Superparamagnetic Iron Oxide Labeling and Transplantation of Adipose-Derived Stem Cells in Middle Cerebral Artery Occlusion-Injured Mice
Henry E. Rice1,
Edward W. Hsu2,
Huaxin Sheng3,
Debra A. Evenson1,
Alex J. Freemerman1,
Kristine M. Safford1,
James M. Provenzale4,
David S. Warner3 and
G. Allan Johnson4
1 Department of Surgery, Duke University Medical Center, Box 3815, Durham, NC
27710.
2 Department of Biomedical Engineering, Duke University Medical Center, Durham,
NC.
3 Department of Anesthesiology, Duke University Medical Center, Durham,
NC.
4 Department of Radiology, Duke University Medical Center, Durham, NC.
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Fig. 3A —Phantoms of various numbers of ferumoxide-poly-L-lysine
(PLL) labeled adipose-derived stem cells implanted in agar in Petri dish,
which was placed on top of 3.0-cm-diameter radiofrequency surface coil. Series
of multislice T2-weighted and T2*-weighted 7-T MR images (TR/TE,
250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field
of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo
sequences. T2-weighted images reveal clear differences in signal intensity
between area without cells (A) and areas with increasing number of
superparamagnetic iron oxide (SPIO)-labeled adipose-derived stem cells.
Numbers of SPIO-labeled adipose-derived stem cells correlated well with
decrease in MR signal intensity. Low signal intensity at all cell numbers
greater than 10 cells suggests MRI can depict at least as few as 10 labeled
adipose-derived stem cells with nominal pixels. Number of cells, 0.
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Fig. 3B —Phantoms of various numbers of ferumoxide-poly-L-lysine
(PLL) labeled adipose-derived stem cells implanted in agar in Petri dish,
which was placed on top of 3.0-cm-diameter radiofrequency surface coil. Series
of multislice T2-weighted and T2*-weighted 7-T MR images (TR/TE,
250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field
of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo
sequences. T2-weighted images reveal clear differences in signal intensity
between area without cells (A) and areas with increasing number of
superparamagnetic iron oxide (SPIO)-labeled adipose-derived stem cells.
Numbers of SPIO-labeled adipose-derived stem cells correlated well with
decrease in MR signal intensity. Low signal intensity at all cell numbers
greater than 10 cells suggests MRI can depict at least as few as 10 labeled
adipose-derived stem cells with nominal pixels. Number of cells, 10.
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Fig. 3C —Phantoms of various numbers of ferumoxide-poly-L-lysine
(PLL) labeled adipose-derived stem cells implanted in agar in Petri dish,
which was placed on top of 3.0-cm-diameter radiofrequency surface coil. Series
of multislice T2-weighted and T2*-weighted 7-T MR images (TR/TE,
250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field
of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo
sequences. T2-weighted images reveal clear differences in signal intensity
between area without cells (A) and areas with increasing number of
superparamagnetic iron oxide (SPIO)-labeled adipose-derived stem cells.
Numbers of SPIO-labeled adipose-derived stem cells correlated well with
decrease in MR signal intensity. Low signal intensity at all cell numbers
greater than 10 cells suggests MRI can depict at least as few as 10 labeled
adipose-derived stem cells with nominal pixels. Number of cells, 100.
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Fig. 3D —Phantoms of various numbers of ferumoxide-poly-L-lysine
(PLL) labeled adipose-derived stem cells implanted in agar in Petri dish,
which was placed on top of 3.0-cm-diameter radiofrequency surface coil. Series
of multislice T2-weighted and T2*-weighted 7-T MR images (TR/TE,
250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field
of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo
sequences. T2-weighted images reveal clear differences in signal intensity
between area without cells (A) and areas with increasing number of
superparamagnetic iron oxide (SPIO)-labeled adipose-derived stem cells.
Numbers of SPIO-labeled adipose-derived stem cells correlated well with
decrease in MR signal intensity. Low signal intensity at all cell numbers
greater than 10 cells suggests MRI can depict at least as few as 10 labeled
adipose-derived stem cells with nominal pixels. Number of cells, 500.
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Fig. 3E —Phantoms of various numbers of ferumoxide-poly-L-lysine
(PLL) labeled adipose-derived stem cells implanted in agar in Petri dish,
which was placed on top of 3.0-cm-diameter radiofrequency surface coil. Series
of multislice T2-weighted and T2*-weighted 7-T MR images (TR/TE,
250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field
of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo
sequences. T2-weighted images reveal clear differences in signal intensity
between area without cells (A) and areas with increasing number of
superparamagnetic iron oxide (SPIO)-labeled adipose-derived stem cells.
Numbers of SPIO-labeled adipose-derived stem cells correlated well with
decrease in MR signal intensity. Low signal intensity at all cell numbers
greater than 10 cells suggests MRI can depict at least as few as 10 labeled
adipose-derived stem cells with nominal pixels. Number of cells, 1,000.
