The authors RR and CT acknowledge support from the M&#228;xi Foundation and the 3R Competence Center.

NOTE: All experiments involving mice were conducted in accordance with governmental, institutional, and ARRIVE guidelines and were approved by the Cantonal Veterinary Office of Zurich. Adult male and female non-obese diabetic SCID gamma (NSG) mice (10-14 weeks, 25-35 g) were used. Mice were housed in regular Type II/III cages in groups of at least two animals per cage in a humidity- and temperature-controlled room with a constant 12/12 h light/dark cycle. .).
1. Cell culture and viral transduction
<ol>
	<li>Differentiate neural progenitor cells (NPCs) from iPSCs using small molecule inhibitors as previously described14.</li>
	<li>Culture NPCs15 from passage 2 onwards in Neural Stem cell Maintenance Medium (NSMM; Table 1) supplemented with small molecules (Table 1) in 6-well plates (2 mL of medium per well) coated with poly-ornithine/laminin521 (pLO/L521). Change the medium daily. 
	NOTE: To passage NPCs, add 1 mL of cell dissociation reagent per well (see the Table of Materials) and incubate at 37 &#176;C for 1 min until most cells detach.
	<ol>
		<li>For coating, incubate 150 &#181;L of pLO in 1 mL of 0.1 M phosphate-buffered saline (PBS) per well for 2h at room temperature (RT). After three washes with PBS, incubate 10 &#181;g of L521 in 1 mL of PBS per well for 2 h at RT.</li>
	</ol>
	</li>
	<li>For viral transduction, plate 50,000 cells per well in a 24-well plate coated with pLO/L521 and add prepackaged viral vectors (pLL-EF1a-rFLuc-T2A-GFP-mPGK-Puro, LL410PA-1) to each well. 
	NOTE: Total infectious units (IFU) provided are &#62;2 &#215; 106 IFU and are enough to infect 100,000 cells at a multiplicity of infection (MOI) of 20. Cell counting has been performed with an automated cell counter. The transduction efficiency strongly depends on the used cell line. Work with lentivirus requires compliance with the local guidelines for biosafety level 2 (BSL-2) products.</li>
	<li>Incubate the cells at 37 &#176;C for 72 h, while continuing daily medium changes.</li>
	<li>Confirm successful transduction by checking the cells for GFP expression in a fluorescence microscope. Set the microscope magnification to 10x or 20x and use the appropriate excitation (460-480 nm) and emission range (490-520 nm) to detect transduced GFP-expressing cells.</li>
	<li>Quantify the ratio of GFP-transduced to total cells to estimate general transduction efficacy. 
	NOTE: Transduction rates of 65-95% were achieved with this protocol. A transduction efficacy of &#62;50% is recommended as a go/no-go criterion before transplantation. If 50% transduction efficacy cannot be achieved, perform puromycin selection or sort the cells using flow cytometry to increase the yield of transduced cells.</li>
	<li>Optional: Freezing of cells
	<ol>
		<li>Spin the cells down (300 &#215; g, 5 min) and discard the supernatant. Resuspend the pellet in 1 mL of freezing medium (see the Table of Materials) and transfer the suspension into vials to obtain 106 cells/vial. Transfer the cells to freezing boxes for 24 h at -80 &#176;C and then to -150 &#176;C for long-term storage.</li>
	</ol>
	</li>
</ol>
2. Cell preparation for transplantation
<ol>
	<li>Collect a vial of cells from -150 &#176;C storage and transfer it to the laboratory. Count the cells using an automated cell counter. 
	NOTE: The vial contains 1.5-2 &#215; 106 cells.</li>
	<li>Quickly transfer the vial to a 37 &#176;C water bath until no ice crystals remain (2-3 min). 
	NOTE: It is important to thaw rapidly to minimize any damage to cell membranes. Do not immerse the vial completely in the water bath as it can increase the risk of contamination.</li>
	<li>Transfer the vial to the biosafety cabinet and pipette the whole content (~1 mL) into a sterile 15 mL conical tube. 
	NOTE: Work with lentivirally transduced cells requires BSL-2. However, washing and passaging cells remove viral particles from the medium. Information about when a transfer from BSL-2 to BSL-1 is permitted should be obtained from the local authorities.</li>
	<li>Add 9 mL of sterile 1x PBS and centrifuge for 5 min at 300 &#215; g, RT.</li>
	<li>Remove the supernatant by aspiration using a pipette (1-10 mL); gently tilt the suspension towards the pipette tip and start aspirating. Be careful not to disturb the pellet.</li>
	<li>Wash the cells by resuspending in 10 mL of sterile 1x PBS. 
	NOTE: Gently tab the tube to resuspend cells in the residual volume. Slowly triturate the cell suspension using a 1 mL pipette until it does not contain clumps or aggregates.</li>
	<li>Count the cells before the final spin using an automated cell counter.</li>
	<li>Centrifuge for 5 min at 300 &#215; g, RT.</li>
	<li>Remove the supernatant (step 2.5) and resuspend the cell pellet in the required volume of sterile PBS to a concentration of 8 &#215; 104 cells/&#181;L. Place the cells on ice and use them for transplantation within the next 5h. 
	&#8203;NOTE: A volume of 1.6 &#215; 105 cells/2 &#181;L of PBS was used in this protocol.</li>
</ol>
3. Transplantation procedure
<ol>
	<li>Preparation for surgery
	<ol>
		<li>Clean and sterilize the surgery equipment.</li>
		<li>Prepare the stereotaxic device and the microinjection pump system. 
		NOTE: It is critical to test the Hamilton syringe and the 30 G, 2 inches needle before starting. Insert the needle into a tube containing sterile 0.9% NaCl and slowly draw the solution in and out.</li>
		<li>Set up the anesthesia machine. Test the machine before involving any animals. Clean the induction chamber with 70% ethanol.</li>
	</ol>
	</li>
	<li>Preparation of animals
	<ol>
