This work was supported by the Ministry of Science and Technology Grants, Taiwan (MOST 104-2923-B-038-004 -MY2, MOST 107-2314-B-038-063, and MOST 107-2314-B-038-042) and Taipei Medical University (TMU 105-AE1-B03, TMU 106-5400-004-400, TMU 106-5310-001-400, DP2-107-21121-01-N-05 and DP2-108-21121-01-N-05-01).

All procedures involving animals in this protocol were approved by the Institutional Animal Care and Use Committee, with the approval of the Ethics Committee for animal use in Taipei Medical University (approval no. LAC-2018-0574; 15.03.2019).
1. Labeling of MSCs with SPIO Nanoparticles
<ol>
	<li>To label MSCs with SPIO, add 6 mL of labeling media (25 &#181;g/mL of SPIO in Dulbecco&#39;s modified Eagle medium [DMEM] with no fetal bovine serum [FBS]) to a T75 flask containing MSCs (80% confluency).</li>
	<li>Incubate the cells with the labeling media in a CO2 incubator (37 &#176;C, 5% CO2) without shaking. After 24 h, gently remove the labeling media using a sterile Pasteur pipette with a plastic tip attached to a vacuum. Wash the cells monolayer 2x with 6 mL of phosphate buffered saline (PBS) to remove any traces of uninternalized SPIO.
	<ol>
		<li>To determine whether the cells have been successfully labeled, check the labeled cells under a fluorescent microscope or confocal microscope if the SPIO are tagged with a fluorophore (i.e., like those used here; Figure 1B,C). Alternatively, perform Prussian blue staining for the cells (see steps 6.2&#8211;6.4 for the staining protocol).</li>
	</ol>
	</li>
	<li>Harvest adherent cells by treatment with 3 mL of trypsin and incubate at 37 &#176;C. After 5 min of incubation, add 7 mL of pre-warmed DMEM media with 10% FBS (v/v) to inactivate the trypsin. Collect the cell suspension using a pipette into a 15 mL conical tube.</li>
	<li>Centrifugate the cell suspension at 300 x g for 5 min. Discard the supernatant and resuspend the cell pellet in PBS. Count the viable cells using trypan blue dye and a hemocytometer. 
	NOTE: The cell pellet of the labeled cells will appear as a dark color due to iron loading (Figure 1D). This protocol is relevant for MSC labeling and MRI imaging. The procedure for MSC labeling6 has been previously optimized, and only the steps to prepare labeled MSCs to track in vivo are included here, since in vivo tracking is the focus. The protocol for culturing and labeling of other cell types should be optimized by the researcher.</li>
	<li>Adjust the cell concentration using PBS to 150,000 cells (or a number that results in a sufficient MRI signal) in 18 &#181;L of PBS (or the total volume that will be used in the intranasal delivery procedure). 
	NOTE: It was noticed that a cell concentration higher than 150,000 cells/18 &#181;L of PBS leads to cell aggregation, which may affect the efficiency of intranasal delivery. If a higher number of cells is needed for intranasal delivery, increase the total volume of cell suspension and increase the number of intranasal administrations, as intranasal administration is a non-invasive procedure, and multiple dosing is possible.</li>
</ol>
2. Controlled Cortical Impact (CCI) Injury
NOTE: In this protocol, male C57 BL/6 mice (7&#8211;8 weeks old) were kept in a 12/12 h light/dark cycle with ad libitum access to food and water.
<ol>
	<li>To prepare each mouse for CCI injury, administer the zolazepam (50 mg/kg) and xylazine (20 mg/kg) anesthetizing cocktail via intraperitoneal (i.p.) injection (1 mL/kg). Ensure that the depth of anesthesia is sufficient by a lack of toe-pinch response. Alternatively, place the mouse in a chamber supplied with 2%&#8211;4% isoflurane for 60 s. </li>
	<li>Shave the fur of the dorsal surface of the skull between the ears using an electronic hair clipper. Clean the shaved area several times using a sterile cotton swab soaked in iodine. Use a cotton swab soaked in 70% ethanol to clean off the iodine.</li>
	<li>Place the anesthetized mouse in the stereotactic frame and secure the mouse using ear bars and nose bars. Make a midsagittal incision (approximately 2.5 cm) in the shaved skin using sterile scissors to access the surface of the skull.</li>
	<li>Remove the tissue on the bone using a cotton pad to expose the skull. Clean the skull surface using a cotton swab soaked in a 3% H2O2 for 10 s, then clean it with a dry cotton pad. 
	NOTE: The skull sutures and both bregma and lambda can now be easily identified.</li>
	<li>Identify the coordinates of choice on the skull surface for the CCI injury and draw a circle (4 mm diameter) around the coordinates using a pencil or proper marker. 
