PET Demonstrates Functional Recovery After Transplantation of Induced Pluripotent Stem Cells in a Rat Model of Cerebral Ischemic Injury

Experimental Design and Animal Groups

Each of 24 adult Sprague–Dawley male rats (body weight, 240–270 g) was randomly assigned to 1 of the following 3 experimental groups (8 per group): the iPSC transplantation group, the ESC transplantation group, and the phosphate-buffered saline (PBS) injection group. All experimental animals underwent middle cerebral artery occlusion (MCAO), and reperfusion was performed 90 min after MCAO. The stem cell transplantation or PBS injection was performed 3 d after reperfusion. Before stem cell transplantation and at weeks 1, 2, 3, and 4 after cell transplantation, all rats underwent neurologic function testing followed by ¹⁸F-FDG small-animal PET scanning. Autoradiographic detection and immunohistochemical staining were done after the last ¹⁸F-FDG small-animal PET scan (Supplemental Fig. 1; supplemental materials are available online only at http://jnm.snmjournals.org).

All procedures associated with the care of animals were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (23) and were approved by the Institutional Animal Care and Use Committee.

Induction of Middle Cerebral Artery Occlusion

Animal preparation and induction of anesthesia for the MCAO procedure and stem cell transplantation followed the institutional guidelines and the advice of veterinary staff. Briefly, after initial induction of anesthesia with a ketamine–xylazine mixture (a 60 mg/kg dose of ketamine plus a 10 mg/kg dose of xylazine, intraperitoneally) (24), the rat was shaved, transported to the surgery table in a dedicated surgery room, and fixed on the platform with a warm pad (model 69001; RWD Life Science) to maintain the body temperature throughout the procedure. The animal received an intraperitoneal injection of 10% chloral hydrate (350 mg/kg) (24). Focal brain ischemia was induced by the intraluminal suture technique. The midline skin incision was made in the neck with subsequent exploration of the left common carotid artery, the external carotid artery, and the internal carotid artery. MCAO was achieved by advancing a 3-0 monofilament nylon suture with a rounded tip to the left internal carotid artery via the external carotid artery until slight resistance was felt. After 90 min of MCAO, reperfusion was performed by withdrawal of the suture until the tip cleared the internal carotid artery, and the wound was closed. The animals were allowed to recover until awake and were returned to their cages. Rats were treated for pain for the first 24 h after operation, by receiving an injection of buprenorphine (0.05 mg/kg, subcutaneously) every 8–12 h (25). More detailed postoperative care is described in the supplemental data. The rats were housed 3 per cage in environmentally enriched conditions under a 12-h light–dark cycle throughout the experiments and were allowed free access to food and water.

Stem Cell Preparation

Enhanced green fluorescent protein (EGFP)–labeled mouse iPSCs and ESCs were cultured as described previously (4). Briefly, mouse iPSCs and ESCs were maintained on a mitotically inactivated (mitomycin C) (Roche) mouse embryonic fibroblast feeder layer in knockout Dulbecco modified Eagle medium (Gibco) containing 10% fetal bovine serum (Hyclone), 10% knockout serum replacement (Invitrogen), 2 mM l-glutamine (Invitrogen), ×100 nonessential amino acids (Invitrogen), ×1,000 β-2-mercaptoethanol (Invitrogen), 50 units of penicillin and a 50 mg/mL dose of streptomycin (Invitrogen), and mouse leukemia inhibitory factor (Invitrogen). Before stem cell transplantation, iPSC and ESC colonies were passaged up to 4 times without feeder cells on 60-mm culture dishes coated with 0.1% gelatin to eliminate contamination of the mouse embryonic fibroblasts.

Transplantation Procedure

After all the rats had been prepared, they were placed in a stereotactic instrument. A midline skin incision was made in the skull, with subsequent drilling for a burr hole. About 1.0 × 10⁶ suspended iPSCs, ESCs, or PBS was stereotactically injected into the left lateral ventricle (0.9 mm anterior to the bregma, 1.5 mm lateral to the midline, and 3.5 mm beneath the dura) in a volume of 20 μL over 10 min with the use of a Hamilton microsyringe (26). The needle was left in place for an additional 10 min and then removed slowly. All surgical procedures were conducted under aseptic conditions. Postoperative pain control and care were performed as described in the section on the MCAO procedure.

Neurologic Function Tests

Testing of the Modified Neurologic Severity Score (mNSS) was performed before and after MCAO; at 3 d (immediately before stem cell transplantation); and at 1, 2, 3, and 4 wk after cell transplantation by the same investigator, who was masked to the experimental groups, as described previously (27). Neurologic function was graded on a scale of 0–18 (0 = normal score; 18 = maximal deficit score). The mNSS is a composite of motor, sensory, reflex, and balance tests. In the severity scores of injury, 1 score point is awarded for the inability to perform the test or for the lack of a tested reflex; thus, the higher the score, the more severe the injury.