		<li>Keep the mice for at least 7 days prior to the experiments in standard conditions to acclimate them. 
		NOTE: The following animals were used for this protocol: female NOD/SCID/IL2r&#947;null (30-35 g, also known as NSG) and female C57BL/6J (20-25 g, also known as B6). The procedure can also be performed with male mice.</li>
		<li>Measure the mouse body weight and adjust the dose of the pain killer to be injected. Administer carprofen (5 mg/kg body weight) intraperitoneally to reduce pain and/or prevent an inflammatory response.</li>
		<li>Anesthetize the animals using isoflurane (3% in the induction phase and 1.5-2% in the maintenance phase during surgery) vaporized in oxygen. 
		NOTE: Gaseous anesthesia is preferred due to a quick wake-up after the surgical procedure and because the levels of anesthetic gas can be easily adjusted.</li>
		<li>Use nociceptive reflexes to ensure the animal is deeply anesthetized (e.g., toe pinches). When deep anesthesia is reached, transport the animal from the induction chamber to the stereotaxic frame. Maintain anesthesia using a face mask. 
		NOTE: The breath rate needs to be monitored visually throughout the procedure (40-60 breaths per minute). Use a warming pad to avoid hypothermia during the procedure.</li>
		<li>Apply ophthalmic lubricant to prevent the eyes from drying out.</li>
		<li>Shave the mouse scalp with an electric razor and disinfect the skin with 5% betadine solution using cotton swabs.</li>
		<li>Secure the mouse head and insert the ear bars into the external meatus. 
		NOTE: Be careful not to damage the eardrums. Apply lidocaine ointment to both ear canals before inserting the ear bars. To check if the animal&#39;s head is in a stable position, carefully push down on the head to see if there is movement. If movement is noted, either the ear bar, nosepiece placement, or both are incorrect and need to be readjusted.</li>
	</ol>
	</li>
	<li>Craniotomy
	<ol>
		<li>Use a surgical blade to make a cut along the midline big enough to reveal the lambda and bregma landmarks. 
		NOTE: Skin retractors can be applied to keep the skull exposed.</li>
		<li>Retract the periosteum and fascia with a scalpel and use sterile cotton swabs to dry the skull surface.</li>
		<li>Adjust the ear and mouth bars to standardize the head position. 
		NOTE: The vertical coordinates for bregma and lambda need to be identical for anteroposterior positioning.</li>
		<li>Place the needle at the bregma and calculate the coordinates of the desired injection points (the coordinates of interest chosen for this protocol: anterior-posterior (AP): + 0.5 mm, medial-lateral (ML): + 1.5 mm). Move the needle to that point and mark it with ink. 
		NOTE: The coordinates were chosen based on the Franklin and Paxinos Mouse Brain Atlas 16. Distances are mm from the bregma.</li>
		<li>Apply sterile 0.9% NaCl to the skull and drill a hole with a diameter of 2-3 mm through the skull with a surgical, dental drill.</li>
		<li>Move the needle to the surface of the dura and calculate the depth coordinates.</li>
	</ol>
	</li>
	<li>Transplantation procedure
	<ol>
		<li>Resuspend the cells in the tube (step 2.9) and draw 2 &#181;L of cell suspension into a syringe (5 &#181;L or 10 &#181;L). 
		NOTE: Make sure that no air bubbles are present in the cell suspension. The syringe needs to be kept in a horizontal position until mounted into the stereotactic device to avoid cell sedimentation.</li>
		<li>Place the syringe above the target site (calculated coordinates: AP: + 0.5 mm, ML: + 1.5 mm) and slowly move the needle to the surface of the dura. 
		NOTE: If unsure about the correct coordinates, perform injections with a dye and histological evaluation of the injection site before transplanting cells (for more details, see 17).</li>
		<li>Guide the needle at a rate of 0.02 mm/s into the brain up to the proper depth (the coordinate chosen for this protocol is dorsal-ventral (DV) - 0.8 mm). Overshoot the depth by 0.1 mm and withdraw the needle over the same distance to create a pocket for the injected cells.</li>
		<li>Apply tissue adhesive around the needle using forceps to prevent leakage of cells.</li>
		<li>Inject 2 &#181;L of the prepared cell suspension at a constant rate of 3-5 nL/s. 
		NOTE: The injection procedure will last between 7 and 12 min.</li>
		<li>Following injection, leave the needle in place for at least 5 min before slowly withdrawing it. Apply tissue adhesive to seal the hole in the skull and wait for another 2 min.</li>
	</ol>
	</li>
	<li>Sutures and post-care
	<ol>
		<li>Apply sterile 0.9% NaCl solution to the exposed skull to avoid dehydration.</li>
		<li>Close up the wound with a 5/0 silk suture thread.</li>
		<li>Hydrate the animal with 0.5 mL of ringer lactate solution subcutaneously injected in the lower back.</li>
		<li>Interrupt anesthesia delivery and carefully remove the mouse from the stereotaxic apparatus and place it back in a cage kept on a heating pad.</li>
		<li>Monitor the animals during the acute phase postinjury. Check the suture, the animal weight, and overall health at least twice a day.</li>
	</ol>
	</li>
</ol>
4. In vivo imaging
<ol>
	<li>Preparation of luciferin
	<ol>
		<li>Thaw D-luciferin potassium salt at RT and prepare a fresh stock solution of D-luciferin at 30 mg/mL in PBS.</li>
		<li>Sterilize the stock solution through a 0.22 &#181;m syringe filter. 
		NOTE: Immediate use of the working solution is recommended. If necessary, dissolved luciferin can be stored at -20 &#176;C. However, prolonged storage may result in the degradation of signal. Luciferin is a light-sensitive reagent; keep it out of direct light whenever possible. Alternative substrates may also be considered, e.g., cycluc, to improve the resolution limit18.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Initial setup 