	NOTE: In this protocol, the coordinates at anteeroposterior (AP) -2.0 mm and mediolateral (ML) +1.5 mm were used for CCI induction.</li>
	<li>Use a microdrill and round burr (0.5 mm diameter) to thin the skull at the marked circle. Avoid applying pressure while drilling, as drilling through the bone may cause damage to the brain parenchyma. Clean bone dust away using a clean and dry cotton swab.</li>
	<li>Gently remove the bone flap using sterile fine forceps to expose the dura mater while keeping it intact. Remove the mouse from the stereotactic frame that was used for pre-injury preparation and place it into the stereotactic frame of the CCI device.</li>
	<li>Stabilize the head of the mouse using the ear bars and nose bars. Make sure the head of the mouse is level in the rostral-caudal direction and adjust the nose bars, if needed.</li>
	<li>Follow the instructions on the control box to zero the impactor tip to the exposed cortical surface. Make sure that the impactor tip is aligned directly above the desired cortex coordinates to be impacted using the X and Y control wheels on the base of the impactor.</li>
	<li>Set the experiment parameters using the control box with a velocity of 5 m/s, dwell time of 250 ms, and injury depth of 1 mm to induce mild injury in the mouse.</li>
	<li>Induce injury by pressing the &#8216;&#8217;impact&#8217;&#8217; button on the control box. Swab any bleeding that occurs using a sterile cotton swab.</li>
	<li>Remove the mouse from the stereotactic frame and close the incision using silk surgical sutures. Do not use metal clips to close the surgical site, since the mouse will be subjected to a magnetic field for MRI.</li>
	<li>Apply topical antibiotics (bacitracin neomycin) to the surgical site to prevent infections. Keep the mouse on the heating pad and monitor it closely during the recovery phase.</li>
	<li>Administer ketoprofen (2.5 mg/kg, IP) daily for 3 days after surgery, unless the ketoprofen administration contradicts with the study goals.</li>
</ol>
3. Intranasal Delivery
<ol>
	<li>At 1 day post-CCI induction, administer the zolazepam (50 mg/kg) and xylazine (20 mg/kg) anesthetizing cocktail via i.p. injection. Ensure that the mouse is deeply anesthetized by lack of toe-pinch response.</li>
	<li>Prepare the mouse for intranasal delivery of MSCs by hyaluronidase treatment.
	<ol>
		<li>Grab the mouse&#8217;s scruff and turn on its back firmly while immobilizing the skull. Place the tip of a pipette that contains hyaluronidase in sterile PBS (4 U/&#181;L) near the nostril of the mouse at a 45&#176; angle.</li>
		<li>Administer 3 &#181;L of hyaluronidase suspension in each nostril. Keep the mouse immobilized and facing upward on a clean pad for 5 min. Repeat hyaluronidase treatment 4x (total of 100 U hyaluronidase suspension).</li>
	</ol>
	</li>
	<li>After hyaluronidase treatment, keep the treated mouse on a clean pad facing up for 30 min.</li>
	<li>To deliver MSCs into the brain, hold the mouse firmly, as described in step 3.2.1. Administer 3&#8201;&#181;L/nostril of MSC suspension with a 3&#8201;s interval. Keep holding the mouse in the same position for 30 s until the sample drops have completely disappeared. 
	NOTE: Avoid forming air bubbles during administration.</li>
	<li>Repeat the administration with a 2 min interval up to 3x. 
	NOTE: The total number of cells to be delivered is 150,000, such that 18&#8201;&#181;L of the cell suspension can be delivered at a 3 &#181;L dosage for each nostril, 3x each.</li>
	<li>Return the mouse to its cage and monitor it closely until it fully recovers from anesthesia.</li>
</ol>
4. In Vivo Magnetic Resonance Imaging
NOTE: Histological staining of brain tissue has previously been used to confirm the successful delivery of stem cells after intranasal administration. However, this method can only be used as an endpoint of a study, not longitudinally. Using MRI probes to label therapeutic stem cells will allow for longitudinal, non-invasive, in vivo tracking of the cells using MRI. Importantly, this protocol efficiently reduces the number of animals required. In this protocol, MRI scanning was performed at days 1, 7, and 14 post-delivery of MSCs.
<ol>
	<li>To prepare the mouse for MRI scanning, anesthetize the mouse with isoflurane (5% isoflurane in 1 L/min of O2&#160;for induction, 1.5%&#8211;2% isoflurane for maintenance). Perform a toe-pinch to ensure that the mouse reaches the required anesthesia level.</li>
