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Model Robustness – application of Axol hiPSC-derived microglia in high-throughput 384-well format in real-time phagocytosis and efferocytosis assays

 The data presented in this application note is kindly provided by Essen BioScience, a Sartorius Company

Introduction

Microglia are commonly described as the immune cells of the brain. Under physiological conditions, they are vigilant guard keepers of their microenvironment, seeking out invading pathogens and clearing up cell debris, apoptotic cells, and misfolded proteins by migrating, phagocytosing, and producing cytokines and neurotrophins; all to maintain a homeostatic balance in the CNS.

Recent advances in stem cell technologies have led to the successful generation of microglia from human induced pluripotent stem cells (hiPSCs). To date, much of microglia research has been conducted using animal models, immortalized microglia-like cell lines (e.g. BV-2) or primary human microglia. Whilst these models have shed light on many aspects of microglial biology, hiPSC-derived microglia can offer several advantages over such traditional models, overcoming issues with limited cell number and availability, variability in homogeneity and unrepresentative inflammatory responses. Consequently, there is a pressing need for more practical, physiologically-relevant models to study microglial behaviour, especially in the context of complex neurodegenerative diseases.

Aims

  • 1. To describe the functional characteristics of hiPSC-derived microglia in phagocytosis and efferocytosis assays

Characterisation will include monitoring of phagocytosis and efferocytosis, two key microglial processes. Specifically, phagocytosis of S. aureus and efferocytosis of induced-apoptotic neurons.

2. To evaluate hiPSC-microglia robustness as a cellular model

Robustness will be tested by assaying in 384-well format to increase biological replicate number and variety of conditions tested


Figure 1. Plate view of masked fluorescence area plotted over time for each individual well in the 384-well plate

 Microglia were seeded into a 384-well plate at 100,000/cm2 and differentiated for 16 days to maturity. The plate was segmented based upon activation state, target material to be added, and presence of inhibitor (figure 1).

The plate was divided into 3 columns: in the first column the microglia were left untreated (resting state), in the second the microglia were polarized to the M1 state (100ng/mL LPS, 100ng/mL IFN𝛾) and in the third, the cells were polarized to M2 (100ng/mL IL-4, 100ng/mL IL-13).

The plate was also split lengthways into 4 equal rows. In the top two rows, pHrodo®-labelled S. aureus bioparticles (top row) and apoptotic Neuro-2a cells (second row) were added to cells pre-polarized to each of the activation states in increasing concentrations going from right to left.

In the bottom two rows, Cytochalasin D (Cyto D) was added in increasing concentrations (4.12 x E3 – 3 μM) going from right to left. Cyto D is an inhibitor of actin polymerization and therefore impedes uptake processes such as phagocytosis and efferocytosis. S. aureus bioparticles (1ug/mL) and apoptotic Neuro-2a (10,000 cells/well) were added at a constant density of along the entire third (S. aureus) and forth (Neuro-2a) rows.


 

 

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Figure 2. Mechanism of pH-sensitive pHrodo dye 
Target material labelled with the pH-sensitive fluorophore IncuCyte® pHrodo® reagent undergoes a dramatic increase in fluorescence upon engulfment, when encountering the acidic microenvironment of the phagolysosome (figure 2, right). In the video (figure 2, left), a microglial cell is captured over 40 minutes phagocytosing an pHrodo®-labelled apoptotic Neuro-2a cell. 


Methods

1. Seeding and generation of mature hiPSC-derived microglia

Microglial precursors were seeded at 100,000/cm2 into a 384-well plate (Corning, 3701) using a FluidX XPP-720 liquid handling system fitted with a 96-channel offset head. On Days 2 and 3, a 50% media change was performed using Microglia Maintenance Medium (Axol Bioscience, ax0660), and every other day thereafter until maturity.

2. Target material preparation

Apoptotic Neuro-2A cells were labelled using the IncuCyte® pHrodo® Orange Cell Labelling kit (Essen BioScience). To induce apoptosis, Neuro-2A cells were treated with 600 nM staurosporine for 24 hours, washed to remove the compound, and incubated with IncuCyte® pHrodo® Orange for 1 hour. After labelling, the cells were spun down and washed to remove excess dye.

For measurement of phagocytosis, pre-labelled IncuCyte® pHrodo® Red S. aureus Bioparticles® (Essen Bioscience, #4619) were used according to manufacturer’s instructions.