		NOTE: Bioluminescence imaging was performed using an in vivo imaging system (see the Table of Materials) consisting of a dark chamber and a cooled charge-coupled device (CCD) camera.
		<ol>
			<li>Double-click the Living Image software icon and select a user ID from the drop-down list.</li>
			<li>Click Initialize in the control panel that appears. Once the initialization process is completed, the temperature box in the control panel will turn green.</li>
			<li>In the control panel, check the Luminescent and Photograph boxes and select Auto exposure (~60 s). Select a field of view (D/12.5cm was chosen for this protocol). Enter the subject height (1.5 cm) and select the use subject height focus option. Manually set the following parameters: large binning, f/2, blocked excitation filter, and open emission filter.</li>
		</ol>
		</li>
		<li>Determine the injection amount of D-luciferin at 300 mg/kg body weight.NOTE: The standard recommended dose is 150 mg/kg of D-luciferin. This procedure was adjusted according to a protocol reporting higher sensitivity using 300 mg/kg18.</li>
		<li>Inject the luciferin intraperitoneally (i.p.). 
		&#8203;NOTE: If the animal needs to be sedated before injection, be aware that it may extend the peak luciferase expression time.</li>
		<li>Wait for 5 min, then anesthetize animals with a continuous supply of isoflurane (4.5% in the induction phase and 1.5-2% in the maintenance phase during imaging procedure).</li>
		<li>Apply ophthalmic lubricant to the eyes, then shave the sedated animals on the head region using a conventional hair shaver. Place the animals in the imaging chamber and start imaging 15 min after the luciferin injection by clicking Acquire in the control panel.</li>
	</ol>
	</li>
</ol>
5. Perfusion
<ol>
	<li>Anesthetize the animals by an i.p. injection of sodium pentobarbital (150 mg/kg body weight). Wait until the mouse no longer responds to painful stimuli, such as toe pinches.</li>
	<li>Lay the mouse on its back and use tweezers and scissors to open the chest cavity.</li>
	<li>Use standard scissors to open the diaphragm.</li>
	<li>Expose the heart and insert a needle (from the tubing with ringer/4% paraformaldehyde solution (PFA)) into the apex of the left ventricle. 
	NOTE: Be careful to keep the tip of the needle in the lumen of the ventricle.</li>
	<li>Cut the right ventricle using scissors.</li>
	<li>Perfuse with ringer solution (can be kept at RT) for 3-4 min (flow rate: 17 mL/min). Continue until the heart is clean.</li>
	<li>Switch the stopcock to allow for the flow of PFA (store at 4 &#176;C; keep on ice during the procedure) and perfuse for another 5 min (~100 mL).</li>
	<li>Stop the pump and remove the needle from the left ventricle. 
	&#8203;NOTE: The perfusion with PFA preserves tissue integrity uniformly. It also facilitates the preservation of GFP signal in the transplants that otherwise may be lost due to diffusion.</li>
</ol>
6. Processing
<ol>
	<li>Tissue collection
	<ol>
		<li>Remove the head using standard scissors and make a midline incision in the skin.</li>
		<li>Turn the skin over the eyes to expose the skull.</li>
		<li>Start from the caudal part at the point of the parietal bone and make a small incision using spring scissors. Advance the scissors rostrally along the midsagittal suture up to a point between the eyes. Start again from the caudal part and make two cuts parallel and ~4 mm apart in the sagittal plane. 
		NOTE: Be careful not to damage the brain by pressing the scissors against the interior surface of the skull.</li>
		<li>Use forceps to carefully tilt one side of the parietal bone and break it off. Do the same with the other side. 
		NOTE: Use a microspatula to free the bone from the meninges; otherwise, they may damage the brain while breaking off the skull. If parts of the frontal bone remain, make a small cut to tilt and break off the bone plate.</li>
		<li>To release the brain, carefully slide the microspatula under the brain (olfactory bulbs) and tilt it gently upward.</li>
		<li>After collecting, keep the brain in 4% PFA solution for 4-6 h at 4 &#176;C. Transfer it to sterile 1x PBS afterward.</li>
	</ol>
	</li>
	<li>Immunohistochemistry
	<ol>
		<li>Transfer the brain to a 30% sucrose solution for at least 48 h at 4 &#176;C to prevent the formation of crystals during freezing.</li>
		<li>Use a sliding microtome to cut coronal sections with a thickness of 40 &#181;m. Collect and store the sections as free-floating sections (in a 24-well-plate) in a cryoprotectant solution (Table 1) at -20 &#176;C until further processing.</li>
		<li>Rinse the sections with 450 &#181;L of 1x PBS for each well (3 times, 5 min each, RT).</li>
		<li>Block non-specific sites with 450 &#181;L of blocking solution for each well (Table 1) for 1 h at RT.</li>
		<li>Incubate each well with 450 &#181;L of primary antibodies at 4 &#176;C overnight. Dilute the antibodies 1:200 in 3% donkey serum; 0.1% Triton-X-100 in PBS. To identify donor material in the host environment, use an antibody to human-specific nuclei (Anti-Human Nuclei Antibody, clone 235-1).</li>
		<li>Wash the sections with 450 &#181;L of 1x PBS for each well (3 times, 5 min each, RT).</li>
		<li>Incubate each well with 450 &#181;L of corresponding fluorescent secondary antibodies for 2-3 h (RT). Dilute the antibodies in 3% donkey serum; 0.1% Triton-X-100 in 1x PBS.</li>
		<li>Wash the sections with 450 &#181;L of 1x PBS for each well (3 times, 5 min each, RT).</li>
		<li>Stain the nuclei with 450 &#181;L of 0.1 &#181;g/mL 4&#39;,6-diamidino-2-phenylindole (DAPI).</li>
	</ol>
	</li>
</ol>

