Target Audience

Stem cell biologist, tissue engineer

Introduction

Human embryonic stem cells (hESCs) have unlimited proliferation potential and can be differentiated into all different human cell types, making them ideal for cell therapy applications in regenerative medicine. Cell tracking is vital for optimizing cellular therapies, from ensuring accurate cell delivery to monitoring engraftment and evolving tissue function. MRI-based cell tracking with SPIOs has been widely used but has limited specificity, as magnetic susceptibility artefacts also lead to a loss of signal. Herein, we investigate the use of a highly efficient, cell-permeable and biocompatible T1 contrast agent for tracking hESCs.

Methods

Human ESCs from line ESI-017 (ESI Bio, SKU: ES-700) were cultured on T-75 tissue culture flasks coated with Corning™ Matrigel™ Membrane Matrix (Fisher Scientific Cat. No. 08-774-552). Cells were maintained in mTeSR™1 (STEMCELL Technologies Cat. No. 85850) and microscopy was done daily to assess cell morphology. Cells were grown to 70% confluency and were labeled with varying concentrations of MnEtP for either 30 min or 24 hours. The colonies were detached by use of a gentle cell dissociation reagent followed by cell scraping and collection. Cells were centrifuged at 300g for 5 minutes and then were resuspended with 1 mL D-PBS; 0.9 ml of the cell suspension was transferred to 115 x 5 mm Wintrobe sedimentation tubes (Kimble Chase, Vinelad, NJ) and pelleted at 300 g for 10 min. MRI was done on a 3T scanner (Achieva 3.0 T TX, Philips Medical Systems) using a 8-channel wrist coil. The subcellular agent distribution was determined by cell fractionation and quantification by high performance liquid chromatography and atomic emission spectroscopy. The total intracellular Mn content was analyzed by atomic emission spectroscopy. Viability and proliferation of labeled cells were assessed. The differentiation potential of labeled cells was confirmed by formation of embryoid bodies. An in-vivo study was done on a female adult Sprague-Dawley rat with approximately 20 million labeled cells, in 200 µL mTeSR™1, injected subcutaneously on the dorsal side below the shoulder blades. A 3D T1-weighted turbo field echo (TFE) sequence was acquired to detect the labeled cells: TR = 6.2 ms, TE = 3.3 ms, FA=20°, NSA = 16, FOV = 100 mm, and 0.6 x 0.6 mm in-plane resolution.

Results

The labeling conditions were optimized. Figure 1 shows the T1 relaxation times of labeled cells demonstrating efficient T1 reduction after labeling. Viability and proliferation assays confirmed no detrimental effects from the optimized labeling conditions of 10 µM MnEtP for 24 hours and 40 µM MnEtP for 30 min. Differentiation potential was confirmed by formation of embryoid bodies post-labeling. Detection sensitivity of labeled cells was demonstrated by MRI with an in-vivo rat study (Fig 2).

Discussion

MnEtP is a very efficient T1 contrast agent for labeling hESCs. Even at low labeling concentrations of 10 µM for 24 hours, there was a five-fold reduction in T1. Viability, proliferation, and differentiation capacity of labeled cells remained unaffected. An in-vivo study clearly demonstrated enhancement where the labeled cells were injected compared to unlabeled cells that were isointense against native tissue. MnEtP is biocompatible, non-toxic and provides sensitive detection of labeled cells in vivo.

Conclusion

We have developed a new contrast agent for sensitive in-vivo tracking of hESCs useful for optimizing cell therapy protocols for applications in regenerative medicine.

Acknowledgements

No acknowledgement found.

References

No reference found.