Differentiation of Induced Pluripotent Stem Cells (iPSCs) into Microglia

If you're looking to generate microglia from iPSCs, you're in the right place. Microglia are a critical component of the central nervous system (CNS) and have important roles in synaptic plasticity, neurogenesis and immune defense of the brain. Microglial activation has been widely observed in neurodegenerative diseases, neuroinflammation and neuropathic pain. iPSC derived microglia are therefore a vital tool for researchers studying these diseases. This guide will walk you through a streamlined, weekend-free method to successfully differentiate your iPSCs into fully mature microglia. Let's get started!

Featured Key Products

Materials

iPSCs
Accutase
Matrigel (0.5 mg/ml)
6-well culture plates
E8-like medium
ROCK inhibitor Y-27632 (HB2297)
DMEM/F12 medium
Sodium bicarbonate

Sodium chloride
L-Ascorbic acid (HB1238)
Insulin-Transferrin-Selenium (ITS-G)
BMP-4
StemPro™-34 SFM
GlutaMax™ Supplement
FGF2 or TCB Small Molecule FGF2 Replacements
SCF

VEGF 165
IL-3
M-CSF
Flt3L
Thrombopoietin (TPO)
4% Paraformaldehyde (PFA)
Anti-IBA1 Antibody (HB7847)

Media Recipes

Aseptically combine components and filter the media before use.

Media A - Day 0 (iPSC Plating)

Component	Final Concentration
Essential 8 Medium	1x
ROCK inhibitor Y-27632 (HB2297)	10 µM

Media B - Days 1 - 4 (Mesoderm Induction Media)

Component	Final Concentration
DMEM / F12 Medium	1x
Sodium bicarbonate	543 mg/L
NaCl	1 g/L
L-Ascorbic acid (HB1238)	64 mg/L
Insulin-Transferrin-Selenium	1x
BMP-4	80 ng/ml

Media C - Days 5 - 6 (Hemapoietic Specification Media)

Component		Concentration
StemPro-34 SFM		1x
GlutaMax		1x
SCF		100 ng/ml
VEGF		80 ng/ml
One of:	FGF2-G3	4 ng/ml
	TCB-32 (HB12632)	400nM
	TCB-541 (HB11050)	200nM
	TCB-621 (HB17550)	100nM

Please note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs. TCB compounds have not been optimised yet for this assay therefore concentrations may need adjusting.

Media D - Days 7 - 14 (Myeloid Progenitor Expansion Media)

Component	Final Concentration
StemPro™-34 SFM	1x
GlutaMax™	2 mM
IL-3	50 ng/ml
SCF	50 ng/ml
M-CSF	50 ng/ml
Flt3L	50 ng/ml
Thrombopoietin (TPO)	5 ng/ml

Media E - Days 15 - 25 (Microglial Progenitor Maturation Media)

Component	Final Concentration
StemPro™-34 SFM	1x
GM-CSF	25 ng/ml
M-CSF	50 ng/ml
Flt3L	50 ng/ml

Media F - Day 26 onwards (Microglial Maturation Media)

Component	Final Concentration
RPMI-1640	1x
GlutaMax™	2 mM
GM-CSF	10 ng/ml
IL-34	100 ng/ml

Protocol

Passage iPSCs twice a week using 0.5mM EDTA and then seed in vitronectin-coated (5 µg/ml) 6-well plates using a 1:6 dilution into fresh E8 media. The following day to passage, replace the medium with fresh 5ml of E8 media.

Day 0 (iPSC Plating): Detach the iPSCs utilizing Accutase and plate them at a density of 3.15 x 10⁵ cells per well into a 6-well plate previously coated with Matrigel (0.5 mg/ml). Culture the cells in Media A.

Days 1 - 4 (Mesoderm Induction): To begin differentiation into the mesodermal lineage, exchange the medium in each well every day with 2 ml of Media B.

Days 5 - 6 (Hematopoietic Specification): Daily, replace the medium with 2 ml of fresh Media C to encourage the transition of mesodermal cells into hemangioblasts. Please note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs.

Days 7 - 14 (Myeloid Progenitor Expansion): Refresh the medium each day with 2 ml of Media D to support differentiation into myeloid progenitors. From day 10 through day 14, harvest the supernatant daily and centrifuge it at 300g for 6 minutes to capture detached cells undergoing maturation. Carefully remove the supernatant, resuspend the resulting cell pellet in fresh medium, and return the cells back to their initial well.

Days 15-25 (Microglial Progenitor Maturation): Every 4 days (days 15, 19 & 23) replace the medium with 2ml of fresh Media E to promote myeloid progenitors to mature into microglial progenitors. As in stage 4 it is important to capture detached cells by centrifuging at 300g then resuspending the pellet in fresh medium and returning to the original well.

Day 26 (Isolating Microglial Progenitors): Collect the supernatant from each well and centrifuge cells at 300g for 6 minutes. Resuspend the pellet in fresh Media F and count the number of cells. Seed the cells at 9.5 x 10⁵ cells / well in Matrigel coated (0.5 mg/ml) coated 6-well plates.

Days 27 - 40 (Terminal differentiation of microglia): Continue culturing cells with media changes (Media F) every 3 - 4 days until microglia progenitors mature into terminally differentiated microglia.

Days 41 - 42 (Confirmation of phenotype): Fix cells in 4% paraformaldehyde and carry out immunocytochemistry using markers of mature microglia such as IBA1 and P2RY12. For more information please see our Immunocytochemistry (ICC) protocol.

References

Abud et al. 2017.iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. PMID: 28426964

Chen et al. 2021. Efficient conversion of human induced pluripotent stem cells into microglia by defined transcription factors. Stem Cell Reports PMID: 33836143

Douvaras et al. 2017. Directed Differentiation of Human Pluripotent Stem Cells to Microglia.Stem Cell Reports PMID: 28528700

Muffat et al. 2016. Efficient derivation of microglia-like cells from human pluripotent stem cells.Nature Medicine PMID: 27668937