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T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-function+injectable+angiogenic+biomaterial+for+the+repair+of+brain+tissue+following+stroke.">Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke.</a> Nature Materials. 17 (7), 642 (2018).</li><li>Montagne, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+degeneration+causes+white+matter+dysfunction+in+the+mouse+central+nervous+system.">Pericyte degeneration causes white matter dysfunction in the mouse central nervous system.</a> Nature Medicine. 24 (3), 326-337 (2018).</li><li>Montagne, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APOE4+leads+to+blood-brain+barrier+dysfunction+predicting+cognitive+decline.">APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline.</a> Nature. 581 (7806), 71-76 (2020).</li><li>George, P. M., Steinberg, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+stroke+therapeutics:+unraveling+stroke+pathophysiology+and+its+impact+on+clinical+treatments.">Novel stroke therapeutics: unraveling stroke pathophysiology and its impact on clinical treatments.</a> Neuron. 87 (2), 297-309 (2015).</li><li>Weber, R. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+learning+based+behavioral+profiling+of+rodent+stroke+recovery.">Deep learning based behavioral profiling of rodent stroke recovery.</a> bioRxiv. , (2021).</li><li>Nair, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+for+humanised+mouse+models+in+precision+medicine+for+neurodegenerative+disease.">Uses for humanised mouse models in precision medicine for neurodegenerative disease.</a> Mammalian Genome. 30 (7), 173-191 (2019).</li><li>Kirabali, T., Rust, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS-derived+pericytes+for+neurovascular+regeneration.">iPS-derived pericytes for neurovascular regeneration.</a> European Journal of Clinical Investigation. 51 (9), 13601 (2021).</li><li>Weber, R. Z., Perron, P., Rust, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+for+brain+repair:+More+than+just+a+neuron's+sidekick.">Astrocytes for brain repair: More than just a neuron's sidekick.</a> Brain Pathology. 31 (5), 12999 (2021).</li><li>Johansen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Tet-on+system+to+avoid+transgene+down-regulation+in+ex+vivo+gene+transfer+to+the+CNS.">Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS.</a> Gene Therapy. 9 (19), 1291-1301 (2002).</li><li>Vogel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+graft+size+and+not+the+innate+immune+response+limit+survival+of+engrafted+neural+stem+cells.">Initial graft size and not the innate immune response limit survival of engrafted neural stem cells.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (3), 784-793 (2018).</li></ol>

