Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Summary

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Abstract

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i³LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Introduction

Microglia are the resident immune cells in the central nervous system (CNS) and play a crucial role in developing the CNS. Microglia are also important in the adult brain for maintaining homeostasis and actively responding to trauma and disease processes. Cumulative evidence shows that microglia are key contributors to the pathogenesis of multiple neurodevelopmental and neurodegenerative diseases^1,2. Although current knowledge about microglial biology has been predominately derived from mouse models, recent studies have elucidated important differences between murine and human microglia, underscoring the need ....

Protocol

NOTE: All the reagents used in this protocol must be sterile, and all the steps must be performed in a biosafety cabinet under sterile conditions. All the iPSC lines, as well as maintenance and differentiation media, are described in the Table of Materials. The microglia differentiation method illustrated below is based on previously published protocols^7,8,12 with new modifications described herein.

1. Microglia differentiation

NOTE: An overview of the protocol is summarized in

Results

To generate iMGs using this protocol, it is important to start with undifferentiated iPSCs that show compact colony morphology with well-defined edges (Figure 2A). Dissociated iPSCs maintained as described in the EB formation section will form spherical aggregates, termed EBs, which will grow in size until day 4 of differentiation (Figure 2B). Once the EBs are collected and plated in the appropriate conditions for PMP generation, they attach to the Matrigel-coat.......

Discussion

The differentiation protocol described here provides an efficient method to obtain iPSC-derived microglia-like cells in ~6-8 weeks with high purity and in a sufficient yield to perform immunofluorescence experiments and other assays that require a higher number of cells. This protocol has yielded up to 1 × 10⁶ iMGs in 1 week, which allows for protein and RNA extraction and corresponding downstream analyses (e.g., RNASeq, qRT-PCR, western blot, mass spectrometry). That said, a limitation of this protocol i.......

