Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

1. Cell Preparation

1. Experiments should be initiated using ex vivo cultured stem cell populations genetically engineered to express the Luciferase and eGFP reporter proteins. Here we use Luciferase/eGFP-expressing murine bone marrow-derived stromal cells (BMSC-Luc/eGFP) as previously described by Bergwerf et al.^{2, 5}.
2. Two days before cell labeling, plate BMSC-Luc/eGFP cells at a density of 8 x 10⁵ cells per T75 culture flask in 15 ml complete expansion medium (CEM) supplemented with 1 μg/ml Puromycine.
3. Allow cells to grow for 48 hr at 37 °C and 5% CO₂.
4. Add 5 x 10⁶ Glacial Blue micron-sized iron oxide particles (GB MPIO) per ml culture medium to the growing cell culture (total: 75 x 10⁶ particles per 15 ml culture medium in a T75 culture flask).
5. Incubate for 16 hr to allow particle uptake via endocytosis.
6. Wash away remaining particles and allow cells to grow for an additional 24 hr to obtain cell confluency and homogeneous distribution of the GB MPIO.
7. Validate particle uptake visually using fluorescence microscopy by counting the ratio of eGFP+ and GB MPIO+ cells to the total amount of eGFP+ cells.
8. Wash GB MPIO-labeled BMSC-Luc/eGFP cells twice with 10 ml PBS and harvest cells following trypsin-EDTA treatment (5 ml per T75 culture flask for 5 minutes).
9. Wash harvested cells and resuspend at final concentration of 133 x 10⁶ cells/ml in PBS.
10. Keep cells on ice until transplantation.

2. Cell Implantation in the CNS of Mice

1. Anaesthetize mice by an intraperitoneal injection of a ketamine (80 mg/kg) + xylazine (16 mg/kg) mixture.
2. Wait for 5 minutes to allow mice to fall asleep.
3. Shave the mouse head to allow for sterile manipulations.
4. Place the mouse in a stereotactic frame.
5. Desinfect the skin and wet the eyes of the mice to prevent dehydration.
6. Make a midline scalp incision to expose the skull, and drill a hole in the skull using a dental drill burr at the given coordinates: 2 mm posterior and 2 mm lateral to Bregma.
7. Vortex the cell suspension briefly and aspirate the cell suspension in the syringe.
8. Place the syringe needle at a depth of 2.5 mm under the dura and allow pressure equilibration for 1 minute.
9. Inject a 3 μl cell suspension (4 x 10⁵ cells) at a depth of 2 mm under the dura and at a speed of 0.70 μl/min using an automated micro-injection pump.
10. Wait for an additional 5 minutes to prevent backflow of the injected cell suspension.
11. Slowly retract the needle and disinfect the skin borders before suturing the skin.
12. Inject 300 μl 0.9% NaCl solution subcutaneously in order to prevent dehydration, and place the mouse under a heating lamp to recover from anaesthesia. Administer post-operative analgesic in accordance with institutional standards.

3. In vivo Bioluminescence

1. Anaesthetize the mice by a mixture of 3% isoflurane and oxygen.
2. Place the mice in the Photon Imager and reduce anaesthesia level to 1.5% isoflurane and oxygen.
3. Inject 150 mg D-luciferin per kg body weight intravenously.
4. Acquire image for 5 minutes using the Photo Vision software.
5. Perform image processing using the M3vision software. Quantify the observed signal using fixed regions of interest.

4. In vivo Magnetic Resonance Imaging

1. Anaesthetize the mice with 3% isoflurane in a mixture of O₂:N₂O (3:7).
2. Place the mice in the restrainer of a horizontal 9.4T MR system and reduce anaesthesia level to 1% isoflurane in a mixture of O₂:N₂O (3:7).
3. Wet the eyes of the mice to prevent dehydration, attach a rectal probe to monitor the body temperature and monitor the breathing rate by placing a sensor underneath the mouse belly.
4. Maintain breathing rate at 110 ± 10 breaths per minute and keep body temperature constant within a narrow range of 37 ± 0.5 °C.
5. Place the surface RF coil on top of the mouse head and position the mouse in the middle of the magnet.
6. Acquire a set of 10 coronal T2-weighted spin echo (SE) images to obtain specific anatomical information and T2*-weighted gradient echo (GE) images in order to study stem cell migration with an in-plane resolution of 70 μm². Set sequence parameters as follows: repetition time (TR): 500 ms, echo time (TE): 8 ms (GE sequence) and repetition time (TR): 4200 ms, echo time (TE): 12.16 ms (SE sequence); field of view (FOV): 18×18 mm², matrix: 256×256, 1 mm slice thickness and 1 mm slice separation (Paravision 5.1 software).
7. Perform data processing using Amira 4.0 software.