The authors have no potential conflicts of interest to declare.

Regenerating the injured brain to allow for functional recovery remains an unmet challenge. Many innovative preclinical approaches have evolved targeting, for example, immune modulation19,20, angiogenesis1,21,22,23, blood-brain-barrier integrity2,3,24,25, and cell replacement5,26. Especially in recent years, cell-based therapies have emerged as a promising treatment strategy for the brain due to major advancements in stem cell technology and efficient differentiation protocols15,28. This paper provides a valuable protocol for transplanting and tracking neural cells in the mouse brain. The method is applicable for all transducable cell lines for in vivo applications in the mouse brain.
The presented setup uses transplants of human origin in a mouse. These transplants are not viable in the long-term in immunocompetent wild-type mice due to immunogenicity. Hence, immunodeficient NSG mice were used to overcome this limitation. Alternatively, the use of mouse transplants may be preferred to overcome the immunogenic aspects. If transplantation of human cells is required, humanized mouse models represent an emerging alternative to reduce the probability of graft rejection29.
A commercial dual-reporter viral vector consisting of firefly luciferase and eGFP under the EF1&#945; promotor was used to visualize the transplants. This promotor was selected to achieve a high signal intensity15. However, apart from NPCs, other cell types have been shown to promote brain function after injury, including pericytes30&#160;and astrocytes31; hence, depending on the cell line used, other promotors might be more suitable to achieve high expression levels. Additionally, the use of transgene promoters, such as CMV, may lead to downregulation, especially in long-term experiments32. The transduction efficiency of the lentiviral vector strongly depends on the used cell line and may vary between single experiments. Therefore, transduction efficiency must be evaluated before starting the in vivo experiments and to correct variations in transduction efficacy between experiments. The brain region of transplantation also influences the signal strength. Although a detection limit of &#60;6,000 cells was achieved for cortical transplantations, it may require more cells to detect a signal in deeper brain regions, for example, striatum or hippocampus.
Transplantation volumes in the mouse brain are limited to 1-2 &#181;L. Therefore, it is important to identify a suitable cell number for the experiments. It has been previously observed that increasing cell numbers leads to decreased survival rate, most likely due to limited availability of nutrients and oxygen in the region of transplantation33. In vivo bioluminescence imaging provides a relatively low spatial resolution compared to other in vivo imaging methods such as MRI or CT. Therefore, short migratory paths of grafted cells can only reliably be assessed in the subsequent post-hoc analysis.
The absolute signal strength of the bioluminescence is generally proportional to the transplanted cell number. However, the signal strength might be reduced if grafts are transplanted in deeper brain structures or if the signal strength is outside the linear detection spectrum of the in vivo imaging system. Currently, novel substrates are developed to ensure more efficient penetration across the blood-brain barrier than D-luciferin, including cycluc1. These substrates may further improve the detection limit of the grafted cells in the future18. Overall, this protocol allows a straightforward, minimally invasive procedure to transplant and observe grafts in the mouse brain.