Materials

Name	Company	Catalog Number	Comments
Antibodies for immunofluorescence analysis
anti-IBA1 rabbit antibody	Wako Chemical USA	NC9288364	1:350 dilution
anti-P2RY12 rabbit antibody	Sigma-Aldrich	HPA014518	1:50 dilution
anti-TMEM119 rabbit antibody	Sigma-Aldrich	HPA051870	1:100 dilution
Antibodies for Western blot analysis
anti-β-Tubulin rabbit antibody	Abcam	ab6046	1:500 dilution
anti-Synaptophysin (SYP) rabbit antibody	Abclonal	A6344	1:1,000 dilution
anti-PSD95 mouse antibody	Millipore	MAB1596	1:500 dilution
Borate buffer components
Boric acid (100 mM)	Sigma	B6768
Sodium bicarbonate (NaHCO3) BioXtra	Sigma-Aldrich	S6297-250G
Sodium chloride (75 mM)	Sigma	S7653
Sodium tetraborate (25 mM)	Sigma	221732
Cell culture materials
6-well plates	Greiner Bio-One	657160
40 μm Cell Strainers	Falcon	352340
100 mm x 20 mm Tissue Culture Treated	CELLTREAT	229620
Cell Lifter, Double End, Flat Blade & Narrow Blade, Sterile	CELLTREAT	229305
low adherence round-bottom 96-well plate	Corning	7007
Primaria 24-well Flat Bottom Surface Modified Multiwell Cell Culture Plate	Corning	353847,
Primaria 6-well Cell Clear Flat Bottom Surface-Modified Multiwell Culture Plate	Corning	353846
Primaria 96-well Clear Flat Bottom Microplate	Corning	353872
Cell dissociation reagents
Accutase	Corning	25058CI	dissociation reagents used for lower motor neuron differentiation
TrypLE reagent	Life Technologies	12-605-010	dissociation reagents used for microglia differentiation
UltraPure 0.5 M EDTA, pH 8.0	Invitrogen	15575020
Coating reagents for cell culture
Matrigel GFR Membrane Matrix	Corning™	354230	Referred as to extracellular matrix coating reagent
CellAdhere Laminin-521	STEMCELL Technology	77004	Referred as to laminin 521
Poly-D-Lysine	Sigma	P7405	Reconstitute to 0.1 mg/mL in borate buffer
Poly-L-Ornithine	Sigma	P3655	Reconstitute to 1 mg/mL in borate buffer
Components of iPSC media
mTeSR Plus Kit	STEMCELL Technology	100-0276	To prepare iPSC media mixed the components to 1x
Components of EB media
BMP-4	Fisher Scientific	PHC9534	final concentration 50 ng/mL
iPSC media			final concentration 1x
ROCK inhibitor Y27632	Fisher Scientific	BD 562822	final concentration 10 µM
SCF	PeproTech	300-07	final concentration 20 ng/mL
VEGF	PeproTech	100-20A	final concentration 50 ng/mL
Components of PMP base media
GlutaMAX	Gibco	35050061	final concentration 1x
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122	final concentration 100 U/mL
X-VIVO 15	Lonza	12001-988	final concentration 1x
Components of PMP complete media
55 mM 2-mercaptoethanol	Gibco	21985023	final concentration 55 µM
IL-3	PeproTech	200-03	final concentration 25 ng/mL
M-CSF	PeproTech	300-25	final concentration 100 ng/mL
PMP base media			final concentration 1x
Components of iMG base media
Advanced DMEM/F12	Gibco	12634010	final concentration 1x
GlutaMAX	Gibco	35050061	final concentration 1x
N2 supplement, 100x	Gibco	17502-048	final concentration 1x
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122	final concentration 100 U/mL
Components of iMG complete media
55 mM 2-mercaptoethanol	Gibco	21985023	final concentration 55 µM
IL-34	PeproTech or Biologend	200-34 or 577904	final concentration 100 ng/mL
iMG base media			final concentration 1x
M-CSF	PeproTech	300-25	final concentration 5 ng/mL
TGF-β	PeproTech	100-21	final concentration 50 ng/mL
Components of Induction base media
DMEM/F12 with HEPES	Gibco	11330032	final concentration 1x
GlutaMAX	Gibco	35050061	final concentration 1x
N2 supplement, 100x	Gibco	17502-048	final concentration 1x
Non-essential amino acids (NEAA), 100x	Gibco	11140050	final concentration 1x
Components of Complete induction media
Compound E	Calbiochem	565790	final concentration 0.2 μM and reconstitute stock reagent to 2 mM in 1:1 ethanol and DMSO
Doxycycline	Sigma	D9891	final concentration 2 μg/mL and reconstitute stock reagent to 2 mg/mL in DPBS
Induction base media			final concentration 1x
ROCK inhibitor Y27632	Fisher Scientific	BD 562822	final concentration 10 μM
Components of Neuron media
B-27 Plus Neuronal Culture System	Gibco	A3653401	final concentration 1x for media and suplemment
GlutaMAX	Gibco	35050061	final concentration 1x
N2 supplement, 100x	Gibco	17502-048	final concentration 1x
Non-essential amino acids (NEAA), 100x	Gibco	11140050	final concentration 1x
iPSC lines used in this study
KOLF2.1J: WT clone B03	The Jackson Laboratories
WTC11 hNIL	National Institute of Health
Synaptosome isolation reagents
BCA Protein Assay Kit	Thermo Scientific Pierce	23227
dimethyl sulfoxide (DMSO)	Sigma	D2650
Syn-PER Synaptic Protein Extraction Reagent	Thermo Scientific	87793	Referred as to cell lysis reagent for isolation of synaptosomes
Phagocytosis assay dyes
NucBlue Live Ready reagent	Invitrogen	R37605
pHrodo Red, succinimidyl ester	ThermoFisher Scientific	P36600	Referred as to pH-sensitive dye
Other cell-culture reagents
Trypan Blue, 0.4% Solution	AMRESCO INC	K940-100ML
Bovine serum albumin (BSA)	Sigma	22144-77-0
BrdU	Sigma	B9285	Reconstitute to 40 mM in sterile water
Cytochalasin D	Sigma		final concentration 10 µM
DPBS with Calcium and magnesium	Corning	21-030-CV
DPBS without calcium and magnesium	Corning	21-031-CV	Referred as to DPBS
KnockOut  DMEM/F-12	Gibco	12660012	Referred as to DMEM-F12 optimized for growth of human embryonic and induced pluripotent stem cells
Laminin Mouse Protein, Natural	Gibco	23017015	Referred as to laminin
Software and Equipment
Centrifuge	Eppendorf	Model 5810R
Cytation 5 live cell imaging reader	Biotek
Gen5 Microplate Reader and Imager Software	Biotek	version 3.03
Multi-Therm Heat-Shake	Benchmark		refer as tube shaker
Water sonicator	Elma	Mode Transsonic 310