Jul 23, 2025

Public workspaceMicroglia differentiation from human pluripotent stem cells

Forked from a private protocol
DOI
https://dx.doi.org/10.17504/protocols.io.14egnr8ezl5d/v1
  • Arun Thiruvalluvan1,2,
  • Agnete Kirkeby1,2
  • 1Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.;
  • 2Novo Nordisk Foundation Center for Stem Cell Medicine (renew), University of Copenhagen, 2200 Copenhagen, Denmark
Laura Castilla-Vallmanya
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DOI: https://dx.doi.org/10.17504/protocols.io.14egnr8ezl5d/v1
Protocol Citation: Arun Thiruvalluvan, Agnete Kirkeby 2025. Microglia differentiation from human pluripotent stem cells . protocols.io https://dx.doi.org/10.17504/protocols.io.14egnr8ezl5d/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: July 16, 2025
Last Modified: July 23, 2025
Protocol Integer ID: 222627
Keywords: human pluripotent stem cells, Generation of EBs, Harvesting EBs, pMacpre and microglia, ASAPCRN, microglia differentiation from human pluripotent stem cell, microglia differentiation, protocol details about microglia differential, microglia differential, human pluripotent stem cell, microglia
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-024296
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Abstract
This protocol details about microglia differential from human pluripotent stem cells.
Attachments
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Guidelines
Reference

A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific
Expression Profile and Inflammatory Response.
Walther Haenseler, Stephen N. Sansom, Julian Buchrieser, Sarah E. Newey, Craig S. Moore, Francesca J. Nicholls, Satyan Chintawar, Christian Schnell, Jack P. Antel, Nicholas D. Allen, M. Zameel Cader, Richard Wade-Martins, William S. James, Sally A. Cowley.

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in
Alzheimer’s disease.
Sudha R. Guttikonda, Lisa Sikkema, Jason Tchieu, Nathalie Saurat, Ryan M. Walsh, Oliver Harschnitz, Gabriele Ciceri, Marjolein Sneeboer, Linas Mazutis, Manu Setty, Paul Zumbo, Doron Betel, Lot D. de Witte, Dana Pe’er and Lorenz Studer.
Materials
Reagents required

ABC
ReagentsSupplierCat no
BMP4Miltneyi130-111-165
SCFMiltenyi130-096-692
VEGFMiltneyi130-109-385
X-VIVO 15LonzaBE04-418
M-CSFMiltneyi130-096-492
IL-3Miltneyi130-095-069
Advanced DMEM/F12Life Technologies12634-010
N2 supplementLife Technologies17502-048
GlutaMAXLife Technologies35050-061
2-mercaptoethanolLife Technologies31350-010
Pen/StrepLife Technologies17502-048
IL-34Miltneyi130-108-977
GM-CSFMiltneyi130-093-864
Aggrewell 800 well (24 wells)STEMCELL Technologies34811
Anti-Adherence Rinsing SolutionSTEMCELL Technologies7010
6 well ULA platesCorning®CLS3471
Nunc™ EasYFlask™ Cell Culture FlasksThermo Fisher156499
Strainers, 40 mmCorning352340
RPMI mediumThermo Fisher11875093
IMDM mediumThermo Fisher12440053
B-27 supplement minus vitamin AThermo Fisher12587010
AccutaseThermo FisherA1110501
D-PBS −Ca2+/−Mg2+ CTSThermo FisherA1285601
Y-27632 dihydrochlorideMiltenyi130-106-538
KnockOut™ Serum ReplacementThermo Fisher10828028
ReagentHuman BMP-4Miltenyi BiotecCatalog #130-111-165
ReagentHuman SCFMiltenyi BiotecCatalog #130-096-692
ReagentHuman VEGF (165) ISMiltenyi BiotecCatalog #130-109-385
ReagentHuman M-CSFMiltenyi BiotecCatalog #130-096-492
ReagentHuman IL-3Miltenyi BiotecCatalog #130-095-069
ReagentAdvanced DMEM/F-12Thermo Fisher ScientificCatalog #12634010
ReagentGibco™ N-2 Supplement (100X)Thermo Fisher ScientificCatalog #17502048
ReagentGlutamaxGibco - Thermo Fisher ScientificCatalog #35050-061
ReagentGibco™ 2-Mercaptoethanol (50 mM)Thermo Fisher ScientificCatalog #31350010
ReagentPenicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140148
ReagentHuman IL-34Miltenyi BiotecCatalog #130-108-977
ReagentHuman GM-CSFMiltenyi BiotecCatalog #130-093-864
ReagentAggreWell™800 24-well Plate, 1 pack 1 Plate STEMCELL Technologies Inc.Catalog #34811
ReagentAnti-Adherence Rinsing Solution 100 mL STEMCELL Technologies Inc.Catalog #7010
ReagentCorning® Costar® Ultra-Low Attachment Multiple Well PlateMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS3471
ReagentNunc™ EasYFlask™ Cell Culture Flasks, T75, filterThermo FisherCatalog #156499
ReagentFalcon 40 µm Cell StrainerCorningCatalog #352340
ReagentRPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
ReagentIMDMGibco - Thermo Fisher ScientificCatalog #12440053
ReagentB-27™ Supplement (50X), minus vitamin AThermo FisherCatalog #12587010
ReagentAccutase cell dissociation reagentGibco - Thermo Fisher ScientificCatalog #A1110501
ReagentGibco™ CTS™ DPBS calcium magnesiumThermo Fisher ScientificCatalog #A1285801 1
ReagentStemMACS™ Y27632Miltenyi BiotecCatalog #130-106-538
ReagentKnockOut™ Serum ReplacementThermo FisherCatalog #10828028
ReagentAPC/Fire™ 750 anti-human CD11b AntibodyBioLegendCatalog #301351
ReagentFITC Mouse Anti-Human CD45 Clone HI30 (RUO)Becton Dickinson (BD)Catalog #560976
ReagentAnti-Iba1 antibodyAbcamCatalog #ab5076
ReagentPU.1 (9G7) Rabbit mAbCell Signaling TechnologyCatalog #2258S
ReagentAnti-CD11b antibody [M1/70]AbcamCatalog #ab8878
ReagentPurified anti-human CD45 AntibodyBioLegendCatalog #304002
ReagentAnti-TMEM119 antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog # HPA051870

