Name: human microglia-like cells: differentiation from induced pluripotent stem cells and in vitro live-cell phagocytosis assay using human synaptosomes
Uploaded: 2022-08-18T14:30:00
Description: Watch this Scientific Journal Video about Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes at JoVE.com

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.

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.
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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 &#215; 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...

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 for developing technologies to study the genetics and biological functions of human microglia3,4. Isolation of microglia from dissected primary tissue can severely modify microglia properties5, potentially confounding results acquired with such cells. The overall goal of this method is to differentiate human iPSCs into iMGs, thereby providing a cell culture system to study human microglia under basal conditions. Furthermore, a phagocytosis assay using a fully human model system is included herein as a means to study the functionality of iMGs, both as a quality control measure and to assess iMG dysfunction in the context of disease.
Multiple protocols for microglia differentiation from iPSCs have recently emerged in the literature6,7,8,9,10. Potential disadvantages of some protocols include extended or long periods of differentiation, the addition of multiple growth factors, and/or complex experimental procedures6,9,10. Here, a &#34;user-friendly&#34; differentiation method is demonstrated that recapitulates aspects of microglia ontogeny through differentiation of iPSCs into precursor cells termed primitive macrophage precursors (PMPs)7,11. PMPs are generated as described previously, with some optimizations presented herein12. The PMPs mimic MYB-independent yolk-sac-derived macrophages, which give rise to microglia during embryonic development by invading the brain before blood-brain barrier closure13. To terminally differentiate PMPs into iMGs, we used a fast and simplified monoculture method based on protocols by Haenseler et al. and Brownjohn et al., with some modifications to generate an efficient microglia differentiation method in which iMGs robustly express microglia-enriched markers7,8. This differentiation method can be reproduced in laboratories with expertise in the culture of iPSCs and with research goals aiming to study microglia biology using a human model system.
iPSC-derived microglia represent a biologically relevant source of human microglia for in vitro experimentation and are an important tool to investigate microglial canonical functions, including phagocytosis. Microglia are the professional phagocytes of the brain and CNS, where they clear cell debris, aggregated proteins, and degraded myelin14. Microglia also function in synaptic remodeling by engulfing synapses and in the defense against external infections through phagocytosis of pathogens15,16. In this protocol, phagocytosis by iMGs is assessed using human synaptosomes as material for iMG engulfment. To this end, a description for isolating synaptosomes derived from human i3LMNs is described. The i3LMN-derived human synaptosomes are labeled with a pH-sensitive dye that allows for quantification of synaptosomes localized within acidic compartments during phagosome processing and degradation in vitro. A phagocytosis assay using live-cell microscopy is shown for monitoring the dynamic process of microglia engulfment in real-time. This functional assay establishes a basis to investigate possible defects in microglial phagocytosis in health and disease using a complete human system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies for immunofluorescence analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA1 rabbit antibody</td>
			<td>Wako Chemical USA</td>
			<td>NC9288364</td>
			<td>1:350 dilution</td>
		</tr>
		<tr>
			<td>anti-P2RY12 rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA014518</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>anti-TMEM119 rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA051870</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Antibodies for Western blot analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-Tubulin rabbit antibody</td>
			<td>Abcam</td>
			<td>ab6046</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>anti-Synaptophysin (SYP) rabbit antibody</td>
			<td>Abclonal</td>
			<td>A6344</td>
			<td>1:1,000 dilution</td>
		</tr>
		<tr>
			<td>anti-PSD95 mouse antibody</td>
			<td>Millipore</td>
			<td>MAB1596</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Borate buffer components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid (100 mM)</td>
			<td>Sigma</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3) BioXtra</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (75 mM)</td>
			<td>Sigma</td>
			<td>&#160;S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate (25 mM)</td>
			<td>Sigma</td>
			<td>221732</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m Cell Strainers</td>
			<td>&#160;Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm x 20 mm Tissue Culture Treated</td>
			<td>CELLTREAT</td>
			<td>229620</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter, Double End, Flat Blade &#38; Narrow Blade, Sterile</td>
			<td>CELLTREAT</td>
			<td>229305</td>
			<td></td>
		</tr>
		<tr>
			<td>low adherence round-bottom 96-well plate</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 24-well Flat Bottom Surface Modified Multiwell Cell Culture Plate</td>
			<td>Corning</td>
			<td>353847,</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 6-well Cell Clear Flat Bottom Surface-Modified Multiwell Culture Plate</td>
			<td>Corning</td>
			<td>353846</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 96-well Clear Flat Bottom Microplate</td>
			<td>Corning</td>
			<td>353872</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell dissociation reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>&#160;Corning</td>
			<td>25058CI</td>
			<td>dissociation reagents used for lower motor neuron differentiation</td>
		</tr>
		<tr>
			<td>TrypLE reagent</td>
			<td>Life Technologies</td>
			<td>12-605-010</td>
			<td>dissociation reagents used for microglia differentiation</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating reagents for cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel GFR Membrane Matrix</td>
			<td>Corning&#8482;</td>
			<td>354230</td>
			<td>Referred as to extracellular matrix coating reagent</td>
		</tr>
		<tr>
			<td>CellAdhere Laminin-521</td>
			<td>STEMCELL Technology</td>
			<td>77004</td>
			<td>Referred as to laminin 521</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P7405</td>