	<li>Place the mouse on the imaging holder and secure its position using taps or any other proper method. Move the holder to the center of the MRI coil (7 T/40 cm magnet) and connect the monitoring connection.</li>
	<li>To acquire T2*-weighted scans using a spin-echo sequence, set the repetition time (TR) to 1500 ms and echo time (TE) to 2.8 ms.</li>
	<li>Use a 16 mm x 16 mm field of view (FOV), 128 x 128 acquisition matrix (MTX), and slice thickness of 0.75 x 0.8 mm2 with four signal averages and a 90&#176; flip angle (FA).</li>
	<li>Retract the mouse holder from the MRI coil center after completing the scans. Return the mouse to its cage and monitor it closely until it completely recovers from anesthesia.</li>
	<li>To track and quantify the labeled MSCs on the T2*-weighted images, use ITK-SNAP software (version 3.8.0)7.
	<ol>
		<li>Transfer the raw data of the MRI scans from the MRI machine&#8217;s computer to the computer used for analysis in a DICOM (digital imaging and communications in medicine) format.</li>
		<li>Run the ITK-SNAP software and load the MRI images by clicking on the File button. Then, click on Open Main Image in the menu. Press on the Open Image button in the display window, then locate and open the MRI images using the Browse button. 
		NOTE: The visualization of labeled cells in the MRI images will appear as hypointense areas. If the image contrast needs to be adjusted, select Tools | Image Contrast.</li>
		<li>Create segmentations of the hypointense areas and lesion or other brain parts by selecting Active Label in the segmentation labels section. Use different label colors for different segments (if the segmentation of more than one part is needed).</li>
		<li>Use the Polygon tool in the Main Toolbar to draw around the hypointense areas representing the SPIO-labeled MSCs. Select Accept, located below the MRI image. The segmented areas will appear as the same color of the active label assigned to that particular segment. Repeat this segmentation step for all MRI slices.</li>
		<li>Develop a 3D map of the segmented areas to represent the MSC distribution in the whole brain by selecting the Scalpel Tool at the bottom of the 3D Toolbar, located in the Segmentation Labels section at the bottom of TheITK-SNAP toolbox. Then, press Accept at the bottom of the created 3D map.</li>
		<li>To perform a quantitative analysis (volume and intensity mean) of the segmented hypointense areas representing labeled cells, press the Segmentation button in the top panel and select Volume and Statistics.</li>
	</ol>
	</li>
</ol>
5. Fixation of the Mouse Brain and Cryosectioning
<ol>
	<li>To fix the mouse brain, perform transcardiac perfusion with 4% paraformaldehyde (PFA) following the last MRI scan, as previously described8.
	<ol>
		<li>Decapitate the head and extract the brain8. Fix the brain with 4% PFA for at least 48 h at 4 &#176;C.</li>
		<li>Dehydrate the brain by immersing it into a 30% sucrose solution at 4 &#176;C until the brain sinks to the bottom of the solution.</li>
	</ol>
	</li>
	<li>Embed the brain in the optimal cutting temperature (OCT) solution and freeze at -20 &#176;C. Section the brain with a cryostat microtome into slices with 14 &#956;m thickness and mount them onto slides. Store the sections slides at -20 &#176;C until further use.</li>
</ol>
6. Prussian Blue Staining
NOTE: Prussian blue staining is commonly used to detect the iron content in SPIO-labeled cells. Here, Prussian blue staining is used to confirm that the hypointense signals in the MRI images correspond to the SPIO-labeled MSCs and not to artifacts. Prussian blue staining is one of the most sensitive histochemical methods used to detect iron in tissues and can be used to identify even a single granule of iron in the cells.
<ol>
	<li>Wash the slides of brain sections with distilled water for 5 min.</li>
	<li>Perform&#160;Prussian blue staining by immersing the slides for 30 min in the staining solution, which contains equal parts hydrochloric acid (10%) and potassium ferrocyanide (10%) prepared immediately before use.</li>
	<li>Wash 3x with distilled water, for 5 min each. Counterstain the sections with nuclear fast red for 5 min. Rinse the slides 2x with distilled water.</li>
	<li>Dehydrate the sections gradually by immersing the slides in 95% and 100% alcohol for 2 min each. Add coverslip with a resinous mounting medium.</li>
	<li>Use a light microscope to detect the stained cells in the brain sections. 
	NOTE: The iron in the labeled cells will appear as blue colored deposits.</li>
</ol>

