutional animal care and use committee. Murine ESCs were stably transfected with a mutant version of herpes simplex virus type 1 thymidine kinase, HSV1-sr39tk, and also were labeled with superparamagnetic iron oxide (SPIO) particles. Cells were injected directly in the border zone of the infarcted heart or in corresponding regions of normal hearts in athymic rats. PET and MR imaging were performed longitudinally for 4 weeks in the same animals. RESULTS: ESCs survived and underwent proliferation in the infarcted and normal hearts, as demonstrated by serial increases in 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine PET signals. In parallel, the hypointense areas on MR images at the injection sites decreased over time. Double staining for host macrophages and SPIO particles revealed that the majority of SPIO-containing cells were macrophages at week 4 after injection. Left ventricular ejection fraction increased in the ESC-treated rats but decreased in culture media-treated rats, and border-zone function was preserved in ESC-treated animals; however, cardiac differentiation of ESCs was less than 0.5%. CONCLUSION: Dual-modality imaging permits complementary information in regard to cell survival and proliferation, graft formation, and effects on cardiac function. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/250/3/821/DC1.