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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 diseases1,2. 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 protocols7,8,12 with new modifications described herein.

1. Microglia differentiation

NOTE: An overview of the protocol is summarized in

Representative 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.......

Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

The authors thank Michael Ward for providing the WTC11 hNIL iPSC line for motor neuron differentiation and the Jackson Laboratories for supplying the KOLF2.1J WT clone B03 iPSC line used for microglia differentiation. We also thank Dorothy Schafer for her support during the implementation of the protocols, Anthony Giampetruzzi and John Landers for their help with the live-cell imaging system as well as Hayden Gadd for his technical contributions during revisions and Jonathan Jung for his collaboration in this study. This work was supported by the Dan and Diane Riccio Fund for Neuroscience from UMASS Chan Medical School and the Angel Fund, Inc.

....

Materials

NameCompanyCatalog NumberComments
Antibodies for immunofluorescence analysis
anti-IBA1 rabbit antibodyWako Chemical USANC92883641:350 dilution
anti-P2RY12 rabbit antibodySigma-AldrichHPA0145181:50 dilution
anti-TMEM119 rabbit antibodySigma-AldrichHPA0518701:100 dilution
Antibodies for Western blot analysis
anti-β-Tubulin rabbit antibodyAbcamab60461:500 dilution
anti-Synaptophysin (SYP) rabbit antibodyAbclonalA63441:1,000 dilution
anti-PSD95 mouse antibodyMilliporeMAB15961:500 dilution
Borate buffer components
Boric acid (100 mM)SigmaB6768
Sodium bicarbonate (NaHCO3) BioXtraSigma-AldrichS6297-250G
Sodium chloride (75 mM)Sigma S7653
Sodium tetraborate (25 mM)Sigma221732
Cell culture materials
6-well platesGreiner Bio-One657160
40 μm Cell Strainers Falcon352340
100 mm x 20 mm Tissue Culture TreatedCELLTREAT229620
Cell Lifter, Double End, Flat Blade & Narrow Blade, SterileCELLTREAT229305
low adherence round-bottom 96-well plateCorning7007
Primaria 24-well Flat Bottom Surface Modified Multiwell Cell Culture PlateCorning353847,
Primaria 6-well Cell Clear Flat Bottom Surface-Modified Multiwell Culture PlateCorning353846
Primaria 96-well Clear Flat Bottom MicroplateCorning353872
Cell dissociation reagents
Accutase Corning25058CIdissociation reagents used for lower motor neuron differentiation
TrypLE reagentLife Technologies12-605-010dissociation reagents used for microglia differentiation
UltraPure 0.5 M EDTA, pH 8.0Invitrogen15575020
Coating reagents for cell culture
Matrigel GFR Membrane MatrixCorning™354230Referred as to extracellular matrix coating reagent
CellAdhere Laminin-521STEMCELL Technology77004Referred as to laminin 521
Poly-D-LysineSigmaP7405Reconstitute to 0.1 mg/mL in borate buffer
Poly-L-OrnithineSigma P3655Reconstitute to 1 mg/mL in borate buffer
Components of iPSC media
 mTeSR Plus KitSTEMCELL Technology100-0276To prepare iPSC media mixed the components to 1x
Components of EB media
BMP-4Fisher ScientificPHC9534final concentration 50 ng/mL
iPSC mediafinal concentration 1x
ROCK inhibitor Y27632Fisher ScientificBD 562822final concentration 10 µM
SCFPeproTech300-07final concentration 20 ng/mL
VEGFPeproTech100-20Afinal concentration 50 ng/mL
Components of PMP base media
GlutaMAXGibco35050061final concentration 1x
Penicillin-Streptomycin (10,000 U/mL)Gibco15140122final concentration 100 U/mL
X-VIVO 15Lonza12001-988final concentration 1x
Components of PMP complete media
55 mM 2-mercaptoethanolGibco21985023final concentration 55 µM
IL-3PeproTech200-03final concentration 25 ng/mL
M-CSFPeproTech300-25final concentration 100 ng/mL
PMP base mediafinal concentration 1x
Components of iMG base media
Advanced DMEM/F12Gibco12634010final concentration 1x
GlutaMAXGibco35050061final concentration 1x
N2 supplement, 100xGibco17502-048final concentration 1x
Penicillin-Streptomycin (10,000 U/mL)Gibco15140122final concentration 100 U/mL
Components of iMG complete media
55 mM 2-mercaptoethanolGibco21985023final concentration 55 µM
IL-34PeproTech or Biologend200-34 or 577904final concentration 100 ng/mL
iMG base mediafinal concentration 1x
M-CSFPeproTech300-25final concentration 5 ng/mL
TGF-βPeproTech100-21final concentration 50 ng/mL
Components of Induction base media
DMEM/F12 with HEPESGibco11330032final concentration 1x
GlutaMAXGibco35050061final concentration 1x
N2 supplement, 100xGibco17502-048final concentration 1x
Non-essential amino acids (NEAA), 100xGibco11140050final concentration 1x
Components of Complete induction media
Compound ECalbiochem565790final concentration 0.2 μM and reconstitute stock reagent to 2 mM in 1:1 ethanol and DMSO
DoxycyclineSigmaD9891final concentration 2 μg/mL and reconstitute stock reagent to 2 mg/mL in DPBS
Induction base mediafinal concentration 1x
ROCK inhibitor Y27632Fisher ScientificBD 562822final concentration 10 μM
Components of Neuron media
B-27 Plus Neuronal Culture SystemGibcoA3653401final concentration 1x for media and suplemment
GlutaMAXGibco35050061final concentration 1x
N2 supplement, 100xGibco17502-048final concentration 1x
Non-essential amino acids (NEAA), 100xGibco11140050final concentration 1x
iPSC lines used in this study
KOLF2.1J: WT clone B03The Jackson Laboratories
WTC11 hNILNational Institute of Health
Synaptosome isolation reagents
BCA Protein Assay KitThermo Scientific Pierce23227
dimethyl sulfoxide (DMSO)SigmaD2650
Syn-PER Synaptic Protein Extraction ReagentThermo Scientific87793Referred as to cell lysis reagent for isolation of synaptosomes
Phagocytosis assay dyes
NucBlue Live Ready reagentInvitrogen R37605
pHrodo Red, succinimidyl esterThermoFisher Scientific P36600Referred as to pH-sensitive dye
Other cell-culture reagents
Trypan Blue, 0.4% SolutionAMRESCO INCK940-100ML
Bovine serum albumin (BSA)Sigma22144-77-0
BrdUSigmaB9285Reconstitute to 40 mM in sterile water
Cytochalasin DSigmafinal concentration 10 µM
DPBS with Calcium and magnesiumCorning21-030-CV
DPBS without calcium and magnesiumCorning21-031-CVReferred as to DPBS
KnockOut  DMEM/F-12Gibco12660012Referred as to DMEM-F12 optimized for growth of human embryonic and induced pluripotent stem cells
Laminin Mouse Protein, NaturalGibco23017015Referred as to laminin
Software and Equipment
CentrifugeEppendorfModel 5810R
Cytation 5 live cell imaging readerBiotek
Gen5 Microplate Reader and Imager SoftwareBiotekversion 3.03
Multi-Therm Heat-ShakeBenchmarkrefer as tube shaker
Water sonicatorElmaMode Transsonic 310

References

  1. Heider, J., Vogel, S., Volkmer, H., Breitmeyer, R. Human iPSC-derived glia as a tool for neuropsychiatric research and drug development. International Journal of Molecular Sciences. 22 (19), 10254 (2021).
  2. Muzio, L., Viotti, A., Martino, G.

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