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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S., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+promotes+systemic+endothelial+inflammation+and+atherosclerosis.">Stroke promotes systemic endothelial inflammation and atherosclerosis.</a> Trends in Molecular Medicine. 24 (7), 593-595 (2018).</li><li>Rust, R., Gantner, C., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-+and+antiangiogenic+therapies:+current+status+and+clinical+implications.">Pro- and antiangiogenic therapies: current status and clinical implications.</a> FASEB Journal. 33 (1), 34-48 (2018).</li><li>Rust, R., Gr&#246;nnert, L., Weber, R. Z., Mulders, G., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refueling+the+ischemic+CNS:+guidance+molecules+for+vascular+repair.">Refueling the ischemic CNS: guidance molecules for vascular repair.</a> Trends in Neurosciences. 42 (9), 644-656 (2019).</li><li>Nih, L. R., Gojgini, S., Carmichael, S. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Viral Transduction</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>Consumables</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 &#181;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>Equipment</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>In-Vivo 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 &#181;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>Pharmaceuticals and Reagents</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 &#8211; 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&#246;sung)</td>
			<td>Mundipharma Medical Company</td>
			<td>All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland</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 &#8211; 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>All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland</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>&#160;PHG6015</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (100x)</td>
			<td>Gibco</td>
			<td>A12860-01</td>
			<td></td>
		</tr>
		<tr>
			<td>hLif (10 &#181;g/mL &#8211; 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&#173;ether) 99.9%)</td>
			<td>Provet AG</td>
			<td>All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland</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>All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland</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>&#160;75202-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>&#160;21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophtalmic lubricant (Retinol palmitat: 15,000 UI)</td>
			<td>Bausch &#38; Lomb Swiss AG</td>
			<td>All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland</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>All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland</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 &#8211; 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/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/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 ...

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2.9K Views.  University of Zurich. 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. 

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

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...