Troubleshooting
Preparation of aggrewell 800 plates
Warm basal medium (DMEM-F12 with KOSR) and complete medium (Desired medium to plate cells).
Open AggreWell plates in a biosafety cabinet.
Note
NOTE: Do not expose AggreWell plates to organic solvents, including ethanol or isopropanol.

Pre-treat wells with Anti-Adherence Rinsing Solution as described below. Add Anti-Adherence Rinsing Solution to each well to be used, as follows:
AB
24-well plate500 μL
6-well plate2 mL
Pipetting
Centrifuge plate at Centrifigation1300 x g, 00:05:00 in a swinging bucket rotor fitted with plate holders.
Note
NOTE: Plates must be well balanced. Prepare a balance plate using a standard plate filled with water to match the weight and position of the AggreWell plate.


5m
Centrifigation
Observe the plate under a microscope to ensure that bubbles have been removed from microwells. If bubbles remain trapped in any microwells, centrifuge at Centrifigation1300 x g, 00:05:00 . Aspirate Anti-Adherence Rinsing Solution from the wells.

5m
Rinse each well with warm basal medium, as follows:


AB
24-well plate2 mL

Aspirate medium from the well.
Add warm complete medium to each well to be used, as follows:
AB
24-well plate1 mL

Pipetting
Generation of EBs
Prepare a single-cell suspension in the desired medium.
To achieve EBs size of 10,000 cells per EB, plate 3.0 x 106 in each well and add complete medium to each well to achieve a final volume as follows:

AB
24-well plate2 mL/well
Prepare a centrifuge balance plate using a standard plate filled with water to match the weight and position of the AggreWell plate.
Pipette cells up and down gently several times to ensure even distribution of cells throughout the well.
Note
Be careful not to introduce bubbles into the microwells.

Immediately centrifuge the AggreWell plate at Centrifigation100 x g, 00:03:00 to capture cells in the microwells, using the balance plate prepared.

3m
Centrifigation
Observe the plate under a microscope to verify that cells are evenly distributed among the microwells.
Imaging
Incubate the plate at Temperature37 °C with 5% CO2 and 95% humidity for Duration24:00:00 . Observe the cells under a microscope.

1d
Incubation
Imaging
Changing medium in aggrewell plate
Warm complete medium.
Perform a 50-75% medium change as follows: 24-well plate: Slowly remove 1-1.5 mL of medium from each well.
Replace with 1-1.5 mL of the fresh complete medium by slowly pipetting down the wall of the well. Slowly dispensing the medium helps to prevent displacement of EBs/spheroids from the microwells.
Harvesting EBs from aggrewell plates
Warm basal medium and complete medium.
Using a serological pipette:
Note
Alternative option: This step can be achieved by using P1000 cut tips.

Remove approximately half of the culture medium from the well.
Dispense the medium firmly back onto the surface of the plate to dislodge the EBs/spheroids from the microwells. Do not triturate.
Select the appropriate strainer and conical tube for separation of EBs/spheroids from single cells: 24-well plate, For harvesting from a single well, use 37-μm Reversible Strainer, Small and a 15-mL conical tube.
Place strainer on top of the tube with the arrow pointed upward. Add Amount1 mL of PBS to wet the surface of the strainer.

