Korean J Radiol.  2015 Jun;16(3):575-585. 10.3348/kjr.2015.16.3.575.

Evaluation of Engraftment of Superparamagnetic Iron Oxide-Labeled Mesenchymal Stem Cells Using Three-Dimensional Reconstruction of Magnetic Resonance Imaging in Photothrombotic Cerebral Infarction Models of Rats

• Shim J
• Kwak BK
• Jung J
• Park S

• KMID: 2155527
• DOI: http://doi.org/10.3348/kjr.2015.16.3.575

Abstract

OBJECTIVE
To evaluate engraftment by visualizing the location of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) three-dimensionally in photothrombotic cerebral infarction (PTCI) models of rats.
MATERIALS AND METHODS
Magnetic resonance imaging (MRI) of an agarose block containing superparamagnetic iron oxide (SPIO)-labeled hBM-MSCs was performed using a 3.0-T MRI, T2-(T2WI), T2*-(T2*WI), and susceptibility-weighted images (SWI). PTCI was induced in 6 rats, and 2.5 x 10(5) SPIO-labeled hBM-MSCs were infused through the ipsilateral internal carotid artery (ICA group) or tail vein (IV group). MRI was performed on days 1, 3, 7, and 14 after stem cell injection. Dark signal regions were confirmed using histology. Three-dimensional MRI reconstruction was performed using the clinical workflow solution to evaluate the engraftment of hBM-MSCs. Volumetric analysis of the engraftment was also performed.
RESULTS
The volumes of SPIO-labeled hBM-MSCs in the phantom MRI were 129.3, 68.4, and 25.9 microL using SWI, T2*WI, and T2WI, respectively. SPIO-labeled hBM-MSCs appeared on day 1 after injection, encircling the cerebral infarction from the ventral side. Dark signal regions matched iron positive cells and human origin (positive) cells. The volume of the engraftment was larger in the ICA group on days 1, 3, and 7, after stem cell injection (p < 0.05 on SWI). SWI was the most sensitive MRI pulse sequence (p < 0.05). The volume of infarction decreased until day 14.
CONCLUSION
The engraftment of SPIO-labeled hBM-MSCs can be visualized and evaluated three-dimensionally in PTCI models of rats. The engraftment volume was larger in the ICA group than IV group on early stage within one week.

Keyword

MRI; Mesenchymal stem cell; Superparamagnetic iron oxide; Photothrombotic cerebral infarction; 3D reconstruction

MeSH Terms

Animals
Cerebral Infarction/pathology/*radiography
Contrast Media
Dextrans
Humans
Imaging, Three-Dimensional/methods
Magnetic Resonance Imaging/*methods
Magnetite Nanoparticles
Male
*Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells/radiography
Nanoparticles
Neuroimaging/*methods
Random Allocation
Rats
Rats, Sprague-Dawley
Tomography, X-Ray Computed
Contrast Media
Dextrans
Magnetite Nanoparticles

Figure

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  Fig. 1 Three-dimensional reconstruction of MR images of photothrombotic cerebral infarction and superparamagnetic iron oxide (SPIO)-labeled stem cells in rat model. A. Using Philips clinical workflow solution, infarction area was assigned color yellow, and dark region due to SPIO-labeled human bone marrow-derived mesenchymal stem cells was assigned color red. Three-dimensional reconstruction was performed using volume-rendering mode. B. Subsequently, volumes of infarction and SPIO were obtained (mm3) using calculate-volume tool.

• 

  Fig. 2 Confirmation of labeling stem cells with superparamagnetic iron oxide (SPIO) nanoparticles. Un-labeled human bone marrow-derived mesenchymal stem cells (hBM-MSCs) (A) and SPIO-labeled hBM-MSCs at 50 µg Fe/mL (B) stained with Perls' reagent. All cells in B were sufficiently labeled without any abnormal changes in morphology (× 400, Prussian blue staining).

