Transplanting iPSC Derived Neural Stem Cells into the Cerebral Cortex of Neonatal Rats

Cell transplantation is a frequently seen technique in neuroscience research and several approaches and methods can be utilized according to research interests. Variations in the survival and integration of grafted cells are problematic in transplantation research.

The immune response to any kind of transplant is a critical consideration, as such, many investigators treat transplanted animals with immunosuppressant drugs or employ an immunocompromised host for example nude rats to reduce the chances of immune-mediated cell death of the grafted cells.

In the event that transplantation into an adult host is not required, grafting can be carried out in neonatal hosts before the full development of the immune response to avoid immunosuppression. This article outlines how to perform transplantation of Axol Human iPSC-Derived Neural Stem Cells into a neonatal (0 to 2 days old) rat host.

Anticipated Results

  • The cell survival of Axol hNSCs into neonatal hosts is estimated to be high at one month post-transplantation.
  • Survival beyond one month post-transplantation is also estimated, although there could be a slight decrease over time.
  • Implanted cells can be recognized utilizing antibodies specific to humans, such as the human nuclear marker HuNuclei (Millipore cat no: MAB1281), or other markers as outlined in the Axol ImmunoCytoChemistry Application Protocol.

Coronal sections from adult rat cortex six weeks after human iPSC-derived cortical NSCs grafting. Cells immunostained with human-specific NCAM, demonstrating both the injection site within the corpus callosum, but also the varied axonal projections emanating around the injection site. The insert in middle panel shows the central area of the brain where the section is captured.

Materials Required

Culturing and Differentiation of Axol Human iPSC-Derived Neural Stem Cells

Please read the ‘Culture and Differentiation of Axol hNSCs’ instruction manual for a full outline of cell preparation and the materials required.

Please note: Axol hNSCs should be grown in culture for seven to eight days ideally once thawing has taken place for this procedure.

  • D-PBS
  • Conical tube (15 mL)
  • Eppendorf tubes
  • Axol Unlock (ax0044)
  • Axol Neural Maintenance Medium (ax0031a&b)
  • Axol Sure Boost (ax0045)
  • Haemocytometer
  • Tissue culture centrifuge
  • Vertical laminar flow hood certified for Level II handling of biological materials
  • Incubator with humidity and gas control to uphold 37 °C and >90% humidity in an atmosphere of 5% CO2 in air
  • Inverted microscope with 4X, 10X and 20X objectives

Transplantation of Axol Human iPSC-Derived Neural Stem Cells

  • Axol hNSCs resuspended in a concentration of 50,000-100,000 cells per μL
  • Ice bucket filled with wet ice
  • Hamilton syringe with a 30 gauge needle (Custom made. Other sizes may be used, but must be optimized)
  • Black fine-tip marker
  • Timer
  • 26 gauge needle
  • Blue-tack or equivalent material that can be conveniently molded into shape
  • Platform to hold pup for visualization under microscope and injection. (Platforms can be made from two to three 100 mm cell culture dishes filled with ice. Blue tack in the shape of a head holder or stabilizer is put on top of the platform)
  • Transfer cage
  • Recovery cage with heat source (heat blanket can be placed under the cage)
  • Stereotaxic frame with needle or syringe holder
  • Side lamps for surgical illumination (if not provided with microscope)
  • Surgical microscope

Procedures

Collection of Axol hNSCs for Transplantation

Please read the ‘Culture and Differentiation of Axol hNSCs’ instruction manual for a full outline. Modified from Passaging Axol hNSCs.