5. Post Mortem Histology

1. Anaesthetize the mice very deeply by inhalation of an isofluorane (4%), oxygen (0.5 L/min) and nitrogen (1 L/min) mixture for 2 minutes. Sacrifice the mice by cervical dislocation.
2. Remove the mouse brain from the skull and fixate the brain tissue in 4% paraformaldehyde in PBS for 2 hours.
3. Dehydrate the brain tissue by placing the brain subsequently in different gradients of sucrose: 2 hr in 5% sucrose in PBS, 2 hr in 10% sucrose in PBS, overnight in 20% sucrose in PBS.
4. Freeze the brain tissue using fluid nitrogen and store the tissue at -80 °C until sectioning.
5. Section the brain tissue in 10 μm thick sections using a cryostat.
6. Screen unstained cryosections for blue fluorescence from the GB MPIO particles and green fluorescence from the eGFP expressing cells using a fluorescence microscope.
7. Screen unstained cryosections for green/red background fluorescence from inflammatory cells.
8. Perform further immunohistochemical and immunofluorescent stainings to identify endogenous cell populations (e.g. microglia, astrocytes, T cells,…) interacting with cellular grafts.

6. Representative Results

We here visually presented an optimized sequence of events for successful multimodal imaging of Luciferase/eGFP-expressing (stem) cell populations in the CNS of mice. At first, the described GB MPIO labeling procedure results in highly efficient labeling of BMSC-Luc/eGFP cells, which can easily be validated using fluorescence microscopy (Figure 2A). Next, upon grafting of GB MPIO labeled BMSC-Luc/eGFP in the CNS of mice, survival of the cellular graft can be monitored by in vivo BLI based on luciferase activity (Figure 2B). Additionally, the exact localization of grafted cells can be monitored by in vivo MRI based on the iron content of the GB MPIO (Figure 2C). Finally, histological analysis allows validation of the results obtained by BLI and MRI. Stable survival was confirmed by a stable eGFP expression in time (Figure 2D). For this, colocalization of eGFP-expressing cells with blue fluorescent MPIO allows for exact determination of localization and survival of grafted cells. In addition, this dual fluorescent labeling of the stem cells reduces the possibility to observe false positive results based on background fluorescence created by inflammatory cells⁵.

Figure 1. Overview of the handling sequence

1. Cell labeling: BMSC-Luc/eGFP are labeled with 5 x 10⁶ GB MPIO particles per ml culture medium. After 16 hr of incubation and a following rest period of 24 hr, cells are harvested for intracerebral implantation.
2. Cell implantation: GB MPIO labeled BMSC-Luc/eGFP are grafted in mouse brain at the following coordinates: bregma -2 mm, 2 mm right from the midline and 2 mm under dura.
3. In vivo BLI: Following intravenous administration of 150 mg D-luciferin/kg body weight, the generated light was acquired during 5 minutes using the Photon Imager.
4. In vivo MRI: A set of 10 coronal T2-weighted spin echo (SE) images and T2*-weighted gradient echo (GE) images with an in-plane resolution of 70 μm² were acquired. Sequence parameters were as follows: repetition time (TR): 500 ms, echo time (TE): 8 ms (GE sequence) and repetition time (TR): 4200 ms, echo time (TE): 12.16 ms (SE sequence); field of view (FOV): 18×18 mm², matrix: 256×256, 1 mm slice thickness and 1 mm slice separation.
5. Post-mortem histology: 10μm thick cryosection were screened for eGFP expressing and blue fluorescently labeled BMSC using a fluorescence microscope.

Figure 2. Multimodal imaging of stem cell grafts

1. Direct immunofluorescence microscopy of Glacial blue (GB) MPIO labeled BMSC-Luc/eGFP.
2. In vivo BLI of mice injected with 4×10⁵ GB MPIO labeled BMSC-Luc/eGFP cells at week 1 and week 2 after implantation. Statistical analysis of detected BLI signals at week 1 and week 2 post-implantation. Data are expressed as average number of photons per second per steradian per square centimeter (ph/s/sr/cm²) from a 5 minute time period +/- standard error calculated from a fixed region of interest in mice showing a clear BLI signal at the injection site.
3. Coronal two-dimensional MRI of GB MPIO labeled BMSC-Luc/eGFP (left panel: (i) T2-weighted spin echo image and (ii) T2*-weighted gradient echo image) and unlabeled BMSC-Luc/eGFP (right panel: (iii) T2-weighted spin echo image and (vi) T2*-weighted gradient echo image) grafts in mouse brain.
4. Histological analysis of GB MPIO labeled BMSC-Luc/eGFP implants at week 2 post-implantation. (i) Direct immunofluorescence analysis for localization of eGFP-expressing and GB MPIO labeled BMSC-Luc/eGFP grafts. (ii) High magnification detail of (i). (iii) Immunofluorescent staining for GFAP antigen, indicating the development of astrocytic scar tissue in the surrounding of GB MPIO labeled BMSC-Luc/eGFP. (vi) Immunofluorescent staining for Iba-1 antigen, indicating the invasion and surrounding of GB MPIO labeled BMSC-Luc/eGFP by microglia.