Jun 17, 2025

Public workspaceDifferentiation of hiPSC-derived microglia-like cells

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpdrp5gzp/v1
  • Alexis Penverne1,
  • James Evans2
  • 1Gandhi Lab;
  • 2University College London, University of London
  • Alexis Penverne: University College London, University of London;
Alexis Penverne
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpdrp5gzp/v1
Protocol Citation: Alexis Penverne, James Evans 2025. Differentiation of hiPSC-derived microglia-like cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpdrp5gzp/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: April 03, 2025
Last Modified: June 17, 2025
Protocol Integer ID: 126085
Keywords: like cells from hipsc, differentiation of hipsc, like cells protocol for generation, hipsc, like cells protocol, derived microglia, microglia
Abstract
Protocol for generation of microglia-like cells from hiPSCs, based on protocol from Garcia-Reitboeck, Pablo et al., 2018.
Troubleshooting
Day 0 - Embryoid Body generation
8m
hiPSCs are maintained on Geltrex (ThermoFisher) in mTeSR (STEMCELL), and passaged using Concentration0.5 Mass Percent EDTA.

Once hiPSCs are healthy and ~80% confluent, incubate with Amount1 mL Accutase (STEMCELL) for Duration00:05:00 at Temperature37 °C Dissociate using Amount1 mL of PBS and resuspend in a Falcon tube in Amount5 mL of PBS.

5m
Centrifuge at 300xg for Duration00:03:00 and aspirate supernatant. Resuspend pellet in Amount1 mL of Embryoid Medium (Amount1 mL of media per well of hiPSC initially dissociated).


Note
How to prepare Embryoid Medium:
  • mTeSR (STEMCELL)
  • Concentration10 micromolar (µM) Rock Inhibitors (Tocris)
  • 50 ng/mL BMP-4 (Peprotech)
  • 50 ng/mL VEGF (Peprotech)
  • 20ng/mL SCF (Peprotech)




Count cells and dilute in Embryoid medium to have suspension at 100,000 cells/mL.
Add Amount100 µL of Embryoid Medium to each well of a low-attachment U-bottom 96-well plate.

Using a multi-channel pipette, add 100ul of cell suspension in each well of the 96-well plate.

Note
A full-well of embryoid bodies is preferable to ensure sufficient quantity of microglia-like cells downstream, therefore ~1 million hiPSCs are needed per induction.
Centrifuge the plate at 800rpm for Duration00:03:00 and transfer to Temperature37 °C 5% CO2 incubator.

3m
After Duration48:00:00 , supplement each well with Amount50 µL of fresh Embryoid media.

Day 4 - Embryoid Body Suspension and Myeloid differentiation
On day 4 after the start of the induction, collect every single Embryoid Body from each well and transfer in a single 15mL Falcon using a P1000 pipette and wide-bore tips.
Let EBs settle naturally at the bottom of the tube and aspirate Embryoid media, and Add 5mL of Myeloid Differentiation media.

Note
How to prepare Myedloid Differentiation Medium:
  • X-Vivo 15 (Lonza)
  • Concentration1 % volume Glutamax (ThermoFisher)
  • Concentration0.5 % volume Penicilin-Streptomycin (ThermoFisher)
  • Concentration50 micromolar (µM) 2-Mercaptoethanol (ThermoFisher)
  • 100ng/mL MSCF (Peprotech)
  • 25ng/mL IL3 (Peprotech)





Add Amount10 mL of Myeloid differentiation media to a T75 flask, and transfer the Amount5 mL with the EBs inside the flask for a total of Amount15 mL of media.

For the following 2 weeks (until day 18), change media with fresh Myeloid Differentiation media. Change by removing ~Amount10 mL of media from the flask and replacing it with fresh media. Be careful to move the plate gently: the EBs need to stick to the flask as this will improve the microglia-like cells yield.

Day 18 onward - Microglia Harvest
Starting on day 18 microglia-like cells can be harvested weekly by taking 10-12mL of media from T75 flask inside a 15mL Falcon. Replace with fresh myeloid differentiation media in flask.
Centrifuge harvested cells in media at 300xg for Duration00:03:00 . Resuspend pellet in Amount1 mL of Maturation Medium.


Note
How to prepare Maturation Medium:
  • X-Vivo 15 (Lonza)
  • Concentration1 % volume Glutamax (ThermoFisher)
  • Concentration0.5 % volume Penicilin-Streptomycin (ThermoFisher)
  • 100ng/mL MSCF (Peprotech)




Filter resuspended cells through a 40-micron filter, and count. Dilute to a concentration of ~300,000 cells/mL in Maturation media.
Plate microglia-like cells directly onto plasticware (well-plates, imaging chambers...) in maturation media at desired concentration.

Note
The first harvest tends to give a low yield of cells. Typically, from one induction it is possible to obtain roughly 5-6 weekly harvests before the yields decrease.

Perform experiments on microglia-like cells after 7 days from harvest.
Protocol references
Garcia-Reitboeck, Pablo et al. “Human Induced Pluripotent Stem Cell-Derived Microglia-Like Cells Harboring TREM2 Missense Mutations Show Specific Deficits in Phagocytosis.” Cell reports vol. 24,9 (2018): 2300-2311. doi:10.1016/j.celrep.2018.07.094