Gently aspirate the dislodged EBs/spheroids. Pass the EB/spheroid suspension through the strainer.
Note
NOTE: The aggregates will remain on the filter; any unincorporated single cells will flow through.


Using a serological pipette, dispense Amount1 mL (24-well plate) or Amount3 mL (6-well plate) of the warm basal medium across the entire surface of the well to dislodge any remaining EBs/spheroids. Collect wash and pass over the strainer.
Wash
Dispense Amount1 mL (24-well plate) or Amount3 mL (6-well plate) of the warm basal medium across the entire surface of the well to dislodge any remaining EBs/spheroids. (1/3)
Dispense Amount1 mL (24-well plate) or Amount3 mL (6-well plate) of the warm basal medium across the entire surface of the well to dislodge any remaining EBs/spheroids. (2/3)
Dispense Amount1 mL (24-well plate) or Amount3 mL (6-well plate) of the warm basal medium across the entire surface of the well to dislodge any remaining EBs/spheroids. (3/3)
Wash can be repeated until you do not see more EBs under the microscope.

Invert the strainer, and place over a new conical tube of the same size. Collect the EBs/spheroids by washing with 2-5 mL of complete medium per well harvested.
Wash
Differentiation protocol
Maintain the pluripotent hPSCs/IPSCs on Lam-521 (0.5 μg/cm2) in iPS-Brew medium. Passage cells can be every 7 days with Concentration0.5 millimolar (mM) EDTA, followed by seeding at a density of 2,500 cells per cm2 with ROCK inhibitor (Concentration10 micromolar (µM) Y-27632) included in the medium for the first Duration24:00:00 after plating.

1d
Day 1: Seed 3x106 iPSCs or ES cells into an Aggrewell 800 well (STEMCELLTechnologies) to form EBs with ROCK inhibitor (Concentration10 micromolar (µM) Y-27632), in iPS-Brew medium and feed medium daily with BMP4 (Amount50 µL ), VEGF (Amount50 µL ), SCF (Amount20 µL ).

Day 2- 4: Change medium gently in the Aggrewell 800 well plate without agitating the EBs on the plate.
Day 4: Differentiate four-day EBs in either 6-well plates (15 EBs/well), T75 (75 EBs), or T175 flasks (150 EBs) with X-VIVO15 and supplement with Amount100 µL M-CSF, Amount25 µL IL-3, Concentration2 millimolar (mM) GlutaMAX, Amount100 µL penicillin, Amount100 µL streptomycin, Concentration0.055 millimolar (mM) b-mercaptoethanol with fresh medium added weekly.
Note
Maintain cells in this medium for up to 6 months.

Collect weekly pMacpre (Stem cell-derived macrophage/microglia progenitors) emerging into the supernatant after approximately 1 month and replenish differentiation cultures with fresh medium. Strain harvested cells (40 mm, Corning) and use either directly as pMacpre; or plate onto tissue-culture treated plastic or glass coverslips at 100,000 per cm2 and differentiated for 7 days or more.
Note
Note: At this point, pMacpre can be harvested and frozen down for further use.

Collect cells in suspension and either (a) Culture in RPMI with 10% FBS, l-glutamine, and pen/strep with IL-34 (Amount100 µL ) and M-CSF (Amount10 µL ) for 7–11 d until cells were adherent and elongated to transition to primitive macrophages, or (b) Co-culture with DA neurons in Neurobasal medium containing B-27 supplement (1:500), l-glutamine (1:1000) and BDNF (Amount20 µL ), ascorbic acid (Concentration0.2 millimolar (mM) ) GDNF (Amount20 µL ), cAMP (Concentration500 micromolar (µM) ), and IL-34 (Amount100 µL ) and M-CSF (Amount20 µL ) for 7-10 days for direct transition to microglia.
For serum-free differentiation, harvest and culture cells in suspension in 75% IMDM, 25% RPMI
medium containing B-27 supplement (1:500), l-glutamine (1:1000) and IL-34 (Amount100 µL ) and M-CSF
(Amount20 µL ) for 11–14 days.
Quality control for pMacpre and microglia
FAC-sorting for CD11b+ and CD45+ on both pMacpre and Microglia.
ABC
AntibodiesSupplierCat no
CD11bBiolegend301351
CD45 BDPharmingen560976
ICC for IBA-1, PU.1, TMEM119 on differentiated Microglia.
ABC
AntibodiesSupplierCat no
IBA-1AbCamab5076
PU.1Cell Signaling2258S
CD11bAbCamab8878
CD45BioLegend304002
TMEM119SigmaHPA051870