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The authors have nothing to disclose.

The protocol described here represents general procedures for the SPIO labeling of MSCs and MRI tracking of SPIO-labeled MSCs post-intranasal delivery. The protocol allows the opportunity to study the migration and biodistribution of MSCs post-delivery in vivo in the brain, using a non-invasive method.
MSCs are attractive candidates for stem cell-based therapies for CNS disorders and injuries due to their ability to secrete trophic factors that 1) trigger neurorestorative processes and 2) provide neuroprotection, owing to their anti-inflammatory effects within the injury area9,10,11,12. Although long-term MRI tracking and detection of SPIO-labeled MSCs may be limited due to the dilution of intercellular SPIO with cell division, labeled cells can be detected for up to several weeks post-transplantation in the brains of animal models13.
Also described here is the labeling protocol of MSCs with SPIO nanoparticles coated with dextran without transfection agents. Other protocols have been used in the literature14,15,16. However, in all cases, these protocols should be adjusted for cell type, SPIO size, incubation time, and SPIO concentration. MSCs have been shown to have impaired chondrogenic differentiation potential but not adipogenic differentiation upon SPIO labeling17. Therefore, it is highly recommended that differentiation assays be performed prior to stem cell delivery to evaluate the influence of SPIO on the differentiation potency of stem cells. In a previous study, it was demonstrated that MSC labeling with the same SPIO type and concentration used in the here did not affect the osteogenic or adipogenic differentiation potency of MSCs6.
The intranasal route of therapeutic stem cell delivery for brain disorders and injuries is a promising approach for the clinical application of stem cells. However, the intrinsic and molecular mechanisms that dictate the behaviors of stem cells in the nasal cavity remain unclear. Although the intranasal route is widely explored for the delivery of small molecules, the size and biodistribution behavior of the therapeutic stem differ from small molecules. The current protocol demonstrates that MSCs tend to migrate toward the injury site after intranasal delivery.
Here, T2*-weighted images were used to track the SPIO-labeled MSCs. Other reports have used gradient echo imaging. However, susceptibility artefacts are often observed in gradient echo imaging due to intercellular SPIO. In the current protocol, the location of the hypointense areas representing the SPIO-labeled MSCs on T2*-weighted images was the same as the location of the SPIO in brain sections as detected by histological examination (Figure 3). This indicates the adequate sensitivity of T2*-weighted spin echo imaging for SPIO-labeled MSC tracking in the brain.
In summary, the described protocol is beneficial for in vivo stem cell tracking studies of brain injuries and disorders. The&#160;longitudinal tracking of stem cells&#160;in vivo has traditionally been performed by sacrificing animals at multiple timepoints. The current protocol provides a non-invasive and efficient approach for MSCs delivery and tracking, which represents a potential procedure for stem cell-based therapy for brain injuries and disorders in clinical settings.

Mesenchymal stem cells (MSC) are attractive candidates for stem cell-based therapies in treatments of central nervous system (CNS) disorders and injuries in humans. Moreover, MSCs have been used as a vehicle for the delivery of therapeutic proteins at injury sites1,2. In recent years, promising innovations have been developed to establish 1) novel routes of cell delivery and 2) cell tracking for stem cell-based therapies of CNS disorders. The intranasal delivery of stem cells into the brain depends on the ability of cells to bypass the cribriform plate and enter the olfactory bulb partially via a parenchymal route3. The combination of intranasal delivery and the labeling of MSCs with superparamagnetic iron oxide (SPIO) nanoparticles represents a promising approach for clinical applications of MSCs in treating CNS disorders, since SPIO nanoparticles are safe probes for magnetic resonance imaging (MRI) and allow non-invasive sensitive longitudinal tracking of MSCs post-delivery by MRI3,4,5. Furthermore, intranasal delivery is a safe and non-invasive route that allows repeated administration within a short period of time.
This article describes a highly sensitive and non-invasive technique for tracking MSCs in vivo post-intranasal delivery in a mouse model of traumatic brain injury (TBI), which employs SPIO-labeled cells and MRI. One important advantage of the SPIO labeling is the sensitive detection of SPIO in tissue by MRI, which makes it possible to track cells efficiently and non-invasively. The SPIO nanoparticles used here are commercially available and tagged with a fluorescein isothiocyanate (FITC) fluorophore, which allows for the detection of SPIO in tissue without immunostaining or additional processing. Furthermore, it is possible to perform longitudinal real-time tracking and investigate the biodistribution of the delivered MSCs.