			<td>Reconstitute to 0.1 mg/mL in borate buffer</td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Sigma</td>
			<td>&#160;P3655</td>
			<td>Reconstitute to 1 mg/mL in borate buffer</td>
		</tr>
		<tr>
			<td>Components of iPSC media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;mTeSR Plus Kit</td>
			<td>STEMCELL Technology</td>
			<td>100-0276</td>
			<td>To prepare iPSC media mixed the components to 1x</td>
		</tr>
		<tr>
			<td>Components of EB media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>Fisher Scientific</td>
			<td>PHC9534</td>
			<td>final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>iPSC media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Fisher Scientific</td>
			<td>BD 562822</td>
			<td>final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>PeproTech</td>
			<td>300-07</td>
			<td>final concentration 20 ng/mL</td>
		</tr>
		<tr>
			<td>VEGF</td>
			<td>PeproTech</td>
			<td>100-20A</td>
			<td>final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Components of PMP base media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>final concentration 100 U/mL</td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Lonza</td>
			<td>12001-988</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Components of PMP complete media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>final concentration 55 &#181;M</td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>PeproTech</td>
			<td>200-03</td>
			<td>final concentration 25 ng/mL</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td>final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>PMP base media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Components of iMG base media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>N2 supplement, 100x</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>final concentration 100 U/mL</td>
		</tr>
		<tr>
			<td>Components of iMG complete media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>final concentration 55 &#181;M</td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td>PeproTech or Biologend</td>
			<td>200-34 or 577904</td>
			<td>final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>iMG base media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td>final concentration 5 ng/mL</td>
		</tr>
		<tr>
			<td>TGF-&#946;</td>
			<td>PeproTech</td>
			<td>100-21</td>
			<td>final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Components of Induction base media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with HEPES</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>N2 supplement, 100x</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA), 100x</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Components of Complete induction media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound E</td>
			<td>Calbiochem</td>
			<td>565790</td>
			<td>final concentration 0.2 &#956;M and reconstitute stock reagent to 2 mM in 1:1 ethanol and DMSO</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D9891</td>
			<td>final concentration 2 &#956;g/mL and reconstitute stock reagent to 2 mg/mL in DPBS</td>
		</tr>
		<tr>
			<td>Induction base media</td>
			<td></td>
			<td></td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Fisher Scientific</td>
			<td>BD 562822</td>
			<td>final concentration 10 &#956;M</td>
		</tr>
		<tr>
			<td>Components of Neuron media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Plus Neuronal Culture System</td>
			<td>Gibco</td>
			<td>A3653401</td>
			<td>final concentration 1x for media and suplemment</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>N2 supplement, 100x</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA), 100x</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>final concentration 1x</td>
		</tr>
		<tr>
			<td>iPSC lines used in this study</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KOLF2.1J: WT clone B03</td>
			<td>The Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WTC11 hNIL</td>
			<td>National Institute of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synaptosome isolation reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Scientific Pierce</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Syn-PER Synaptic Protein Extraction Reagent</td>
			<td>Thermo Scientific</td>
			<td>87793</td>
			<td>Referred as to cell lysis reagent for isolation of synaptosomes</td>
		</tr>
		<tr>
			<td>Phagocytosis assay dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Live Ready reagent</td>
			<td>Invitrogen</td>
			<td>&#160;R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red, succinimidyl ester</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;P36600</td>
			<td>Referred as to pH-sensitive dye</td>
		</tr>
		<tr>
			<td>Other cell-culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% Solution</td>
			<td>AMRESCO INC</td>
			<td>K940-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>22144-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Sigma</td>
			<td>B9285</td>
			<td>Reconstitute to 40 mM in sterile water</td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Sigma</td>
			<td></td>
			<td>final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>DPBS with Calcium and magnesium</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>Referred as to DPBS</td>
		</tr>
		<tr>
			<td>KnockOut&#160; DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>Referred as to DMEM-F12 optimized for growth of human embryonic and induced pluripotent stem cells</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td>Referred as to laminin</td>
		</tr>
		<tr>
			<td>Software and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 live cell imaging reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 Microplate Reader and Imager Software</td>
			<td>Biotek</td>
			<td>version 3.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Therm Heat-Shake</td>
			<td>Benchmark</td>
			<td></td>
			<td>refer as tube shaker</td>
		</tr>
		<tr>
			<td>Water sonicator</td>
			<td>Elma</td>
			<td>Mode Transsonic 310</td>
			<td></td>
		</tr>
	</tbody>
</table>

human microglia-like cells: differentiation from induced pluripotent stem cells and in vitro live-cell phagocytosis assay using human synaptosomes

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.

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...

Watch this Scientific Journal Video about Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes at JoVE.com

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

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.

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

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Research

JoVE Journal

Neuroscience

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