The mammalian brain has limited regenerative capacities following injury or disease, requiring innovative strategies to promote tissue and functional repair. Preclinical strategies focus on different aspects of brain regeneration, including neuroprotection, neurogenesis, angiogenesis1,2, blood-brain-barrier repair3,4, or cell therapy5,6. Cell therapy has the advantage of being able to promote many of these pro-repair processes simultaneously. In experiments with transplantation of cells, tissue repair has occurred through (1) direct cell replacement and (2) production of cytokines leading to angiogenesis and neurogenesis7. Recent advancements in stem cell technology have further facilitated the development of scalable, well-characterized neural cell sources that are now in the pipeline for clinical trials (reviewed in 7,8,9). Although cell therapies have reached the clinical stage for a few neurological diseases (e.g., Parkinson&#39;s disease10, stroke11, and spinal cord injury12), their efficacy has been variable, and more preclinical research is needed to understand the mechanisms of graft-host interactions.
One major limitation of many preclinical studies is the continuous tracking of the transplanted cells inside the host. Often only end-point measurements are performed, omitting the dynamic migratory and survival processes in the host6,13. These limitations result in the poor characterization of the grafted cells and require high animal numbers to comprehend longitudinal changes. To overcome these limitations, in this study, we transduce induced pluripotent stem cell (iPSC)-derived neural progenitor cells with a commercially available dual-reporter lentiviral vector consisting of red firefly luciferase and enhanced green fluorescent protein (rFluc-eGFP). These cells are transplanted via stereotaxic intraparenchymal injection into the mouse brain and are longitudinally tracked using in vivo bioluminescence imaging over 5 weeks. After brain tissue collection, the GFP-expressing grafted cells are identified and further characterized in histological brain sections. This method can be smoothly adapted to alternative transducable cell sources and routes of transplantation for in vivo applications in the rodent brain. Overall, the procedure is valuable to obtain longitudinal information of graft survival and migration in the mouse brain and facilitates subsequent histological characterization.