<table><tbody><tr><td>Cell culture supplies (Plastics)</td><td>ThermoFisher Scientific</td><td>Varies</td><td>Replaceable with any source</td></tr><tr><td>Disposable Microtome Blade</td><td>VWR</td><td>95057-832</td><td></td></tr><tr><td>D-MEM/F-12 (1x) with GlutaMAX</td><td>GIBCO</td><td>10565-018</td><td></td></tr><tr><td>Embedding medium for frozen tissue specimens (O. C. T.)</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GIBCO</td><td>12662-029</td><td></td></tr><tr><td>Fluorescence Wild Field Microscope</td><td>Olympus</td><td>Olympus BX43</td><td></td></tr><tr><td>Forcept</td><td>Fine Science Tools</td><td>11293-00</td><td>Surgery</td></tr><tr><td>Gentamicin (10 mg/mL)</td><td>GIBCO</td><td>15710-064</td><td></td></tr><tr><td>Hair clipper</td><td>Pet Club</td><td>PC-400</td><td></td></tr><tr><td>Head Trauma Contusion device</td><td>Precision Systems and Instrumentation</td><td>Model TBI-0310</td><td></td></tr><tr><td>Hyaluronidase from bovine testes</td><td>MilliporeSigma</td><td>H3506</td><td></td></tr><tr><td>ITK-SNAP Software</td><td>Penn Image Computing and Science Laboratory (PICSL) at the University of Pennsylvania, and the Scientific Computing and Imaging Institute (SCI) at University of Utah</td><td>ITK-SNAP 3.8.0</td><td></td></tr><tr><td>Ketamine (Ketavet)</td><td>Pfizer</td><td>778-551</td><td></td></tr><tr><td>Mice</td><td>National Laboratory Animal Center, Taiwan</td><td>C57BL6</td><td>Wild type mice strain used in the study</td></tr><tr><td>Microdrill</td><td>Nakanishi</td><td>NE50</td><td>Combine with Burrs for generating the bone window</td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2265</td><td></td></tr><tr><td>Mouse (C57BL/6) Mesenchymal Stem Cells</td><td>GIBCO</td><td>S1502-100</td><td></td></tr><tr><td>MRI scanner</td><td>Bruker Biospec</td><td></td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>Corning Cellgro/ThermoFisher</td><td>21-031-CV</td><td></td></tr><tr><td>Povidone-iodine 7.5%</td><td>Purdue product L.P.</td><td></td><td>Surgical scrub</td></tr><tr><td>Prussian Blue Stain</td><td>Abcam</td><td>ab150674</td><td></td></tr><tr><td>Scissor</td><td>Fine Science Tools</td><td>14084-08</td><td>Surgery</td></tr><tr><td>Stereotaxic frame</td><td>Kopf Instruments</td><td>Model 900</td><td></td></tr><tr><td>Superparamagnetic iron oxide (SPIO) nanoparticles</td><td>BioPAL</td><td>Molday ION EverGreen, CL-50Q02-6A-51</td><td>stem cells labeling for in vivo tracking using MRI</td></tr><tr><td>Suture monofilament</td><td>Ethicon</td><td>G697</td><td>Suture</td></tr><tr><td>Timer</td><td>Wisewind</td><td></td><td>Replaceable with any source</td></tr><tr><td>TrypLE</td><td>GIBCO</td><td>12604-013</td><td></td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer</td><td>QN05 cm92</td><td></td></tr></tbody></table>

