Sep 29, 2025
Version 1
Microglia differentiation V.1
- Riana Lo Bu1,2,
- Frank Soldner1,2
- 1Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA.;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815.
Protocol Citation: Riana Lo Bu, Frank Soldner 2025. Microglia differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22zbjl1y/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: January 08, 2024
Last Modified: September 29, 2025
Protocol Integer ID: 93091
Keywords: ASAPCRN, microglia differentiation this protocol, differentiate microglia cells from hesc, differentiate microglia cell, microglia differentiation
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-024409
Abstract
This protocol has been refined to differentiate microglia cells from hESC adapted to feeder free culture systems as described in the following protocol:
Citation
LINK
Protocol Overview
A. Flask Preparation
B. Poly-D-Lysine (PDL) plate coating
C. Media recipes
D. Cell passage and differentiation
E. Microglia precursor isolation and platting for maturation
General Notes
A list of reagents and relevant vendor information can be found in the table listed under the materials tab.
Attachments
Materials
Reagent table:
| Item | Vendor | Catalog Number | |
| DMEM/F12 | Gibco | 11320082 | |
| Reduced growth factor matrigel | Fisher | CB40230 | |
| T75 Flask | Fisher | 07202000 | |
| mTESR | Stem Cell Tech | 100-0276 | |
| STEMPRO-34 | Gibco | 10639011 | |
| BMP4 | PeProtech | 120-05 | |
| VEGF | PeProtech | 100-20 | |
| FGF | PeProtech | 100-18B | |
| SCF | PeProtech | 300-07 | |
| IL-3 | PeProtech | 200-03 | |
| TPO | PeProtech | 300-18 | |
| M-CSF | PeProtech | 300-25 | |
| FLT-3 | PeProtech | 300-19 | |
| GM-CSF | PeProtech | 300-03 | |
| FLT-3 | PeProtech | 300-19 | |
| M-CSF | PeProtech | 300-25 | |
| TGF-β1 | PeProtech | 100-21 | |
| IL-34 | PeProtech | 200-34 | |
| M-CSF | PeProtech | 300-25 | |
| Primaria Plates - 6w | Corning | 353846 | |
| Primaria Plates - 24w | Corning | 353847 | |
| Poly-D-Lysine | Sigma | P6407 | |
| Neurobasal media | Gibco | 21103049 | |
| N2 Neuroplex | Gemini | 400-163 | |
| GEM21 Neuroplex | Gemini | 400-160 | |
| Albumax I | Gibco | 11020021 | |
| Sodium Chloride | Fisher | BP35810 | |
| Sodium Pyruvate | Gibco | 11360070a | |
| Glutamax I | Gibco | 35050061 | |
| Pen Strep | GIbco | 15140122 |
Troubleshooting
Flask preparation
1h
Thaw matrigel Overnight on ice inside a 4 °C fridge. Chill all flasks, pipettes and materials to be used for flask preparation under the same conditions and keep everything as cold as possible throughout the procedure (do not use a freezer as it can cause the matrigel solution to freeze while platting).
Dilute matrigel in cold DMEM/F12 as described by the manufacturer and add into the T75 flasks (~6 mL /flask total volume). Incubate at 37 °C for at least 01:00:00 before use. Flasks can be stored in the incubator for several weeks before use.
1h
Poly-D-Lysine (PDL) plate coating
6h
Prepare PDL (0.5 µg/ml) in sterile water and add it to each well and incubate 06:00:00 to Overnight at 37 °C . The day after, wash each well 4 times with sterile water to remove excess PDL (at this point the plates are ready to be used for culture - do not let the wells dry at any step as it can cause PDL crystallization (cytotoxic)).