In Vivo PET Study and Image Analysis

At 3 d after reperfusion and at 1, 2, 3, and 4 wk after stem cell transplantation, rats were anesthetized with isoflurane (2%) and injected with approximately 18.5 MBq (500 μCi) of ¹⁸F-FDG via the tail vein. At 40 min after ¹⁸F-FDG injection, the rats were anesthetized with isoflurane (2%) and positioned prone in the microPET R4 scanner (Siemens Medical Solutions) for a 10-min static acquisition with the mid skull in the center of the field of view. The images were reconstructed with use of a modified backprojection algorithm. ¹⁸F-FDG uptake was calculated as the percentage injected dose per gram of tissue using the AMIDE software package (version 9.2; Stanford University). In each image plane, 3-dimensional regions of interest 2 mm in diameter were drawn over the infarcted area of the cerebral cortex and the contralateral normal tissue, and the mean percentage injected dose per gram of tissue was averaged. The lesion-to-normal homologous contralateral (L/N) ratio was used for semiquantitative analysis. The L/N ratio was calculated with use of the following formula: L/N ratio = mean counts per pixel of lesion region of interest/mean counts per pixel of contralateral normal area. The average radioactivity concentration within the infarcted area was obtained from the mean pixel values, normalized to that of nonischemic cortex, and expressed as a percentage.

Autoradiographic Imaging

Immediately after the last small-animal PET study, 6 animals (2 per group) were decapitated under deep anesthesia; the brains were removed rapidly into a rodent brain matrix and placed into the cryostat (Bright Instrument Co. Ltd.) for about 60 min at −20°C. Coronal brain slices (1-mm thick) were cut and kept in contact with an imaging plate (BAS-IP MS 2040; Fujifilm) for 20 min. The exposed plates were scanned with a bioimaging analyzer (FLA-7000; Fujifilm), and the images were displayed with a Dell computer (Image Reader FLA-7000, version 1.12; Fujifilm) for qualitative evaluation.

Immunohistochemical and Immunofluorescent Staining

After the last small-animal PET scan, 18 animals (6 per group) were transcardially perfused with saline followed by 200 mL of 4% paraformaldehyde in phosphate buffer under deep anesthesia (supplemental data). The brains were dissected and fixed in paraformaldehyde for 6–8 h and then dehydrated with 30% sucrose in 0.1 M PBS for 2 d. The brains were frozen on powdered dry ice, and then coronal sections (20 μm thick) were processed with a cryostat (Leica) at 20°C throughout the entire ischemic damaged brain.

For immunohistochemical staining, the sections were permeabilized with 0.04% Triton X-100 and blocked with 10% normal goat serum and 0.5% bovine serum albumin in PBS for 1 h and then treated overnight at 4°C with primary antibodies: Rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP) (1:300 dilution; Abcam), mouse monoclonal antibody against neuronal nuclei (NeuN) (1:200 dilution; clone: A60, Millipore), or rabbit polyclonal antibody against von Willebrand factor (vWF) (1:200 dilution; DAKO). The sections were washed 3 times in PBS for 10 min each and incubated in biotinylated secondary antiserum (Vectastain ABC kit, Vector Lab) for 30 min at room temperature. The sections were rinsed and incubated in ABC reagents (Vectastain ABC kit) for 30 min and then washed thoroughly and incubated in 0.05% diaminobenzidine (DAB) and 0.03% H₂O₂ for 3–5 min until a brown reaction product was observed.

Immunohistochemical studies were performed to determine whether transplanted stem cells can induce neurogenesis and angiogenesis. NeuN was used as a mature neuronal marker, GFAP as the mature astrocyte marker, and vWF as the endothelial cell marker. We counted the number of cells positively stained with NeuN or vWF and calculated the mean values and SDs. The number of positively stained cells with NeuN or vWF in 3 different microscopic fields (×200 magnification) was calculated. To evaluate the average integral optical density (IOD) of GFAP, the hot spots of the section were selected with the use of a microscope (×100 magnification). Individual counts were then made in ×200 fields (BX60; Olympus). Sections (3 microscopic fields per slice, 1 slice per animal) were digitized with the use of a camera. The IOD of GFAP was obtained using Image-ProPlus 5.0 software (Media Cybernetics).

To follow up on the fate of EGFP-labeled transplanted cells, some sections were processed for immunofluorescent analysis of the cell implants. Sections were blocked and incubated overnight at 4°C with primary antibodies as mentioned above; the dilution of all primary antibodies was 1:100. After washes in PBS, sections were incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor 568, 1:500; Invitrogen) for 1 h at room temperature. Sections were washed and counterstained with the nuclear dye 4,6-diamino-2-phenylindole. Fluorescence-labeled sections were viewed with use of a confocal microscope (Olympus).

Statistical Analyses

The data were expressed as mean ± SD. A difference between groups was considered statistically significant if the P value was less than 0.05. The Student t test was used to compare body weight between treatment group and control group after MCAO. ANOVA was used when the groups numbered 3. Statistical analyses were performed with SPSS software (version 15.0, SPSS Inc.).