Dec 12, 2025

Public workspaceDifferentiation of Induced Pluripotent Stem Cells (iPSCs) into Microglia-like cells (iMG)

DOI
https://dx.doi.org/10.17504/protocols.io.ewov11rw2vr2/v1
  • Elisa Perciballi1,
  • Micheal John Dolan2,
  • Martine Therrien1,
  • Beth Stevens3
  • 1University of California, Davis;
  • 2Smurfit Institute of Genetics School, Trinity College Dublin;
  • 3HHMI, Broad Institute, Boston Children's Hospital
Martine Therrien
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov11rw2vr2/v1
Protocol Citation: Elisa Perciballi, Micheal John Dolan, Martine Therrien, Beth Stevens 2025. Differentiation of Induced Pluripotent Stem Cells (iPSCs) into Microglia-like cells (iMG). protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11rw2vr2/v1
Manuscript citation:
Dolan, M.-J., Therrien, M., Jereb, S., Kamath, T., Gazestani, V., Atkeson, T., Marsh, S.E., Goeva, A., Lojek, N.M., Murphy, S., et al. (2023). Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro. Nat. Immunol. 24, 1382–1390. https://doi.org/10.1038/s41590-023-01558-2.
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: December 06, 2025
Last Modified: December 12, 2025
Protocol Integer ID: 234321
Keywords: stem cells, microglia, derived microglia, role of microglia, microglia, microglial state, induced pluripotent stem cell, differentiation of induced pluripotent stem cell, immune alterations during development, immune alteration, human cell, response to specific brain challenge, use of human cell, lentiviral transduction, amenable to lentiviral transduction, specific brain challenge
Funders Acknowledgements:
Alzheimer's Association
Cure Alzheimer’s Fund
Howard Hughes Medical Institute
Stanley Center for PsychiatricResearch
Abstract
iPSC-derived microglia are an important tool for studying the role of microglia and neuro-immune alterations during development, adulthood, and aging, both in health and disease. The use of human cells enables the identification of molecular mechanisms driven by an individual's genetic susceptibility that would not be possible with animal models. Here, we described a step-by-step protocol used in our previous manuscript (Dolan, Therrien, et al 2023). This protocol is used to induce a microglial state that arises in response to specific brain challenges and is amenable to lentiviral transduction.
Materials
ReagentACCUTASE™ 100 mL STEMCELL Technologies Inc.Catalog #7920 Equipment
·        Incubator for normoxic conditions (20% O₂, 5% CO₂, 37°C)
·        Humidified tri-gas incubator for hypoxic conditions (5% O₂, 5% CO₂, 37°C)
·        Centrifuge
·        Cell counting equipment
 
iPSCs maintenance list of reagents
 
AB
Reagent Company; Cat #
PBS 1X Thermo Fisher; 14190250
Matrigel VWR; 47743-722
Essential E8 medium Thermo Fisher; A1517001
Cryostor Stem Cell Technologies; 100-1061
Accutase Stem Cell Technologies; 07920
 
Microglia differentiation media composition and reagents
 
ABC
iHPC Base Growth Medium (first 10 days)
Component Final concentration Company; Cat #
IMDM 50% Thermo Fisher; 31980097
F12 50% Thermo Fisher; 11765062
ITSG-X 2% v/v Thermo Fisher; 51500-056
L-ascorbic acid 2-Phosphate 64 µg/ml Sigma-Aldrich; A8960-5G
monothioglycerol 400 µM Sigma-Aldrich; M1753-100ML
PVA 10 µg/ml Sigma-Aldrich; P8136-250G
GlutaMax (1X) 1X Thermo Fisher; 35050079
Chemically defined lipid concentrate (1X) 1X Thermo Fisher; 11905031
Non-essential amino acids (NEAA) (1X) 1X Thermo Fisher; 11140050
iHPC base media: All reagents are combined and filtered in .22uM bottle. Base media is used within 2 weeks. L-ascorbic acid 2-Phosphate and PVA solutions are used within a week.
 
ABC
Day 0 growth factors
Component Final concentration Company; Cat #
FGF2 50 ng/ml Thermo Fisher; PHG0261
BMP4 50 ng/ml Thermo Fisher; PHC9534
Activin-A 12.5 ng/ml Thermo Fisher; PHC9564
Rock Inhibitor (RI) 1 µM Selleckchem; s1049
Lithium Chloride (LiCl) 2 mM Sigma-Aldrich; L7026
Day 0 iHPC media: iHPC base media + growth factors listed. Growth factors are aliquoted in single use aliquots and store at -80C or -20C until expiration date following manufacturer instructions.
 
