Aug 14, 2023

Public workspacePreparation of human iPSC-derived cortical neuronal progenitors for transplantation into the rodent brain

DOI
dx.doi.org/10.17504/protocols.io.x54v9ppeqg3e/v1
  • 1University of Sydney;
  • 2Florey Institute of Neuroscience and Mental Health
courtney.wright Wright
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9ppeqg3e/v1
Protocol Citation: louise.cottle, Clare Parish 2023. Preparation of human iPSC-derived cortical neuronal progenitors for transplantation into the rodent brain. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9ppeqg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86422
Keywords: ASAPCRN, human iPSC, transplantation, cortical progenitors, neurons, human-to-mouse xenograft
Funders Acknowledgement:
Micheal J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes how we prepare human iPSC-derived cells, that have been differentiated into cortical neuronal progenitors, for transplantation into the brain of immunocompromised athymic mice. Neuronal progenitors mature within the mouse brain and are used to study the pathogenesis of Parkinson’s disease.
Materials
Material input

  • Adult (8-10 week) athymic mice (BALBc/Nu)
  • IPSCs differentiated into cortical progenitors (D25-30 of differentiation depending on desired experimental outcomes), typically grown in 48 well plates. Full details of the cortical differentiation protocol: dx.doi.org/10.17504/protocols.io.bu6znzf6.
Equipment

  • Microscope
  • Microscope (brightfield with Phase contrast)
  • Heamocytometer
  • Pipettes (P20, P200, P1000)
Consumables

  • Falcon tubes
  • PCR tubes
  • pipette tips


Key reagents

  • Reagent1X PBS (Phosphate-buffered saline )
  • ReagentACCUTASE™ 100 mL STEMCELL Technologies Inc.Catalog #7920
  • ReagentRock inhibitor Y-27632 dihydrochlorideTocrisCatalog #125410
  • ReagentTrypan Blue 100 mL STEMCELL Technologies Inc.Catalog #7050
Solutions



Safety warnings
Attention
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Preparation of cell suspension
Preparation of cell suspension
  1. Prepare a Falcon tube with Amount2 mL base cortical media plus Rock inhibitor (Ri, 1:1000 dilution)
  2. Wash cells with Amount300 µL PBS -/- (gently run PBS down the side of each well)
  3. Incubate cells in Amount300 µL Accutase (per well) at Temperature37 °C to lift cells off wells in small clumps
- Monitor the Accutase incubation after Duration00:10:00 ; tap the plate to dislodge cells and look for cells lifting in a sheet before proceeding
- Triturate 5 times to break the cells into clumps
- Incubate clumps for a further Duration00:05:00 at Temperature37 °C
- Triturate 10 times to break the cells into single cells and small clumps

4. Transfer cells from the wells into the 15ml Falcon tube containing media + Ri (Step 1)
5. Rinse with plate with a further Amount300 µL of Accutase to ensure all cells are collected
6. Spin cells at Temperature4 °C , Centrifigation1300 rpm for Duration00:04:00
7. Discard supernatant
8. Resuspend cell pellet in 1-2ml base cortical media and add Ri (1:1000)
9. Count cells
- Take 2 x Amount10 µL aliquot of cells in two separate tubes
Dilute cells 1:1 using Amount10 µL of trypan blue
- Count cells in a Amount10 µL aliquot from each tube using a haemocytometer
- Calculate total number of cells (Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4)

10. Calculate the total volume needed to resuspend cells to a final density of 100 000/uL
11. Spin cells at Temperature4 °C , Centrifigation1300 rpm for Duration00:04:00
12. Whilst cells are spinning, make up Amount1 mL media + Ri which will be used to resuspend the cells for implantation surgery
13. At end of the spin, collect tube and discard supernatant
14. Using a P20, add Amount5 µL and resuspend pellet in base cortical media + Ri
15. Transfer resuspended pellet to PCR tube and precisely measure the volume
16. Add the remaining volume needed for cells to reach a density of 100 000 cells/ul as calculated in step 10.
17. Label the PCR tube containing the cells with details of the cell line and concentration
18. Store cells on ice and transport to animal surgical room.

23m
Transplantation
Transplantation
  1. Athymic mice undergo stereotactic surgery to receive a unilateral graft of 100,000 cortical progenitors in Amount1 µL volume. The surgical coordinates from Bregma are AP: +1, ML: +/-1.5, DV: -1.5
For full details of procedures for implantation of cell suspension can be found at https://www.protocols.io/view/transplantation-of-fetal-midbrain-dopamine-progeni-261ge4jq7v47/v1
Protocol references
Cortical differentiation protocol: https://dx.doi.org/10.17504/protocols.io.bu6znzf6