• 

  Fig. 3 Two-dimensional (2D) and three-dimensional (3D) reconstruction of phantom MR images. Solidified agarose phantom had 2.2-mm diameter U-shaped column with 6.0 × 102 superparamagnetic iron oxide (SPIO)-labeled stem cells in total volume of 200-µL. A. Column is seen as U-shaped dark signal region on T2-weighted images (T2WI), T2*-weighted images (T2*WI), and susceptibility-weighted images (SWI). Dark signal region is large and prominent on SWI, followed by T2*WI, and then T2WI. B. In three-dimensionally reconstructed images, color red was assigned to dark signal region due to SPIO-labeled human bone marrow-derived mesenchymal stem cells. Volumes were 25.9, 68.4, and 129.3 µL on T2WI, T2*WI, and SWI, respectively.

• 

  Fig. 4 Time-elapsed MR images of photothrombotic cerebral infarction and superparamagnetic iron oxide (SPIO)-labeled stem cells in rat model. MR images were obtained on day 2 after photothrombotic cerebral infarction (pre, 1 day before cell injection) and on days 1, 3, 7, and 14 after internal carotid arterial injection of SPIO-labeled human bone marrow-derived mesenchymal stem cells. As time progressed, high signal intensity of infarction (⊚) was reduced in size and intensity, and dark signal region (arrowheads) appeared by encircling infarction from day 1 until day 14, indicating engraftment of stem cells to infarction. Dark regions were large and prominent in susceptibility-weighted images (SWI), followed by T2*-weighted images (T2*WI), and then by T2-weighted images (T2WI).

• 

  Fig. 5 Correlation between MR image and histology. Dark signal region (A) encircling lateral margin of cerebral infarction (⊚) on MR image (day 14) was correlated with hematoxylin and eosin (HE) staining (B, × 12.5), Prussian blue (PB) staining (C, × 100), and immunohistochemistry (IHC) with anti-human mitochondria antibody (D, × 100). On HE staining, infarction (⊚) and cystic encephalomalacia (**) were seen in right parietal lobe (A, B). On PB staining and IHC, dark regions on MR image were confirmed as superparamagnetic iron oxide-labeled human bone marrow-derived mesenchymal stem cells (A, C, D).

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  Fig. 6 Correlation between Prussian blue and immunohistochemistry (IHC) staining. Two images of Prussian blue staining (A, × 200) and IHC with anti-human mitochondria antibody (B, × 200) of same slide are superimposed (C). Positive cells can be seen at periphery of cerebral infarction. Blue-color stained iron positive cells (A) were thoroughly matched to green fluorescent human mitochondria positive cells (B). Engraftment of implanted superparamagnetic iron oxide-labeled human bone marrow-derived mesenchymal stem cells was confirmed by matching.

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  Fig. 7 Ventral perspective of three-dimensional reconstruction of MR images of photothrombotic cerebral infarction and superparamagnetic iron oxide (SPIO)-labeled human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in rats. Color yellow was assigned to cerebral infarction, and color red was assigned to SPIO-labeled hBM-MSCs surrounding infarction. Internal carotid arterial (ICA) injection (A) of SPIO-labeled hBM-MSCs shows engraftment of more cells in earlier days than intravenous (IV) injection (B). SWI = susceptibility-weighted images, T2WI = T2-weighted images, T2*WI = T2*-weighted images

• 

  Fig. 8 Volumetric analysis of superparamagnetic iron oxide (SPIO)-labeled human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and cerebral infarction. A. Volumes of engrafted SPIO-labeled hBM-MSCs in internal carotid arterial (ICA) group were larger than those in intravenous (IV) group on days 1, 3, and 7 (not on day 14) after injection on susceptibility-weighted images (SWI) (p < 0.05), but not on T2*-weighted images (T2*WI) and T2-weighted images (T2WI) (p > 0.05). SWI was most sensitive in detecting SPIO-labeled cells among MRI pulse sequences (p < 0.05). B. Volume of infarction decreased as time progressed, i.e., from day 2 after photothrombotic cerebral infarction (1 day before cell injection) until day 14 after stem cell injection in all 6 rats. Between ICA and IV groups, there were no differences in volume of cerebral infarction (p > 0.05).

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