Apr 23, 2026

Differentiation of KOLF2.1J Human iPSCs into Microglia-like Cells (iMGL) via Primitive Macrophage Precursors (PMPs)

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld4xo7l5b/v1
  • 1Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA;
  • 2Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
Pilar Alvarez Jerez
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld4xo7l5b/v1
Protocol Citation: Jae-Hyeon Park, Alexandra Beilina, Mark Cookson 2026. Differentiation of KOLF2.1J Human iPSCs into Microglia-like Cells (iMGL) via Primitive Macrophage Precursors (PMPs). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4xo7l5b/v1
Manuscript citation:

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 23, 2026
Last Modified: April 23, 2026
Protocol  Integer ID: 315635
Keywords: microglia progenitor cell, microglia, primitive macrophage precursor, like cells suitable for flow cytometry, pluripotent stem cell, induced pluripotent stem cell, flow cytometry, phagocytosis assay
Abstract
This protocol describes the differentiation of human induced pluripotent stem cells (iPSCs) into microglia-like cells (iMGLs) using an embryoid body (EB)-based approach that generates primitive macrophage precursors (PMPs) as an intermediate stage. This method is adapted from Brownjohn et al. 2018, based on established PMP derivation approaches (Karlsson et al. 2008; van Wilgenburg et al. 2013). iPSCs are aggregated into EBs in the presence of BMP-4, SCF, and VEGF-121, followed by culture in hematopoietic medium containing M-CSF and IL-3 to generate microglia progenitor cells (MPCs). MPCs are then terminally differentiated in IL-34 and GM-CSF for ~10 days. This method yields microglia-like cells suitable for flow cytometry, scRNA-seq, phagocytosis assays, and functional stimulation experiments.
Guidelines
- All incubations are performed at 37°C, 5% CO₂, unless otherwise stated.
- iPSCs should be healthy and minimally differentiated before starting.
- Use gentle handling throughout EB steps to avoid EB disruption.
- Microglia progenitors typically appear between Day 12–18.
- This protocol follows the method described in Brownjohn et al. 2018, using PMP derivation as the starting point.
Materials
Reagents
ReagentCompanyCatalog #
Essential 8 (E8) MediumThermo Fisher ScientificA1517001
Advanced RPMI 1640Thermo Fisher Scientific12633020
X-VIVO 15LonzaLZBE02-060F
TrypLE ExpressGibco12604013
BMP-4PeproTech120-05
SCFPeproTech300-07
VEGF-121PeproTech100-20A
M-CSFPeproTech300-25
IL-3PeproTech200-03
IL-34PeproTech200-34
GM-CSFPeproTech300-03
ROCK inhibitor (Y-27632)StemCell72307
β-Mercaptoethanol (55 mM stock)Thermo Fisher Scientific21985023
GlutaMAXThermo Fisher Scientific35050-061
Penicillin-StreptomycinThermo Fisher Scientific15140-122
PBSThermo Fisher Scientific20012-027
 Consumables
  • Ultra-low attachment V 96-well plates (Sbio, Cat # MS-9096VZ) 
  • 15 mL and 50 mL conical tubes 
  • T75 flasks 
  • Serological pipettes 
  • Wide-bore P200 tips (recommended) 


