and A.G.E.: conception and design, data analysis and interpretation; M.K.: conception and design, collection and/or assembly of data; D.A.E.: data analysis and interpretation, manuscript writing, final approval of manuscript; R.A.: conception and design, financial support, manuscript writing, provision of study material or patients, data analysis and interpretation, final approval of manuscript. Disclosure of Potential Conflicts of Interest The Ziyuglycoside I authors indicated no potential conflicts of interest.. 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 explained 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 usually a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was utilized for cell Ziyuglycoside I 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 power in future cardiac stem cell therapy trials. = 3, imply SEM). Views of unlabeled Ziyuglycoside I control (green) and positive control (yellow) representing 100 g/ml real ferumoxytol suspended in 50-l agarose plugs are shown. Mass spectrometry data (in atom counts) comparing iron retention between Ziyuglycoside I cells treated with different iron concentrations (50, 100, 200, and 300 g/ml) (D) and at different days of differentiation (day ?1, day 0, and day 3) (E). (F): Circulation cytometry analysis showing PDGFR, CD56, and CD13 expression in corresponding ferumoxytol-labeling conditions (= 3, mean SEM). (G): Circulation cytometry analysis showing PI and Annexin V expression in corresponding ferumoxytol-labeling conditions (= 3, mean SEM). Percentage of viable cells depicted graphically. (H): Field-of-view images showing NKX2-5 (green) expression in cells labeled at day 0 with 100 g/ml, 200 g/ml, and 300 g/ml ferumoxytol. Level bars = 100 m. Abbreviations: CHIR, CHIR99021; d, day; hESC, human embryonic stem cell; PI, propidium iodide; Th, Thurston measurement. In Vitro MRI Cell Preparation To determine the imaging potential Ziyuglycoside I and transmission attenuation of ferumoxytol-labeled hESC-CPCs, the cells were harvested at days 4 and 10 of differentiation and resuspended in 50-l agarose gel plugs for in vitro MRI. Post-Sort Culture Freshly sorted day 3 CD13+/ROR2+ cells were recultured on Matrigel-coated plates in Roswell Park Memorial Institute plus B27 for any recovery period of 24 hours before injection into the healthy pig heart (supplemental online Fig. 1). Cell Injection and Animal Maintenance Animal housing, maintenance, and experimentation were approved by, and performed in accordance with the guidelines set by, the Institutional Animal Care and Use Committee of the University or college of California and the National Institutes of Health Guideline for the Care and Use of Laboratory Animals. A total of 3 Yorkshire pigs weighing approximately 40 kg underwent thoracotomy and transplantation of ferumoxytol-labeled hESC-CPCs under direct visualization. Two injection sites were selected around the left ventricular free wall and marked with suture. Site 1 was injected with ferumoxytol-labeled CPCs. Site 2 was injected with unlabeled CPCs. A suspension of 4 107 cells (determined by hemocytometer) in approximately 300 l Rabbit polyclonal to KBTBD7 of conditioned media was injected in each site using a 27-gauge needle. The pigs were imaged using T2-based MRI on the day of transplantation and again 40 days later. The pigs were immunosuppressed with cyclosporine (serum level of 100C120 ng/ml) and treated with ketoconazole (20 mg/kg) and trimethoprim sulfa (40 mg/kg) daily, which began 3 days before cell transplantation and was continued until euthanasia. After 40 days, the pigs were euthanized, and the hearts were harvested and sectioned for histological analysis. Detailed protocols are given in the supplemental online data and used published procedures. Results Variation in Transmission Intensity Is Dependent on Ferumoxytol Exposure Day The differentiation protocol efficiently generated precardiac mesoderm as shown by quantitative polymerase chain reaction and circulation cytometry (supplemental online Fig. 2AC2C). Furthermore, under these conditions, differentiating cells gave rise to cardiomyocytes, easy muscle mass cells, and endothelial cells in vitro (supplemental online.