J Korean Med Sci.  2010 Feb;25(2):211-219. 10.3346/jkms.2010.25.2.211.

In vivo Tracking of Mesenchymal Stem Cells Labeled with a Novel Chitosan-coated Superparamagnetic Iron Oxide Nanoparticles using 3.0T MRI

• Reddy AM
• Kwak BK
• Shim HJ
• Ahn C
• Lee HS
• Suh YJ
• Park ES

• KMID: 1779249
• DOI: http://doi.org/10.3346/jkms.2010.25.2.211

Abstract

This study aimed to characterize and MRI track the mesenchymal stem cells labeled with chitosan-coated superparamagnetic iron oxide (Chitosan-SPIO). Chitosan-SPIO was synthesized from a mixture of FeCl2 and FeCl3. The human bone marrow derived mesenchymal stem cells (hBM-MSC) were labeled with 50 microg Fe/mL chitosan-SPIO and Resovist. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity, phenotype and differentiation potential. Electron microscopic observations and Prussian blue staining revealed 100% of cells were labeled with iron particles. MR imaging was able to detect the labeled MSC successfully. Chitosan-SPIO did not show any cytotoxicity up to 200 microgram Fe/mL concentration. The labeled stem cells did not exhibit any significant alterations in the surface markers expression or adipo/osteo/chondrogenic differentiation potential when compared to unlabeled control cells. After contralateral injection into rabbit ischemic brain, the iron labeled stem cells were tracked by periodical in vivo MR images. The migration of cells was also confirmed by histological studies. The novel chitosan-SPIO enables to label and track MSC for in vivo MRI without cellular alteration.

Keyword

Chitosan; Mesenchymal Stem Cells; Superparamagnetic Iron Oxide; Magnetic Resonance Imaging; Brain Ischemia

MeSH Terms

Animals
Brain Ischemia/chemically induced/pathology/therapy
Cell Differentiation
Chitosan/*chemistry
Coordination Complexes/*chemistry/toxicity
Ferric Compounds/*chemistry
Humans
Magnetic Resonance Imaging
Magnetics
Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells/*chemistry/cytology
Metal Nanoparticles/*chemistry
Phenotype
Rabbits
Coordination Complexes
Ferric Compounds
Chitosan

Figure

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  Fig. 1 Comparative MR phantom study of Resovist and chitosan coated SPIO showing similar relation between signal intensity and iron concentration on T2 weighted image.

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  Fig. 2 In vitro evaluation of iron labeling of mesenchymal stem cells by cellular MRI that show labeled cells indicated by arrows (A), Prussian blue staining of in vitro cultured cells (B) and electron microscopic images that show internalized SPIO nanoparticles (C).

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  Fig. 3 Cytotoxicity assay of SPIO labeled mesenchymal stem cells. (A), A comparative dose response along with control cells (0), in cells treated overnight with PLL only (PL), SPIO only (IO) and 3.1-1,600 µg/mL SPIO (Resovist or chitosan-SPIO) plus PLL. (B), Time course study on day 4, 7, 14, and 21 after labeling MSC with 25 and 100 µg Fe/mL of Resovist (R25, R100) and chitosan-SPIO (C25, C100) including control (C) and, SPIO only (Fe-only) cells.

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  Fig. 4 Phenotypic characterization of SPIO labeled mesenchymal stem cells. Chitosan-SPIO (100 µg Fe/mL) labeled MSC were analyzed for both positive (CD29, CD44, CD73, CD105, CD166) and negative (CD14, CD34, CD45) surface markers of MSC. A little increase in CD34 marker expression is observed (21.3%).

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  Fig. 5 Differentiation potential of Resovist and chitosan-SPIO labeled mesenchymal stem cells. Iron (100 µg Fe/mL) labeled MSC were exposed to adipogenic (A, B), osteogenic (C, D) and chondrogenic differentiation media (E, F) for 3 weeks. Red colored oil granules are detected by Oil red O staining in adipogenic cultures of Resovist (A) and chitosan-SPIO labeled mesenchymal stem cells (B). Calcium mineralization is detected by von Kossa staining in osteogenic cultures of MSC labeled with Resovist (C) or chitosan-SPIO (D). Alcian Blue staining reveals that chitosan-SPIO labeled MSC (F) exhibit chondrogenic differentiation (asterisk, blue color), but not Resovist labeled cells (E).

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  Fig. 6 In vivo MRI of rabbit ischemic brain that received chitosan-SPIO labeled mesenchymal stem cells. Asterisk indicates ischemic area and open arrows indicate chitosan-SPIO-labeled mesenchymal stem cells at the injection site. (A, B) T2WI and diffusion weighted image (DWI) of brain immediately after injection of mesenchymal stem cell at contralateral side of ischemic area (open arrows) on day 4 of ischemia. (C, D) T2WI and susceptibility weighted image (SWI) 16 days after stem cell transplantation. At the ischemic site, dark signal (white arrows) especially on SWI matches mesenchymal stem cells (black arrows) on Prussian staining (E, F). (E, F) Prussian blue staining detected iron labeled stem cells at the liquefied infarct area in a section of 4% paraformaldehyde-fixed brain tissue on day 16.

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