tracking superparamagnetic iron oxide-labeled mesenchymal stem cells using mri after intranasal delivery in a traumatic brain injury murine model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Twenty-four hours following intranasal delivery, the SPIO-labeled MSCs were detected as strong hypointense areas medial to the cortical injury on T2*-weighted images (Figure 2B), indicating the targeted migration of SPIO to the injury site. This migration remained visible up to 14 days post-delivery, as the hypointense signals were found to be visible without significant reduction for this time period (Figure 2B). The injured animals treated with PBS showed no hypointense areas, indicating that the observed hypointense areas correspond to the SPIO labeled MSCs and not to signal artifacts (Figure 2A) The biodistribution of the labeled MSCs that were observed&#160;in vivo&#160;with MRI was visualized using 3D reconstruction (Figure 2C,D). The migration of MSCs to the injured cortex was confirmed histologically by Prussian blue staining and FITC channel detection of the FITC-tagged SPIO in the labeled MSCs (Figure 3A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60450/60450fig1.jpg" /> 
Figure 1: Schematic flowchart of the protocol and in vitro confirmation of SPIO uptake by MSCs. (A) MSCs were incubated with SPIO for 24 h for labeling. Then, the labeled MSCs were delivered into a TBI mouse model via an intranasal (IN) route. MRI at different timepoints was performed to track the labeled MSCs. Confirmation of sufficient labeling of MSCs by SPIO was achieved by (B) fluorescence microscopy and (C) confocal microscopy using the FITC channel, since SPIO nanoparticles were tagged with FITC. (D) The cell pellet of the labeled MSCs appeared dark in color due to iron loading. FITC = fluorescein isothiocyanate; SPIO = superparamagnetic particles of iron oxide; MSCs =&#160;mesenchymal stem cells; MRI = magnetic resonance imaging; IN = intranasal; TBI = traumatic brain injury. <a href="https://www.jove.com/files/ftp_upload/60450/60450fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60450/60450fig2.jpg" /> 
Figure 2: Real-time MRI enables the detection and tracking of SPIO-labeled MSC migration toward injury sites in the brains of TBI-induced mice. (A) Mice were subjected to TBI, followed by treatment with PBS or SPIO-labelled MSCs, administered via an intranasal route 24 h after injury. Coronal sections of T2*-weighted images showed the labeled MSCs as a hypointense area (arrowhead) on the edge of the injury site (outlined area) at 1, 7, and 14 days post-delivery. The PBS-treated mice show no hypointense area. (B) Segmentation process of the injury site area (green) and labeled MSCs (red) based on coronal T2*-MRI images. (C) 3D reconstruction of the mouse brain treatment based on T2*-weighted images illustrating the biodistribution of SPIO-labeled MSCs in the brain 14 days post-delivery. <a href="https://www.jove.com/files/ftp_upload/60450/60450fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60450/60450fig3.jpg" /> 
Figure 3: Histological analysis confirms the presence of SPIO-labeled MSCs in the brains of the treated animals. Prussian blue staining of brain sections of a (A) mouse treated with PBS (control) and (C) mouse treated with SPIO-labeled MSCs. SPIO-positive cells were detected in MSC-treated mouse (boxed cells, blue), while the control mouse showed no positive cells at the injury site in the cortex at 14 days post-delivery, confirming MRI observations. Fluorescence microscopy analysis of the cortex of a (B) control mouse treated with PBS and (D) mouse treated with SPIO-labeled MSCs was conducted 14 days post-delivery. The analysis revealed the presence of FITC-tagged SPIO-positive cells (boxed cells, green) at the injured cortex in the MSC-treated mouse, but no FITC signals were observed in the cortex of the PBS-treated mouse. Scale bars = 50 &#181;m, unless stated otherwise. <a href="https://www.jove.com/files/ftp_upload/60450/60450fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model at JoVE.com

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

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Medicine

7.6K Views.  Taipei Medical University and National Health Research Institutes. Deze methode kan helpen bij het bepalen van de basisverdeling van de lobitic stamcel na intranasale levering aan de hersenen. Het belangrijkste voordeel van deze techniek is dat vergemakkelijkt de niet-invasieve levering en het bijhouden van mesenchymale stamcellen naar de hersenen. Ook, de meerdere doseringen zijn mogelijk om de therapeutische effecten van de getransplanteerde cellen te maximaliseren in het chronische stadium na de verwondingen.Deze techniek maximaliseert het aantal c...

Video: Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Biology

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

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

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Neuroscience

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

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