ABC
Day 2 growth factors
Component Final concentration Company; Cat #
FGF2 50 ng/ml Thermo Fisher; PHG0261
VEGF 50 ng/ml Peprotech; 100-20-50UG
Day 2 iHPC media: iHPC base media + growth factors listed. Growth factors are aliquoted in single use aliquots and store at -80C or -20C until expiration date following manufacturer instructions.
 
ABC
Day 4 growth factors
Component Final concentration Company; Cat #
FGF2 50 ng/ml Thermo Fisher; PHG0261
VEGF 50 ng/ml Peprotech; 100-20-50UG
TPO 50 ng/ml Thermo Fisher; 300-18-50UG
SCF 10 ng/ml Thermo Fisher; PHC2115
IL-6 50 ng/ml Thermo Fisher; 200-06
IL-3 10 ng/ml Thermo Fisher; 200-03-10UG
Day 4-10 iHPC media: iHPC base media + growth factors listed. Growth factors are aliquoted in single use aliquots and store at -80C or -20C until expiration date following manufacturer instructions.
 
ABC
iMG differentiation basal medium
Component Final concentration Company; Cat #
DMEM 50% Thermo Fisher; 10569044
F12 50% Thermo Fisher; 11765062
ITS-G 2x v/v Thermo Fisher; 41400045
B-27 2x v/v Thermo Fisher; 17504044
N2 0.5x Thermo Fisher; 17502048
Monothioglycerol 200 µM Sigma-Aldrich; M1753-100ML
GlutaMax (1X) 1X Thermo Fisher; 35050079
Non-essential amino acids (NEAA) (1X) 1X Thermo Fisher; 11140050
Insulin 5 µg/ml Sigma-Aldrich; I2643
Mg base media: All reagents are combined and filtered in .22uM bottle. Base media is used within 2 weeks. B27 and N2 are frozen in single used aliquots.
 
ABC
Day 10 growth factors
Component Final concentration Company; Cat #
M-CSF 25 ng/ml Thermo Fisher; 300-25-100
IL-34 10 ng/ml Thermo Fisher; 200-34-250UG
TGFβ 50 ng/ml Thermo Fisher; 100-21-100UG
Day 10 Mg media: Mg base media + growth factors listed. Growth factors are aliquoted in single use aliquots and store at -80C or -20C until expiration date following manufacturer instructions.
 
ABC
Day 30 growth factors
Component Final concentration Company; Cat #
CD200 100 ng/ml Thermo Fisher; 103872-010
CX3CL1 100 ng/ml Thermo Fisher; 300-31
M-CSF 25 ng/ml Thermo Fisher; 300-25-100
IL-34 10 ng/ml Thermo Fisher; 200-34-250UG
TGFβ 50 ng/ml Thermo Fisher; 100-21-100UG
Day 10 Mg media: Mg base media + growth factors listed. Growth factors are aliquoted in single use aliquots and store at -80C or -20C until expiration date following manufacturer instructions.
 
Materials for microglia cell growth
AB
Product name Company; Cat #
6-well Ultra-Low Attachment plate Corning Life Sciences; 3471
Cell lifter Life Sciences; 76036-004
 
 
 
Protocol materials
ReagentEssential 8Life TechnologiesCatalog #A1517001
ReagentTeSR™-E8™ Kit for hESC/hiPSC Maintenance 1 Kit STEMCELL Technologies Inc.Catalog #5990
ReagentCorning® Matrigel® Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mLCorningCatalog #356237
ReagentCryoStor CS10-100mlSTEMCELL Technologies Inc.Catalog #07930
ReagentACCUTASE™ 100 mL STEMCELL Technologies Inc.Catalog #7920
ReagentDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
ReagentY-27632 (ROCK inhibitor) (Selleck Chemicals Catalog # S1049)SelleckchemCatalog #S1049
ReagentCostar® 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well PlatesCorningCatalog #3471
ReagentFisherbrand™ Cell LiftersThermo Fisher ScientificCatalog #08-100-240
Troubleshooting
iPSC maintenance
Maintenance of stem cells
Successful differentiation is achieved with high quality stem cells. It is recommended to split iPSCs at least 2 times after thawing before starting differentiation and check frequently at their morphology to avoid spontaneous differentiations that may interfere with mesodermal induction. Make sure iPSCs are growing in ReagentEssential 8Life TechnologiesCatalog #A1517001 or ReagentTeSR™-E8™ Kit for hESC/hiPSC Maintenance 1 Kit STEMCELL Technologies Inc.Catalog #5990 otherwise change accordingly.