3. Polarisation of hiPSC-microglia to M1 and M2

To induce an M1 phenotype (classically-activated), microglia were treated for 24 hours with LPS and IFN𝛾 added to microglia medium at a final concentration of 100ng/mL for both. To polarise to M2/M2a (alternatively-activated), cells were treated with 100ng/mL IL-4 and 100ng/mL IL-13 for 24 hours.

3. Live-cell analysis of phagocytic and efferocytic function

After 16 days of microglial differentiation, target material was added and cells were incubated at 37oC, 5% C02 . Phase and fluorescent images were acquired every 30 minutes for 24 hours at 10x magnification using an IncuCyte® S3 Neuroscience instrument. Increasing concentrations of either IncuCyte® pHrodo®-labelled S. aureus bioparticles® (0.04 - 30 μg/mL) or apoptotic Neuro2A cells (7,000 – 50,000 cells/well) were used to measure phagocytic and efferocytic activity, respectively.

Target material labelled with the pH-sensitive fluorophore IncuCyte® pHrodo® reagent undergoes fluorescence enhancement upon engulfment, when encountering the acidic microenvironment of the phagolysosome. Non-engulfed objects have low intensity and were used to set the threshold for fluorescence change. Phagocytosis and efferocytosis were quantified using IncuCyte® software, in which background subtraction and automated segmentation were applied to accurately quantify the amount of fluorescence in the field of view. Data were expressed as the total fluorescent area recorded per image.

4. Inhibtion of phagocytosis and efferocytosis by Cytochalasin D

Cytochalasin D (Sigma, C2618) was serially diluted in microglia medium and added to cells in increasing concentrations (4.12 x E3 – 3 μM). Cells were incubated with Cyto D at 37oC, 5% C02 for 1 hour and washed once with medium prior to addition of IncuCyte® pHrodo®-labelled S. aureus bioparticles® or pHrodo®-labelled apoptotic Neuro2A cells.


Phagocytosis of S. aureus bioparticles

 

Figure 3. Time course of phagocytosis at several bioparticle densities for each activation state, M0, M1 and M2

Phagocytosis of S. aureus bioparticles
Phagocytosis of S. aureus bioparticles






Figure 4. Effect of activation state on bioparticle uptake. Endpoint fluorescence is plotted for each density in M0, M1 and M2 

Fluorescence increased with time as microglia engulfed the pHrodo®-labelled bioparticles; Uptake of bioparticles was concentration-dependent, where fluorescence increased more at higher bioparticle density (figure 3); Phagocytosis was activation state-dependent. M1 polarised cells phagocytosed less than M2 or non-activated cells (figure 4).  


Efferocytosis of apoptotic Neuro-2a cells

 

Figure 5. Efferocytosis uptake profiles at several N2A  densities for each activation state, M0, M1 and M2

Efferocytosis of apoptotic Neuro-2a cells
Efferocytosis of apoptotic Neuro-2a cells


Figure 6. Effect of activation state on Neuro-2a cell engulfment. Endpoint fluorescence is plotted for each density in M0, M1 and M2.

Fluorescence increased with time as microglia engulfed the pHrodo®-labelled apoptotic neurons. Uptake of apoptotic Neuro-2a cells was concentration-dependent, where fluorescence increased more at higher neuron density (figure 5) ; Efferocytosis was activation state-dependent. M1 polarised cells phagocytosed less than M2 or non-activated cells (figure 6). The rate of efferocytosis was non-linear; the rate is initially fast but then plateaus off. Compared to phagocytosis, efferocytosis was a much more rapid process.


Inhibition of Phagocytosis and Efferocytosis using Cytochalasin D

 


Through titration of Cytochalasin D, inhibition response curves can be accurately plotted to determine IC50 values.

During phagocytosis, microglia polarized to the M2 state are the most easily inhibited (pIC50=-6.2M), whereas the dose requirement to inhibit M1 microglia is much higher (pIC50=-5.6M).

During efferocytosis, M2 microglia are less easily inhibited than M1. 


Discussion

The M1 state is known to be associated with inflammation and clearance of pathogenic material by phagocytosis. While M1-activated cells engulf target material to a lesser extent than M2 and non-activated cells, phagocytosis is more difficult to inhibit, which may reflect a greater commitment to phagocytosis in the M1 phenotype.

The M2 state is associated with homeostasis and clearance of apoptotic neurons and debris by efferocytosis. Equally, as efferocytosis was the most challenging to inhibit in M2-polarized microglia, this may reflect a bias toward efferocytosis in the M2 phenotype.


The low degree of variability displayed between technical replicates over multiple densities of target material strongly indicates Axol’s hiPSC-microglia to be suited to high-throughput screens and assay formats.


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