Equipment
  • Tissue culture hood 
  • 37°C incubator (5% CO₂) 
  • Centrifuge (capable of 200–300 × g) 
  • Cell counter 
Safety warnings
- Avoid harsh pipetting to preserve viability.
- Pipette slowly at the edge of the well to avoid aspirating EBs.
Before start
1. Maintain iPSCs on Matrigel-coated plates in E8 medium.
2. Ensure iPSCs are healthy and ~70–80% confluent before starting differentiation.
3. Avoid cultures with high spontaneous differentiation.
4. Warm all media and reagents to room temperature or 37°C as appropriate.
Before start
Maintain iPSCs on Matrigel-coated plates in E8 medium.
Ensure iPSCs are healthy and ~70–80% confluent before starting differentiation.
Avoid cultures with high spontaneous differentiation.
Warm all media and reagents to room temperature or 37°C as appropriate.
Part 1: Embryoid Body (EB) Formation
1h 5m
Step 1: Dissociate iPSCs into single cells (Day 0)
1. Aspirate iPSC culture medium.
2. Wash cells 1× with PBS.
3. Add TrypLE Express and incubate at 37°C until cells detach.
4. Gently triturate to obtain a single-cell suspension.
00:10:00 5 to 10 minutes
‱ Avoid harsh pipetting to preserve viability.
10m
Step 2: Plate iPSCs for EB formation (Day 0)
  1. Count cells.
  2. Plate cells in ultra-low attachment 96-well plates in 100 µL Embryoid Body Medium (EBM) per well.
  3. Return plate to incubator.
00:15:00
‱Expected result: EBs should be visible within 24 h.
15m
Embryoid Body Medium (EBM)
Prepared in E8 medium:
  • ROCK inhibitor: 10 µM 
  • BMP-4: 50 ng/mL 
  • SCF: 20 ng/mL 
  • VEGF-121: 50 ng/mL 
Step 3: Day 1 media change (remove ROCK inhibitor)
  1. The following day, remove 80 µL medium from each well.
  2. Replace with 100 µL fresh EBM without ROCK inhibitor.
00:20:00
‱ Pipette slowly at the edge of the well to avoid aspirating EBs.
20m
Step 4: Day 2–3 daily media changes
  1. Continue culturing EBs for 2 additional days.
  2. Perform daily full media changes using fresh EBM without ROCK inhibitor.
00:20:00 per day
‱ Expected result: EBs remain compact and increase slightly in size.
20m
Part 2: Primitive Macrophage Precursor (PMP) / Hematopoietic Differentiation
45m
Step 5: Transfer EBs into hematopoietic culture (Day 4)
  1. Collect EBs from the 96-well plate using wide-bore tips.
  2. Transfer EBs into a conical tube and allow them to settle by gravity.
  3. Carefully remove supernatant.
  4. Transfer approximately ~200 EBs into a T75 flask containing 20 mL Hematopoietic Medium.
  5. Return flask to incubator.
00:30:00
‱ This culture will produce primitive macrophage precursors / microglia progenitors.
30m
Hematopoietic Medium (HM)
Prepared in X-VIVO 15:
  • GlutaMAX: 2 mM 
  • β-Mercaptoethanol: 55 µM 
  • M-CSF: 100 ng/mL 
  • IL-3: 25 ng/mL 
Step 6: Feed hematopoietic cultures
  1. Replace hematopoietic medium every 2 days with fresh HM.
00:15:00 per feeding
‱ Expected result: floating progenitor-like cells begin to emerge over time.
15m
Step 7: Monitor emergence of microglia progenitor cells (MPCs)
  1. Observe cultures starting Day 10.
  2. Microglia progenitor cells (MPCs) typically appear between Day 12–18.
‱ Variable ‱ Expected result: large, round, refractive floating cells in the supernatant.
Part 3: Collection of Microglia Progenitor Cells (MPCs)
30m
Step 8: Collect microglia progenitors
  1. Collect culture supernatant containing floating MPCs.
  2. Transfer to conical tubes.
  3. Centrifuge at 250 × g for 5 min.
  4. Aspirate supernatant.
  5. Resuspend pellet in Microglia Maturation Medium (MMM).
00:15:00

15m
Step 9: Seed progenitors for terminal differentiation
  1. Seed approximately 4 million cells per T75 flask into non-coated 75 cm² flasks.
  2. Add MMM to a final volume appropriate for flask culture (typically 15–20 mL).
  3. Return to incubator.
00:15:00
‱  Use non-coated flasks (coating is not required).
15m
Part 4: Terminal Microglia Differentiation
1w 3d
Microglia Maturation Medium (MMM)
Prepared in Advanced RPMI:
  • GlutaMAX: 2 mM 
  • IL-34: 100 ng/mL 
  • GM-CSF: 10 ng/mL 
Step 10: Differentiate progenitors into iMGLs
  1. Culture cells in MMM for 10 days.
  2. Perform full media changes every other day.
240:00:00 10 days total
‱Expected result: progressive microglial morphology with increased adherence and branching.
1w 3d
Timing
StageDuration
iPSC dissociation and EB plating~30–45 min
EB formation phase4 days
Hematopoietic differentiation (PMP/MPC stage)~8–14 days
MPC appearanceDay 12–18
Terminal maturation in MMM10 days
Total protocol time~25–30 days
 
Anticipated Results
  • Floating MPCs appear between Day 12–18.
  • After 10 days in MMM, cells should display microglia-like morphology and express markers such as:
CD11b
CX3CR1
IBA1
P2RY12
TMEM119
  • Mature iMGLs can be used for:
scRNA-seq
flow cytometry
phagocytosis assays
inflammatory stimulation (IFN_γ_, LPS, etc.)
Troubleshooting
Problem: Poor EB formation
Possible cause: low iPSC viability after dissociation
Solution: reduce pipetting stress, use fresh TrypLE or Accutase, confirm iPSCs are healthy before starting

Problem: EBs break apart
Possible cause: harsh pipetting during media changes
Solution: use wide-bore tips and pipette gently from the edge of wells

Problem: Low progenitor (MPC) yield
Possible cause: cytokines degraded or incorrect feeding schedule
Solution: prepare fresh cytokine aliquots, maintain strict every-2-day feeding schedule

Problem: Poor terminal differentiation
Possible cause: progenitors collected too early
Solution: wait until Day 12–18 window when progenitors are robust and abundant
Protocol references