Generation of HPC
47m
Day -1: Generation of embryoid bodies
 
Detach cells
  •  Aspirate the culture media and wash with 1X PBS.
  • Dissociate iPSCs by incubating with ReagentACCUTASE™ 100 mL STEMCELL Technologies Inc.Catalog #7920 Duration00:10:00 (time may vary depending on different cell lines) at Temperature37 °C , 5% CO2. Lightly tap the side of the plate to dislodge the cells.
  • Collect the entire plate volume and transfer the cell suspension to a 15 ml conical tube.
  • Wash the plate again with ReagentDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 to collect cells left behind and transfer to the same 15 ml tube.
  • ·Centrifuge the cells at Centrifigation300 x g, 00:05:00 . Aspirate supernatant to waste and resuspend the pellet to triturate to create a single cell suspension using ReagentEssential 8Life TechnologiesCatalog #A1517001 with Concentration10 micromolar (µM) ReagentY-27632 (ROCK inhibitor) (Selleck Chemicals Catalog # S1049)SelleckchemCatalog #S1049




15m
 Count the cells and adjust cell density to 5 x 105 per well in a ReagentCostar® 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well PlatesCorningCatalog #3471 in ReagentEssential 8Life TechnologiesCatalog #A1517001 + ReagentY-27632 (ROCK inhibitor) (Selleck Chemicals Catalog # S1049)SelleckchemCatalog #S1049 (2 ml in a 6-well plate).
  • Count cells using Trypan blue. Viability should be at least 90%.
  • Plate evenly on growth surface and begin normoxic culture conditions (20% O2, 5% CO2, 37°C).

Note
Because of the different proliferation rates among cell lines, it is recommended to plate different concentrations (2 x 105 and 8 x 105) and start differentiation with the one with 50-100 embryoid bodies (EBs).




Day 0: Change media
  • Carefully collect the entire plate medium with EBs in suspension by using a 5 ml pipette to not dislodge them, and transfer to a 15 ml tube.
  • Centrifuge EBs at Centrifigation100 x g, 00:05:00 Aspirate supernatant to waste and resuspend EBs in iHPC Base Growth medium supplemented with Amount2 mL Day 0 growth factors in a new low-attachment plate well.
  • Plate evenly on growth surface and begin hypoxic culture conditions by incubating cells in a humidified tri-gas incubator with 5% O2, 5% CO2, Temperature37 °C .


5m
Day 2: Making HPC media and change media
  • Carefully collect the entire plate medium with EBs in suspension by using a 5 ml pipette to not dislodge them, and transfer to a 15 ml tube.
  • Centrifuge EBs at Centrifigation100 x g, 00:05:00 . Aspirate supernatant to waste and scroll the tune on the grill of the hood to dislodge the pellet. Resuspend EBs inAmount2 mL iHPC Base Growth medium supplemented with Day 2 growth factors in a new low-attachment plate well.
  • Plate evenly on the growth surface and incubate under hypoxic culture conditions (5% O2, 5% CO2, Temperature37 °C ).


5m
Day 4: Change media
  • Carefully collect the entire plate medium with EBs in suspension by using a 5 ml pipette to not dislodge them, and transfer to a 15 ml tube..
  • Centrifuge EBs at Centrifigation100 x g, 00:05:00 . Aspirate supernatant to waste and scroll the tube on the grill of the hood to dislodge the pellet. Resuspend EBs in Amount2 mL iHPC Base Growth medium supplemented with Day 4 growth factors in a new low-attachment plate well.
  • Plate evenly on growth surface and incubate under normoxic culture conditions (20% O2, 5% CO2, Temperature37 °C ).


5m
Day 6: Feed cells
AddAmount1 mL of iHPC Base Growth medium supplemented with Day 4 growth factors.

Day 8: Feed cells
Add Amount1 mL of iHPC Base Growth medium supplemented with Day 4 growth factors.

