Labeling of mesenchymal stem cells for MRI with single-cell sensitivity

Abstract

Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist^® regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist^® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist^® for improved MRI of MSC with single-cell sensitivity.

Keywords:

• magnetic field microdistortions
• single-cell imaging
• mesenchymal stem cells
• VSOP
• MCP
• Resovist^®

Supplementary materials

Figure S1 Mesenchymal stem cell surface marker expression.

Notes: MSC surface marker expression for cells without nanoparticles (empty cells: dotted gray line histograms) is similar to that of VSOP-labeled cells (continuous black line). In addition, histograms for VSOP-labeled cells without staining for MSC surface markers (broken gray line) show low fluorescent background.

Abbreviations: MSC, mesenchymal stem cells; VSOP, very small iron oxide nanoparticle; APC, Allophycocyanin; FITC, Fluorescein Isothiocyanate; PE, Phycoerythrin.

Figure S2 Agarose phantom of labeled MSC imaged on 7 T Biospec vs 7 T Pharmascan MRI (Bruker).

Notes: MRI of agarose phantoms using a Bruker BioSpec MRI scanner with CryoProbe (A1–D1) has higher resolution and improves the signal-to-noise ratio (SNR: 86) in comparison to 7 T Pharmascan MRI (SNR: 60) (A2–D2). Hence, the detection of magnetic field microdistortions caused by NP-labeled MSC in postprocessed phase images was improved, and the percentage of quantified single cells was two- to three-fold higher for the 7 T BioSpec. The Biospec MRI scanner has recently become available to us, and these results promise improvements for MRI with single-cell sensitivity in future in vivo experiments.

Abbreviations: 7 T, 7 Testla; MCP, multicore carboxy-methyl-dextran-coated iron oxide nanoparticle; MRI, magnetic resonance imaging; MSC, mesenchymal stem cell; NP, nanoparticle; VSOP, very small iron oxide nanoparticle.

Figure S3 MRI detection at 7 T of MSC in mouse brain after carotid injection of MCP-labeled cells.

Notes: Magnitude (A1–C1) and phase images (A2–C2) of mouse brain before injection (A), after injection of nonlabeled cells (B), and after injection of 1,000 MCP-labeled MSC (C). MSC trapped in blood vessels of the left hemisphere are visible as signal reductions in the magnitude image (C) and as dipole figures in the phase image (MFMD) (C2). (FLASH gradient-echo sequence, 80 µm in-plane resolution, slice thickness 300 µm, TE 5.4 milliseconds, TR 400 milliseconds).

Abbreviations: MRI, magnetic resonance imaging; MCP, multicore superparamagnetic nanoparticles; MSC, mesenchymal stem cells; MFMD, magnetic field microdistortion; TE, echo time; TR, repetition time.

Figure S4 Effect of cell density, nanoparticle concentration, and ratio of NP to protamine sulfate on NP uptake by MSC.

Notes: MSC at variable cell densities were labeled with MCP in combination with protamine sulfate (PS) at different ratios and stained for iron using the Prussian Blue stain protocol. Cell incubation with increasing NP concentration with and without PS (25:0) increased cellular NP uptake for MSC at 15,000 cells/cm². Increasing cell density caused high extracellular matrix and high NP aggregation when incubated with MCP-PS complexes as observed for 40,000 cells/cm² with MCP-PS at ratios of 10:12 and 25:30. However, cell recovery was decreased after MSC incubation with high PS concentration (25:30). Overall, more efficient cellular recovery and cellular NP uptake were achieved when MSC were cultured at 15,000 cells/cm² and incubated with MCP-PS complexes at 10:12 ratio or with high NP concentration but without PS (25:0).

Abbreviations: MSC, mesenchymal stem cells; MCP, multicore superparamagnetic nanoparticles; PS, protamine sulfate; NP, nanoparticle.

Figure S5 Example to illustrate the intracellular uptake of VSOP and MCP by MSC by TEM.

Notes: MSC were incubated with nanoparticles (2 mM) for 24 hours, followed by washing steps and extracellular matrix removal (24 hours + ECM) as described in “Methods” section. NP clustering engulfed by phagolysosomes proves intracellular uptake for VSOP and MCP. All scale bars correspond to 100 nm.

Abbreviations: ECM, extracellular matrix; MCP, multicore superparamagnetic nanoparticles; MSC, mesenchymal stem cells; NP, nanoparticle; TEM, transmission electron microscopy; VSOP, very small iron oxide nanoparticles.

Figure S6 Nanoparticle TEM study.

Notes: Nanoparticle size and morphology were analyzed by HRTEM using a TECNAI G2 20 S-Twin (FEI-Company, Hillsboro, OR, USA). TEM samples were prepared by coating copper grids with diluted nanoparticle solutions.

Abbreviations: TEM, transmission electron microscopy; HRTEM, high-resolution transmission electron microscopy.

Acknowledgments

This work was supported by the Clinical Research Unit (KFO 213, TA 166/3-1, TA 166/3-2, TA 166/7-2, and WA 3105/1-2). We thank Bettina Herwig for language editing; Susanne Mueller from the Center for Stroke Research, Charité, Berlin, for assistance with MRI; Petra Schrade from the Core Facility for Electron Microscopy, Charité, Berlin, for cell TEM imaging; Sören Selve from the ZELMI, Technische Universität Berlin, for nanoparticle TEM imaging; Monika Ebert from the Experimental Radiology Division, Charité, Berlin, for assistance with nanoparticle analysis; and Frank Wiekhorst, Physikalisch-Technische Bundesanstalt (PTB), Berlin, for collaboration with magnetic particle spectroscopy.

Disclosure

The authors report no conflicts of interest in this work.