Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

Summary

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Abstract

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

Protocol

Labeling of hMSC with Ferucarbotran

This is the technique for labeling human mesenchymal stem cells with Ferucarbotran, an iron oxide based contrast agent for MR imaging:

1. To begin, plate the cells 18-24 hours before the labeling procedure in T75 flasks at a confluence of 80%. If you use a different culture dish, this is equal to about 10000 cells per cm².
2. The next day, start with preparing the labeling media by adding 30 µl of Resovist to 8 ml of serum free media. This will label one T75 flask at 80% confluency, corresponding to a concentration of 100 µg Fe/ml media.
3. Ta....

Discussion

A non-invasive depiction of stem cells is crucial for monitoring virtually any stem cell based therapy. A better understanding of the in vivo kinetics of labeled stem cells, as visualized on MR images, could lead the way to a rational and a more effective use of stem cell-based therapies. Since we use clinically applicable contrast agents and imaging techniques, our presented protocols should be readily translatable to applications in patients. Potential clinical applications of our methods include comparative investigat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
D-MEM High Glucose	Reagent	Sigma-Aldrich	D5648
Gelatin	Reagent	Sigma-Aldrich	G1890	dilute Gelatin in PBS to final concentration of 0.1 %
PBS (Ca++ Mg++ freed)	Reagent	GIBCO, by Life Technologies	14190-144
Trypsin-EDTA 0.05%	Reagent	Invitrogen	25300-120
Trypsin-EDTA 0.25%	Reagent	Invitrogen	25200-114
FBS characterized	Reagent	Hyclone	SH30071.03
Cell Strainer (40 μm Nylon)	Tool	BD Biosciences	352340
Knockout DMEM	Reagent	GIBCO, by Life Technologies	10829018
Knockout Serum Replacer	Reagent	GIBCO, by Life Technologies	10828028
b-Mercapt–thanol	Reagent	Sigma-Aldrich	7522
non-essential amino acids	Reagent	GIBCO, by Life Technologies	11140050
FGF-2	Reagent	R&D Systems	233-FB-025
Glutamine	Reagent	GIBCO, by Life Technologies	25030081
Penicillin/Streptomycin	Reagent	GIBCO, by Life Technologies	15140-122
Ferumoxides (Endorem)	Reagent	Guerbet
Ferucarbotran (Resovist®)	Reagent	Bayer AG