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Transferrin Receptor Upregulation: In Vitro Labeling of Rat Mesenchymal Stem Cells with Superparamagnetic Iron Oxide¹

¹ From the Institute of Clinical and Experimental Transfusion Medicine (R.S., A.G., H.N.) and Departments of Diagnostic Radiology (R.K., J.W., R.B., J.P., C.D.C.), Pathology (H.W.), and Cardiology (B.R.B.), University Medical Center Tübingen, Hoppe-Seyler-Str 3, D-72076 Tübingen, Germany. Received April 4, 2006; revision requested June 2; revision received July 12; accepted August 23; final version accepted November 13. Address correspondence to R.K. (e-mail: rainer.kehlbach{at}med.uni-tuebingen.de).

View larger version (123K): [in this window] [in a new window] [Download PPT slide]   Figure 1a: (a) Agar matrix with embedded iron contrast medium–containing cells. (b) Wrist coil for measurement with 3.0-T MR imaging unit.

View larger version (141K): [in this window] [in a new window] [Download PPT slide]   Figure 1b: (a) Agar matrix with embedded iron contrast medium–containing cells. (b) Wrist coil for measurement with 3.0-T MR imaging unit.

View larger version (103K): [in this window] [in a new window] [Download PPT slide]   Figure 2: Results of light microscopy of adipogenic, osteogenic, and chondrogenic differentiated labeled (Resovist [R] or Resovist C [RC]) and nonlabeled MSCs (passage 14). SPIO or USPIO could be identified as brown particles. a, Adipogenic differentiation after 21 days of treatment with adipogenic medium. (Oil red O stain, hematoxylin counterstain; original magnification, x400.) Red droplets represent lipid vacuoles. Arrows indicate adipocytes. o, Osteogenic differentiation after 21 days of treatment with osteogenic medium. (Alkaline phosphatase stain, hematoxylin counterstain; original magnification, x400.) Positive cells are stained pink to violet. c, Chondrogenic differentiation after 14 days of treatment with chondrogenic medium. Mucopolysaccharides are stained blue to bluish green. (Alcian blue stain; original magnification, x200.)

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 3: CD71 expression (at passage [P] 9, 12, 15, and 18) of MSCs labeled with Resovist (R) or Resovist C (RC) without TA at passages 7, 12, 13, and 16. The maximum of this effect was observed at passage 12 during a susceptible phase between passage 9 and passage 18. Red indicates isotype control. Green indicates probes. X-axis indicates fluorescence intensity (FL1-H) (100–104). Y-axis indicates counts (0–120).

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 4: Bar graph shows TIL (intracellular iron plus iron coating cellular surface) of cells after labeling with SPIO or USPIO alone or in combination with a TA. Error bars indicate standard deviations. DR = DOSPER plus Resovist (4 hours incubation), DRC = DOSPER plus Resovist C (4 hours incubation), jPR = jetPEI plus Resovist (4 hours incubation), jPRC = jetPEI plus Resovist C (4 hours incubation), R = Resovist, RC = Resovist C.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 5: MR imaging of cell counts from 1000 to 100 000 MSCs in agar solution (1%) at 3.0 T. Experimental parameters were as follows: 10 µL jetPEI per milliliter and 60 µg Resovist (jPR) or Resovist C (jPRC) per milliliter and 4 hours incubation time. Arrow indicates lowest cell count (5 x 103 cells) at which a signal was detectable at sagittal, coronal, and transverse imaging. R = Resovist, RC = Resovist C.

View larger version (190K): [in this window] [in a new window] [Download PPT slide]   Figure 6a: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

View larger version (190K): [in this window] [in a new window] [Download PPT slide]   Figure 6b: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

View larger version (199K): [in this window] [in a new window] [Download PPT slide]   Figure 6c: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

View larger version (104K): [in this window] [in a new window] [Download PPT slide]   Figure 6d: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

View larger version (65K): [in this window] [in a new window] [Download PPT slide]   Figure 6e: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.