6h
Media preparation
The below media recipes are used in the remainder of the protocol:
STEP1 media: mTeSR media + Penicillin-Streptomycin (100U/ml)+ 80ng/ml BMP4
STEP2 media: StemPro-34 SFM media + 2 mM GlutaMAX + 80 ng/ml VEGF, 25 ng/ml FGF + 100 ng/ml SCF
STEP3 media: StemPro-34 SFM media + 50 ng/ml SCF + 50 ng/ml IL-3 + 5 ng/ml TPO + 50 ng/ul M-CSF + 50 ng/ul Flt3
STEP4 media: StemPro-34 SFM media + 50 ng/ml SCF + 50 ng/ml IL-3 + 5 ng/ml TPO + 50 ng/ul M-CSF + 50 ng/ul Flt3
Microglia maturation media: Neurobasal media + N2 Neuroplex (1x final concentration) + GEM21 Neuroplex (1x final concentration) + 20% AlbuMAX I (0.2% final concentration) + NaCl (5M) (50mM final concentration) + sodium pyruvate 100x (1x final concentration) + glutaMAX 100x (1x final concentration) + Penicillin-Streptomycin (100U/ml) + 50 ng/ml TGF-β1 + 100 ng/ml IL-34 + 12.5 ng/ml M-CSF
Cell passage and differentiation
Note
It is important to start the differentiation from pristine, undifferentiated feeder free cultures. For more details consult: https://doi.org/10.17504/protocols.io.b4mcqu2w
Note
When changing media in the flasks, avoid scraping the bottom surface of the flasks to prevent cell and coating loss.
Manually dissect undifferentiated hPSC colonies and transfer 40-60 small aggregates into a matrigel coated flask.
Maintain undifferentiated cells in mTeSR media until undifferentiated colonies reach an average colony size of 1 mm. Usually, 7 mL of media/flask and media change every other day works well to maintain the cells until they reach the right size.
STEP1 (days 0-3): Once the hPSC colonies reach the correct size, microglia differentiation is initiated (day 0) by culturing the undifferentiated cells in 6 mL of STEP1 media. STEP1 media should be replaced with fresh media on day 2.
STEP2 (days 4-5): Culture cells in 6 mL of STEP2 media. No media change until the next step.
STEP3 (days 6-13): Culture cells in 6 mL of STEP3 media. Media should be changed on day 10.
STEP4 (days 14-28): Culture cells in 6 mL of STEP4 media. Media should be replaced every 96:00:00 .
4d
Microglia precursor isolation and platting for maturation
4m
Note
The timepoint of microglial precursor generation as indicated by the appearance of a large number of floating cells with microglial precursor typical morphology in the supernatant is variable between hPSC cell lines and starts usually around day 28 of differentiation. Microglia precursor differentiation can be validated by FACS analysis (described in another protocol in this same collection). We normally only initiate microglial differentiation from cultures with more than 80% CD11b/CD45 and CD14/CD16 double positive microglial precursors.
Note
If properly maintained with STEP4 media changes every 4 days, cultures in STEP4 are capable of generating microglia precursors for several months (as monitored by FACS). Microglia precursors can be collected every 4 days for microglial maturation as described below.
Note
For media changes in STEP4 on days without collecting microglial precursors for maturation (as described below), we usually try to return microglial precursors after media change. Because microglia precursor cells grow mainly in suspension, we collect ~5 mL of microglial containing supernatant in a conical tube, centrifuged at 150 rcf for 00:04:00 , and subsequently resuspend the cells in 5 mL of fresh STEP4 media to return to the same flask.
Note
Microglial precursors can be plated on either Primaria plates or Poly-D-Lysine (PDL) coated cell culture plates or glass cover slips. However, based on our experience, platting on primaria plates results in more consistent differentiation to mature microglia.
Once microglia precursor differentiation is validated on day 28 or later, microglia progenitors are collected from STEP4 flasks by transferring the supernatant to a conical tube followed by centrifugation at 150 rcf, Room temperature for 00:04:00 . The cell pellet is resuspended in microglia maturation media and plated at 75k cells/cm2 (Primaria or PDL-coated plates) in microglia maturation media. Media should be changed every 4 days for at least 2 weeks to allow the cells to mature.
4m
Citations
Douvaras P, Sun B, Wang M, Kruglikov I, Lallos G, Zimmer M, Terrenoire C, Zhang B, Gandy S, Schadt E, Freytes DO, Noggle S, Fossati V. Directed Differentiation of Human Pluripotent Stem Cells to Microglia.
https://doi.org/10.1016/j.stemcr.2017.04.023