stem cell biologist, tissue engineerEmbryonic stem (ES) cells have the unique potential of developing into all the different cell types in our body and offer exciting new potential in the field of regenerative medicine. Non-invasive cell tracking of ES cells can enable us to determine if they survive in vivo, migrate to the desired location, and engraft properly. Conventional cell tracking on MRI usually employs iron oxides (SPIO) that generate negative contrast, an approach with limited specificity, as blood clots and vessels also appear dark, as do macrophages that will ingest the SPIO should the labelled cells die. In this study, we investigate the use of a highly efficient, cell permeable, and cell retentive T1 contrast agent, MnAMP ¹, for labelling mouse ES cells. R1/E mouse ES cells produced from the Nagy lab (Nagy, Rossant and Abramow-Newerly) were cultured on a layer of mouse embryonic fibroblasts until they reached 70% confluency. The stem cells were cultured on the fibroblasts for the first two passages and then transferred to a feeder-free flask with a layer of 0.1% gelatin. Cells were labelled with 0.1 mM of MnAMP for various labelling intervals and kept in contrast-free medium for different retention periods. Cell pellets were prepared in glass vials and imaged on a 3.0T scanner (Achieva 3.0 T TX, Philips Medical Systems) using a 32-channel head coil. T1 mapping was performed using inversion recovery turbo spin echo: TR = 3000 ms, TE = 18.5 ms, 5 cm field-of-view, 3 mm slices, 0.5 x 0.5 mm in-plane resolution, and TI = [50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 and 2500] ms. Atomic absorption spectroscopy was used to assess intracellular manganese content. Viability and proliferation of labelled samples was assessed. The ability of ES cells to differentiate into cardiomyocytes was determined using the hanging drop method ².Viability and proliferation assays confirmed no adverse effect from cell labelling with MnAMP at 0.1 mM. Viability was 84.1% for 24-hour labelled ES cells compared to 86.7% for control; proliferation rates remained constant. Fig. 1 shows a T1-weighted image of labelled cell pellets prepared with different labelling intervals and retention periods. Fig. 2 compares changes in T1 across different labelling and retention intervals. Fig. 3 demonstrates that labelling did not affect the ES cells’ ability to differentiate into cardiomyocytes; spontaneous contractions were observed in both control cells and labelled cardiomyocytes.MnAMP is a highly efficient T1 contrast agent for labelling mouse ES cells. A significant reduction in T1 was achieved from a 2-hour incubation interval, and the significant change in contrast was maintained 24 hours post-labelling. Viability and proliferation were unaffected. Most importantly, the capacity of the ES cells to differentiate into beating cardiomyocytes was preserved.We have presented a promising new method for sensitive MRI tracking of embryonic stem cells to study cellular therapies and tissue engineering and regeneration strategies.