<table><tbody><tr><td>&lt;strong&gt;Viral Transduction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pLL-EF1a-rFLuc-T2A-GFP-mPGK-Puro (Lenti-Labeler virus)</td><td>Systembio</td><td>LL410VA-1</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eppendorf microtubes; 1.5 mL</td><td>Sigma Aldrich</td><td>Z606340</td><td></td></tr><tr><td>Falcon Tubes; 15 mL</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>Microscope cover slips</td><td></td><td></td><td>Product of choice</td></tr><tr><td>Microscope slides</td><td></td><td></td><td>Product of choice</td></tr><tr><td>Sterlie cotton swabs</td><td></td><td></td><td>Product of choice</td></tr><tr><td>Sutures; 5/0 silk with curved needle</td><td>B. Braun</td><td>G0762482</td><td></td></tr><tr><td>Syringe filter; 0.22 &amp;micro;m</td><td>TPP</td><td>99722</td><td></td></tr><tr><td>Syringe; 1 mL and 0.5 mL</td><td>B. Braun</td><td>9166017V</td><td></td></tr><tr><td>Tissue culture plate (24-well)</td><td>TPP</td><td>92024</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Automated cell counter (Vi-CELL XR)</td><td>Beckmann Coulter Life Science</td><td>383721</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11064-07</td><td></td></tr><tr><td>Forceps, fine</td><td>Fine Science Tools</td><td>11412-11</td><td></td></tr><tr><td>Heating pad</td><td></td><td></td><td>Product of choice</td></tr><tr><td>High Speed Brushless Micromotor Kit</td><td>Foredom</td><td>K.1060-22.</td><td></td></tr><tr><td>Ideal Micro Drill Burr Set Of 5</td><td>Cell Point Specific</td><td>60-1000</td><td></td></tr><tr><td>&lt;em&gt;In-Vivo&lt;/em&gt; imaging system (IVIS Lumina III with Living Imaging 4.2 software package)</td><td>Perkin Elmer</td><td>CLS136334</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>Provet AG</td><td>330724</td><td></td></tr><tr><td>Microinjection Syringe Pump system</td><td>World Precision Instruments</td><td>UMP3T-1</td><td></td></tr><tr><td>Microliter syringe; 700-Series; Volume: 5-10 &amp;micro;L</td><td>Hamilton</td><td>7635-01</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>HM430</td><td></td></tr><tr><td>NanoFill-33 G-Needle (removable and reusable)</td><td>World Precision Instruments</td><td>NF33BV-2</td><td></td></tr><tr><td>Needle Holder</td><td>Fine Science Tools</td><td>12001-13</td><td></td></tr><tr><td>Perfusion pump and tubing</td><td>Masterflex</td><td>HV-77120-42</td><td></td></tr><tr><td>Scalpel</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Small bonn scissors, straight</td><td>Fine Science Tools</td><td>14184-09</td><td></td></tr><tr><td>Small spring scissors, straight</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>Spatula</td><td>Merck</td><td>Z243213-2EA</td><td></td></tr><tr><td>Stereotaxic frame for rodents; motorized</td><td>World Precision Instruments</td><td>99401</td><td></td></tr><tr><td>&lt;strong&gt;Pharmaceuticals and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>A11105-01</td><td>Proteolytic and collagenolytic; cell dissociation reagent</td></tr><tr><td>Anti-Human Nuclei Antibody, clone 235-1, Biotin Conjugate</td><td>Merck</td><td>MAB1281B</td><td></td></tr><tr><td>B27 &amp;ndash; Supplement (50x)</td><td>Gibco</td><td>17504-001</td><td></td></tr><tr><td>Betadine (11 mg Iod als Povidon-Iod pro 1 ml L&amp;ouml;sung)</td><td>Mundipharma Medical Company</td><td>&lt;em&gt;All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland&lt;/em&gt;</td><td></td></tr><tr><td>Blocking solution (3% donkey serum; 0.1% Triton-X-100 in PBS)</td><td></td><td></td><td>Product of choice; can be homemade</td></tr><tr><td>CHIR99021 (10 mM &amp;ndash; 2,500x)</td><td>StemMACS</td><td>130-103-926</td><td></td></tr><tr><td>Cryoprotectant solution</td><td></td><td></td><td>Product of choice; can be homemade</td></tr><tr><td>DAPI solution (1 mg/mL)</td><td>Thermo Fisher Scientific</td><td>62248</td><td></td></tr><tr><td>D-Luciferin Potassium Salt</td><td>Perkin Elmer</td><td>122799</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320-074</td><td></td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td><td>Invitrogen</td><td>A-31570</td><td></td></tr><tr><td>Donkey serum</td><td></td><td></td><td>Product of choice</td></tr><tr><td>Esconarcon (Pentobarbitalum natricum 300 mg)</td><td>Streuli Pharma AG</td><td>&lt;em&gt;All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland&lt;/em&gt;</td><td></td></tr><tr><td>Ethanol; 70%</td><td></td><td></td><td>Product of choice</td></tr><tr><td>FGF Basic recombinant human protein, Animal-origin free</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;PHG6015</td><td></td></tr><tr><td>Glutamax (100x)</td><td>Gibco</td><td>A12860-01</td><td></td></tr><tr><td>hLif (10 &amp;micro;g/mL &amp;ndash; 1,000x)</td><td>PeproTech</td><td>AF-300-05-25UG</td><td></td></tr><tr><td>Isoflurane (Isofluran (1-Chlor-2,2,2-trifluorethyl-difluoromethyl&amp;shy;ether) 99.9%)</td><td>Provet AG</td><td>&lt;em&gt;All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland&lt;/em&gt;</td><td></td></tr><tr><td>Laminin-L521 (L-521)</td><td>Biolaminin LN</td><td>LN521</td><td></td></tr><tr><td>Lidocaine ointment (Lidocain: 25 mg , Prilocain: 25 mg)</td><td>Aspen Pharma Schweiz GmbH</td><td>&lt;em&gt;All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland&lt;/em&gt;</td><td></td></tr><tr><td>Mounting Medium</td><td></td><td></td><td>Product of choice; can be homemade</td></tr><tr><td>N2- Supplement (100x)</td><td>Gibco</td><td>&amp;nbsp;75202-001</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>&amp;nbsp;21103-049</td><td></td></tr><tr><td>Ophtalmic lubricant (Retinol palmitat: 15,000 UI)</td><td>Bausch &amp;amp; Lomb Swiss AG</td><td>&lt;em&gt;All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland&lt;/em&gt;</td><td></td></tr><tr><td>Paraformaldehyde solution</td><td></td><td></td><td>Product of choice</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>Can also be homemade</td></tr><tr><td>Poly-L-ornithine Solution (pLO)</td><td>Sigma-Aldrich</td><td>P4957</td><td></td></tr><tr><td>Rimadyl (Carprofen 50 mg)</td><td>Zoetis Schweiz GmbH</td><td>&lt;em&gt;All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland&lt;/em&gt;</td><td></td></tr><tr><td>Ringer lactate</td><td>B. Braun</td><td>3570500</td><td></td></tr><tr><td>Ringer solution</td><td>B. Braun</td><td>3570030</td><td></td></tr><tr><td>Saline (0.9% NaCl)</td><td>B. Braun</td><td>3570160</td><td></td></tr><tr><td>SB431542 (10 mM &amp;ndash; 3,333.3x)</td><td>StemMACS</td><td>130-106-543</td><td></td></tr><tr><td>Tissue Adhesive (Histoacryl)</td><td>B. Braun</td><td>1050060</td><td></td></tr></tbody></table>

intracerebral transplantation and in vivo bioluminescence tracking of human neural progenitor cells in the mouse brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