Day 10: Collect HPC
  • Carefully collect the entire plate medium with EBs in suspension and transfer to a 15 ml tube.
  • Let the EBs precipitate by gravity (around Duration00:02:00 ).
  • Collect the supernatant containing hematopoietic progenitor cells (hpc) in single cells in a tube and filter through a 40 µm screen (to avoid any residual EBs).
  • Centrifuge the single cell suspension at Centrifigation300 x g, 00:05:00 Aspirate supernatant to waste and resuspend the pellet in Mg differentiation basal medium supplemented with Day 10 growth factors.
  • Count the cells using Trypan blue
 

Note
Quality control: To assess that differentiation is successful, on Day 10, it is recommended to run a quality control to check viability, and the expression of CD45 and CD43 markers via flow cytometry. Viaiblity should be > 70%, CD43 positive cells > 95% and CD45 positive cells > 50%

Note
Cells can be frozen at this time in ReagentCryoStor CS10-100mlSTEMCELL Technologies Inc.Catalog #07930 . We recommend freezing at least 300 000 cells/aliquot so they can be thawed in 1 well of a 6-well plate




7m
Cell replating to continue differentiation
  • Resuspend HPC in Mg differentiation basal media with Day 10 growth factors 
  • Adjust cell density to 3 x 105 per well in a ReagentCorning® Matrigel® Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mLCorningCatalog #356237 coated 6-well plate.
  • Incubate under normoxic culture conditions (20% O2, 5% CO2, Temperature37 °C ).

Every 2 days: Feed cells
Add 1 ml of Mg differentiation basal medium supplemented with Day 10 growth factors
Day 22: Collect, count and replate cells
Collect and count cells
  • Collect the entire plate medium and transfer to a 15 ml tube (do not aspirate, as many cells might still be in suspension).
  • Collect early iMGL by addingReagentDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 to the well and let sit for Duration00:05:00 at room temperature.
  • Gently detach cells with a ReagentFisherbrand™ Cell LiftersThermo Fisher ScientificCatalog #08-100-240 . Wash the well with ReagentDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 and transfer to a new 15 ml tube.
  • Centrifuge both tubes (one containing plate medium and one PBS) at Centrifigation300 x g, 00:05:00
  • From the tube with PBS, aspirate supernatant to waste. From the tube with conditioned Mg medium, collect the supernatant, and transfer to a new tube, leaving 1 ml to cover the pellet. Resuspend the pellet with the remaining 1 ml and transfer this cell suspension into the PBS tube.
  • Combine and gently triturate the two pellets together.
  • Count using Trypan blue

10m
Replate cells
  • Adjust cell density to 3 x 105 per well in a ReagentCorning® Matrigel® Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mLCorningCatalog #356237 coated 6-well plate.
  • Plate cells in 50% old conditioned medium + 50% fresh Mg differentiation basal medium supplemented with Day 10 growth factors.

Note
For high-proliferating cell lines, it might be necessary to split cells at Day 20 instead.


Microglia generation and maturation
5m
Starts from either freshly collected iHPC or from frozen iHPC vials. If cells were frozen, thaw rapidly at Temperature37 °C water bath, resuspend cells in warm DMEM-F12, centrifuge at Centrifigation300 x g, 00:05:00 and count to plate at the appropriate density.

5m
Every 2 days: Feed cells
Add 1 ml of Mg differentiation basal medium supplemented with Day 10 growth factors
Day 30: Collect, count and replate cells
Note
Cells at Day 30 must be plated in the final experimental vessel.

Collect and count cells
  • Collect the entire plate medium and transfer to a 15 ml tube (do not aspirate, as many cells might still be in suspension).
  • Collect early iMGL by addingReagentDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 to the well and let sit for Duration00:05:00 at room temperature.
  • Gently detach cells with a ReagentFisherbrand™ Cell LiftersThermo Fisher ScientificCatalog #08-100-240 Wash the well with ReagentDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 and transfer to a new 15 ml tube.
  • Centrifuge both tubes (one containing plate medium and one PBS) at Centrifigation300 x g, 00:05:00
  • From the tube with PBS, aspirate supernatant to waste. From the tube with conditioned Mg medium, collect the supernatant, and transfer to a new tube, leaving 1 ml to cover the pellet. Resuspend the pellet with the remaining 1 ml and transfer this cell suspension into the PBS tube.
  • Combine and gently triturate the two pellets together.
  • Count using Trypan blue.


Replate cells
  • Adjust cell density to 3 x 105 per well in a ReagentCorning® Matrigel® Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mLCorningCatalog #356237 coated 6-well plate.
  • Plate cells in 50% old conditioned medium + 50% fresh Mg differentiation basal medium supplemented with Day 30 growth factors.
Every 2 days: Feed cells
Add 1 ml of Mg differentiation basal medium supplemented with Day 30 growth factors.
Day 40: Collect cells for desired experiments.