  1. Remove the complete medium from the culture vessel.
  2. Rinse the surface of the cell layer gently with D-PBS (2 mL D-PBS per 10 cm²) by adding the DPBS to the side of the vessel and rocking back and forth several times.
  3. Add Axol Unlock to detach the cells (1 mL per 10 cm² culture surface area). Incubate the cells for three to five minutes at 37 °C.
  4. After the cells have been detached, gently pipette the cells up and down to break the cell clumps into a single cell suspension.
  5. Halt the dissociation reaction by adding four volumes of Complete Axol Neural Maintenance Medium (for example, if 1 mL Axol Neural Unlock is utilized, then add 4 mL medium to halt the enzymatic action).
  6. Move the cells to a 15 mL conical tube and centrifuge the tube at 200g for five minutes at room temperature. Discard the supernatant.
  7. Resuspend the cells in 1 mL Complete Axol Neural Maintenance Medium and count cells using a haemocytometer. Once the cells have been counted, centrifuge the tube at 200g for five minutes at room temperature.
  8. Resuspend the cells in the desired amount of Complete Axol Neural Maintenance Medium supplemented with 1000X Axol Sure Boost stock solution. For example, 1 μL Axol Sure Boost per 1 mL medium. Cells should be held at a warm temperature, at 37°C if possible, or room temperature for smaller periods of time (two to three hours maximum) is also adequate.

Note: Optimal concentration for cell transplantation is 50,000 - 100,000 cells/μL.

Neonatal Transplantation Procedure

  1. The entire litter should be removed from the mother’s home cage and placed into a transfer cage. Take some of the bedding from the home cage and put into the transfer cage to maintain a similar smell and surrounding for the pups. This will stop the mother from rejecting the pups when they are taken back to the home cage.
  2. Anesthetize the first animal on ice for approximately five minutes. Once the animal is anesthetized, transfer the animal to the platform (containing ice to continue hypothermic anesthesia).

Note: Hypothermia is highly recommended as the process of anesthesia in this protocol. Inhalant anesthesia can be utilized but it is very variable and unreliable at this age due to the neonates’ breathing patterns.

Anesthesia by injection is also not recommended as it has been known to have a high mortality rate at this age. If unfamiliar with these processes, ask local or national regulation authorities and seek veterinary advice. Position the platform within the stereotaxic frame and under the microscope for visualization of the animal’s head.

Note: Animals of this age cannot be held in the frame with adult ear bars so take care to make sure that the platform is secured into place and doesn’t move. Fill Hamilton syringe (with the 30 gauge needle) with the required amount of cells, taking care not to clog the tip of the needle. Change the needle in the needle holder held in the stereotaxic frame.

Note: 50,000 – 100,000 cells per μL in a 30 gauge needle is the smallest size that should be utilized for the best outcome. Find bregma by visualization through the skin (albino rats are the easiest for visualization). Make a tiny mark on bregma with a black marker.

  1. Focus your needle to be centered on the bregma and continue in finding your desired coordinates by manipulating the needle holder to the preferred injection area.
  2. Note: Coordinates will change according to your intended injection site. Refer to appropriate brain atlases or published literature to gain more information. For the presumed sensorimotor cortex of P0 pups, the following coordinates can be utilized for two unilateral injections:- 1.0mm AP, 1.0mm ML, -0.5mm DV; -0.5mm AP, 1.0mm ML, -0.5MM DV. It helps to first insert a 26 gauge needle into the desired injection site before the main injection to open up an appropriate hole in the skull. Once this is complete, the injection needle can be inserted. Inject around 0.5 to 1 μL per site, or less if using many injection sites. Refer to local and national regulations for injection volume limitations.
  3. Carry out manual injections over the course of one minute per injection, followed by a waiting time of two minutes before needle withdrawal. Repeat for desired number of injections.
  4. Note: An injection pump can be utilized if desired, but keep injection times to a minimum as hypothermia anesthesia is used. For lengthier injection times, perhaps utilize inhalation anesthetics. When injections are complete, transport the animal into a warm recovery cage, with bedding from the original cage to make sure home cage smells are maintained and risk of rejection by the mother is minimized.
  5. Repeat this sequence for the entire litter. Once the procedure is running efficiently, consider anaesthetizing further t animals while injecting another animal. Make sure that the timing is recorded so that the original hypothermia doesn’t exceed five to six minutes before the procedure. As a whole, the total anesthesia session should not be longer than 10 to 14 minutes for the best outcome.
  6. It is hard to mark each individual animal to uniquely identify them at this age. It is also best to not do this to avoid disturbance of the pups and prevent rejection by the mother. Tail marking is not beneficial as the mother will clean the animal of all markings within a day. Ear clipping is a possibility, but again is not preferable as this may stress the mother.

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Nov 18, 2019 at 8:08 AM

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