We aim to longitudinally track transplanted neural progenitor cells in the mouse brain using in vivo bioluminescence imaging and identify the transplanted cells in subsequent histological analysis (Figure 1A). Therefore, neural progenitor cells are transduced with a lentiviral vector consisting of EF1&#945;-rFluc-eGFP. Before transplantation, cells were tested for successful transduction by expression of eGFP in vitro (Figure 1B). The successfully transduced cells were stereotactically transplanted in the mouse brain at the desired coordinates (e.g., in the sensorimotor cortex). Following transplantation, the mice were systemically injected with D-luciferin, the substrate for rFluc, and signal intensities of the transplanted cells were measured to confirm successful transplantation (Figure 1C).
To evaluate the detection limit of the in vivo bioluminescence imaging, a range of 6,000-180,000 cells was transplanted in the right sensorimotor cortex of the mouse (Figure 2A). We detected &#60;6,000 cells and a bioluminescence signal proportional to the transplanted cell count directly after transplantation (Figure 2B). As human cell sources are immunogenic to immunocompetent mice, NOD scid gamma (NSG)&#160;immunodeficient mice were used to observe the long-term survival of the cell grafts. Long-term survival and detection of a bioluminescence signal for up to 5 weeks were confirmed after cell transplantation (Figure 2C,D). The transplanted cells were successfully detected ex vivo in a subsequent histological analysis through the eGFP reporter and immunostaining with anti-human nuclei and anti-human mitochondrial antibodies (Figure 2E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63102/63102fig01.jpg" /> 
Figure 1: Transplantation of neural progenitor cells. (A) Schematic overview of generation and transplantation of rFluc-eGFP NPCs. (B) Representative immunofluorescence image of transduced NPCs (GFP reporter, green) counterstained with DAPI (blue); scale bars = 5 &#181;m. (C) In vivo detection of bioluminescence signal in transplanted cells; color bar = blue (0, min, no signal), red (4 flux, p/s &#215; 105, max signal) Abbreviations: NPCs = neural progenitor cells; GFP = green fluorescent protein; rFluc-eGFP = red firefly luciferase and enhanced green fluorescent protein; DAPI = 4&#39;,6-diamidino-2-phenylindole; p/s = photons/s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63102/63102fig01large.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/63102/63102fig2v2.jpg" /> 
Figure 2: Time course of transplanted cells. (A) Schematic view of cell numbers for transplantation. (B) Detection limit of transplanted cells 1 h after transplantation. (C, D) Time course of transplantation (180,000 cells) for up to 35 days in NSG mice ; color bar = blue (0, min, no signal), red (4 flux, p/s &#215; 105, max signal) Data are mean &#177; SEM (n = 3). (E) Representative fluorescence images of histological sections and transplanted cells 5 weeks following transplantation. Scale bar = 10 &#181;m. Abbreviations: D = day after transplantation; NSG = immunodeficient NOD scid gamma; DAPI = 4&#39;,6-diamidino-2-phenylindole; GFP = green fluorescent protein; HuNu = Anti-Human Nuclei Antibody, clone 235-1; p/s = photons/s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63102/63102fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain at JoVE.com

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

intracerebral-transplantation-vivo-bioluminescence-tracking-human

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.0K Views.  University of Zurich. Celgebaseerde therapie heeft een enorm potentieel voor hersenregeneratie. Het protocol dat we hier demonstreren is waardevol, omdat het longitudinale in vivo beeldvorming van getransplanteerde cellen in het muizenbrein mogelijk maakt. Dit protocol is vooral handig om inzicht te krijgen in de migratie en de overleving van het transplantaat in hetzelfde dier over een bepaalde periode.Deze procedure kan worden toegepast op elke luciferase-expresserende cellijn die naar de hersenen van de ...

Video: Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Medicine

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Biology

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetics

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, ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

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 ...

A Human Cerebral Organoid Model of Neural Cell Transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. 
We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

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