Transplantation of Human Neural Progenitor Cells in the Mouse Brain

Abstract

Source: Weber, R. Z., et al. Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain. J. Vis. Exp. (2022)

This video demonstrates the stereotaxic transplantation of genetically modified human neural progenitor cells (NPCs) into the mouse brain. Researchers inject NPCs expressing luciferase and fluorescent proteins, enabling in vivo bioluminescence imaging and post-mortem fluorescence microscopy to study cell survival, migration, and integration in neurological research.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Transplantation procedure

1. Preparation for surgery
   1. Clean and sterilize the surgery equipment.
   2. Prepare the stereotaxic device and the microinjection pump system.
      NOTE: It is critical to test the Hamilton syringe and the 30 G, 2-inch needle before starting. Insert the needle into a tube containing sterile 0.9% NaCl (sodium chloride) and slowly draw the solution in and out.
   3. Set up the anesthesia machine. Test the machine before involving any animals. Clean the induction chamber with 70% ethanol.
2. Preparation of animals
   1. 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/IL2rγnull (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.
   2. Measure the mouse's body weight and adjust the dose of the painkiller to be injected. Administer carprofen (5 mg/kg body weight) intraperitoneally to reduce pain and/or prevent an inflammatory response.
   3. 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.
   4. 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.
   5. Apply ophthalmic lubricant to prevent the eyes from drying out.
   6. Shave the mouse's scalp with an electric razor and disinfect the skin with 5% betadine solution using cotton swabs.
   7. 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'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.
3. Craniotomy
   1. 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.
   2. Retract the periosteum and fascia with a scalpel and use sterile cotton swabs to dry the skull surface.
   3. 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.
   4. 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. Distances are mm from the bregma.
   5. 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.
   6. Move the needle to the surface of the dura and calculate the depth coordinates.
4. Transplantation procedure
   1. Resuspend the viral transduced human neural progenitor cells in the tube and draw 2 µL of cell suspension into a syringe (5 µL or 10 µL).
      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.
   2. 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.
   3. 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.
   4. Apply tissue adhesive around the needle using forceps to prevent leakage of cells.
   5. Inject 2 µL 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.
   6. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps	Fine Science Tools	11064-07
Forceps, fine	Fine Science Tools	11412-11
Heating pad			Product of choice
High Speed Brushless Micromotor Kit	Foredom	K.1060-22.
Ideal Micro Drill Burr Set Of 5	Cell Point Specific	60-1000
Isoflurane vaporizer	Provet AG	330724
Microinjection Syringe Pump system	World Precision Instruments	UMP3T-1
Microliter syringe; 700-Series; Volume: 5-10 µL	Hamilton	7635-01
Microtome	Leica	HM430
NanoFill-33 G-Needle (removable and reusable)	World Precision Instruments	NF33BV-2
Needle Holder	Fine Science Tools	12001-13
Perfusion pump and tubing	Masterflex	HV-77120-42
Scalpel	Fine Science Tools	10003-12
Small bonn scissors, straight	Fine Science Tools	14184-09
Small spring scissors, straight	Fine Science Tools	15000-03
Stereotaxic frame for rodents; motorized	World Precision Instruments	99401
Ophtalmic lubricant (Retinol palmitat: 15,000 UI)	Bausch & Lomb Swiss AG	All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland
PBS	Thermo Fisher Scientific	10010023	Can also be homemade
Poly-L-ornithine Solution (pLO)	Sigma-Aldrich	P4957
Rimadyl (Carprofen 50 mg)	Zoetis Schweiz GmbH	All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland
Saline (0.9% NaCl)	B. Braun	3570160
Tissue Adhesive (Histoacryl)	B. Braun	1050060