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Fig. 3F —Phantoms of various numbers of ferumoxide-poly-L-lysine
(PLL) labeled adipose-derived stem cells implanted in agar in Petri dish,
which was placed on top of 3.0-cm-diameter radiofrequency surface coil. Series
of multislice T2-weighted and T2*-weighted 7-T MR images (TR/TE,
250/10-40 in increments of 5 milliseconds; matrix size, 128 x 128; field
of view, 4.0 cm; slice thickness, 1 mm) were acquired with standard spin-echo
sequences. T2-weighted images reveal clear differences in signal intensity
between area without cells (A) and areas with increasing number of
superparamagnetic iron oxide (SPIO)-labeled adipose-derived stem cells.
Numbers of SPIO-labeled adipose-derived stem cells correlated well with
decrease in MR signal intensity. Low signal intensity at all cell numbers
greater than 10 cells suggests MRI can depict at least as few as 10 labeled
adipose-derived stem cells with nominal pixels. Number of cells, 5,000.
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Fig. 4 —Mouse 2 weeks after middle cerebral artery occlusion-induced
infarction. In vivo 7-T T2*-weighted MR image of mouse head placed
above 1.0-cm-diameter surface coil. Multislice spin-echo images (TR/TE, 1/35;
matrix size, 256 x 256; field of view, 2.5 cm; resolution, 98 µm;
thickness of interleaved consecutive slices, 1.0 mm; number of signals
averaged, 2) were obtained in anatomic coronal orientation. All sequences were
performed within 40-60 minutes per mouse. Infarct (arrow) is evident
within right cortex. Adipose-derived stem cells were transplanted into
ipsilateral hippocampus.
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Fig. 5 —Middle cerebral artery occlusion-injured mouse sacrificed 24 hours
after transplantation of 1,000 ferumoxide-poly-L-lysine-labeled
adipose-derived stem cells. High-resolution ex vivo coronal 9.4-T proton
density MR image of mouse brain perfused with 10% buffered formalin/5%
gadoteridol shows areas of infarction (solid arrow) and low signal
intensity (dashed arrow) corresponding to donor cell
implantation.
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Fig. 6A —Middle cerebral artery occlusion-injured mouse sacrificed 24 hours
after receiving transplant of 1,000 superparamagnetic iron oxide
(SPIO)-labeled green fluorescent protein (GFP)-positive adipose-derived stem
cells into hippocampus. Coronal MR image shows hypointense region in area of
transplantation. Annular hyperintense ring is thought to represent artifact
from SPIO intensity given absence of corresponding abnormality on histologic
sections in B-E.
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Fig. 6B —Middle cerebral artery occlusion-injured mouse sacrificed 24 hours
after receiving transplant of 1,000 superparamagnetic iron oxide
(SPIO)-labeled green fluorescent protein (GFP)-positive adipose-derived stem
cells into hippocampus. Low-magnification Nissl stain of brain section similar
to that in A shows donor cell deposit (box).
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Fig. 6C —Middle cerebral artery occlusion-injured mouse sacrificed 24 hours
after receiving transplant of 1,000 superparamagnetic iron oxide
(SPIO)-labeled green fluorescent protein (GFP)-positive adipose-derived stem
cells into hippocampus. High-magnification Prussian blue stain of section
adjacent to B shows cell deposit.
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Fig. 6D —Middle cerebral artery occlusion-injured mouse sacrificed 24 hours
after receiving transplant of 1,000 superparamagnetic iron oxide
(SPIO)-labeled green fluorescent protein (GFP)-positive adipose-derived stem
cells into hippocampus. Immunohistochemical results with use of anti-GFP
antibody and 3,3'-diaminobenzidine conjugate of adjacent section show
brown staining of donor cells.
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Fig. 6E —Middle cerebral artery occlusion-injured mouse sacrificed 24 hours
after receiving transplant of 1,000 superparamagnetic iron oxide
(SPIO)-labeled green fluorescent protein (GFP)-positive adipose-derived stem
cells into hippocampus. High-magnification Nissl stain shows iron-laden donor
cells within CA3 region of hippocampus.
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Copyright © 2007 by the American Roentgen Ray Society.
) and percentage iron staining
(
) in presence of ferumoxide (12.5 µg/mL of elemental iron). Error
bars represent SE. Increasing concentration of PLL resulted in both increased
iron staining and decreased cell viability. These data showed PLL
concentration of 0.375 µg/mL as providing acceptable level of iron staining
with minimal loss of cell viability. Findings led to choice of this
concentration for future experiments.
), ferumoxide (
), and PLL-ferumoxide complex
(
). Results show initial decrease in proliferation of cells grown with
PLL-ferumoxide complex, although within 7 days, all experimental groups showed
proliferation equivalent to that of control cells.