Microglia, the resident immune cells of the brain, are intimately involved in safeguarding and fortifying neurons against harm.
However, mounting evidence indicates that chronic activation of microglia can create a neurotoxic environment, potentially driving pathological processes in neurodegenerative diseases like Alzheimer’s.
In this application note, researchers from Medicines Discovery Catapult (MDC) used iPSC-derived microglia derived from opti-ox deterministic reprogramming to study the mechanics of microglia activation, shedding light on their role in neurodegeneration and and their potential as therapeutic targets.
Download this app note to learn about:
- Functional, consistent and scalable iPSC-derived microglia that allow for rapid and accurate modeling of neurodegenerative diseases
- How to measure the chemotaxis of these cells in response to disease-relevant stimuli
- The complex mechanisms of microglial activation and chemotaxis in conditions like Alzheimer’s
Microglia, the resident immune cells of the brain, play a crucial
role in maintaining central nervous system homeostasis through
mechanisms such as phagocytosis and trogocytosis, contributing
to neuronal protection. Toll-like receptors on microglia detect
pathogens or molecules associated with neuronal damage,
triggering a cascade of responses that include the production
of inflammatory molecules and migration towards stimuli via
chemotaxis. However, chronic microglial activation can lead to
neurotoxicity, particularly evident in neurodegenerative diseases
like Alzheimer’s, where microglia are observed to accumulate
around neuritic plaques. The involvement of proteins like
amyloid β, hyperphosphorylated tau, and complement protein
5a (C5a) in driving neuroinflammation underscores their potential
as therapeutic targets. To support the study of microglia’s role in
disease, bit.bio has developed ioMicroglia*: functional, consistent
and scalable human microglia derived from induced pluripotent
stem cells (iPSCs) with opti-ox* precision reprogramming.
In this application note, researchers from Medicines Discovery
Catapult (MDC) assessed whether ioMicroglia are an appropriate
model for studying chemotactic responses to C5a in-vitro, and
showed that the cells display C5a-mediated chemotaxis in-vitro
in a dose-dependent manner.
In this application note,
readers will learn:
About the intricate mechanisms
of microglial activation and
chemotaxis in neurodegenerative
diseases like Alzheimer’s.
The role of C5a and other proteins
in driving neuroinflammation.
The C5a dose-dependant
chemotaxis phenotype of
ioMicroglia, and their suitability
as a model for studying microglial
responses to chemotactic stimuli
in-vitro.
* “io”, “io” and “opti-ox” are trade marks owned and/or registered by Bit Bio Limited
2
C5a gradient
io Microglia
1-100 nM C5a
chemoattractant
io Microglia move through
pores in plate insert towards
C5a chemoattractant
Chemotaxis
measured
every 4 hours
Reservoir
Insert
Introduction
ioMicroglia are seeded into the insert of an Incucyte®
Clearview 96-well microtiter plate, consisting of an
optically clear membrane with 96 laser-etched pores
(sitting in a reservoir plate), and cultured for 7 days,
as per the ioMicroglia user manual.
C5a chemoattractant is then added to the
reservoir plate at various concentrations
ranging from 1 nM to 100 nM.
The plate is scanned for cell migration every
4 hours for up to 60 hours and imaged using the
Incucyte® Live-Cell Analysis System with the
Incucyte® Chemotaxis Analysis Software Module
to quantify chemotactic transmembrane migration
as the cells migrate through pores towards the
chemoattractant.
Microglia are brain-resident immune cells with
an increasingly recognized role in maintaining
homeostasis within the central nervous system.
Through phagocytosis (the engulfment of
large cellular structures) and trogocytosis
(the contact-dependent engulfment of small
structures), microglia support local innate
immune responses and mediate neuronal
plasticity1,2. Microglia are thus intimately
involved in protecting and fortifying neurons
from harm. However, mounting evidence
indicates that chronic activation of microglia
can lead to a neurotoxic environment and
may be a driving pathological process in
neurodegenerative disease3.
One such disease is Alzheimer’s disease,
wherein microglia activation appears
chronic and prolific, with the cells observed
accumulating around disease-defining neuritic
plaques. Studying the mechanics of microglia
activation in Alzheimer’s disease may shed
critical light on disease pathology and reveal
potential therapeutic strategies4.
It is known that, through an arsenal of tolllike receptors, ramified microglia survey the
neuronal microenvironment for molecules with
pathogen- or damage-associated molecular
patterns. Once detected, microglia transition
into an activated state that involves migrating
towards the antagonistic signal (chemotaxis),
producing numerous inflammatory molecules
(TNF-α, IL-6, IL-1), and upregulating proteins
involved in phagocytosis (among other
transcriptional and morphological changes)5.
Multiple proteins are suspected to play a role
in driving microglia activation in Alzheimer’s
disease, including the complement protein
5a (C5a)6. Like other complement proteins,
the interaction of C5a with its cognate
receptor (C5aR) on immune cells elicits an
inflammatory response. Importantly, multiple
complement proteins have been found to be
enriched near neuritic plaques in patients with
Alzheimer’s disease7. While the mechanics
of microglia activation in neurodegenerative
disease are not well understood, it is likely that
proteins like amyloid β, hyperphosphorylated
tau, and C5a are powerful drivers of
neuroinflammation.
A key part of microglial activation is the cells’
ability to migrate towards a stimulus (such
as C5a) through chemotaxis3. Damaged or
infected cells form a signalling epicentre, from
which triggering stimuli diffuse outward into
the surrounding neuronal tissue. Microglia
sense these antagonistic gradients and are
triggered to navigate towards the source
where, when exposed to greater stimulant
concentrations, they mount an immune
response. Precisely how this chemotactic
response is triggered in neurodegenerative
disease is not well understood, though
the involvement of proteins like amyloid
β, hyperphosphorylated tau and C5a are
suspected8. Therefore studying the factors
that trigger and mediate this migration may
shed light on the pathogenesis of microglia in
neurodegeneration and help identify potential
therapeutic targets.
To support the study of microglia’s role in
disease, bit.bio has developed ioMicroglia:
functional, consistent and scalable human
microglia derived from induced pluripotent
stem cells (iPSCs) with opti-ox precision
reprogramming. In order for iPSC-derived
cells to be useful as an in vitro model to
unpick the complexities of neuroinflammation
and neurodegenerative disease, they need
to accurately recapitulate in vivo cellular
functions in vitro. In this application note,
researchers from Medicines Discovery
Catapult (MDC) utilized ioMicroglia and
the Incucyte® Chemotaxis Cell Migration
Assay, which uses an advanced transwell
system, to study whether ioMicroglia are an
appropriate model for studying chemotactic
responses to C5a in vitro. MDC uses
complex cell models and assays to support
drug discovery innovators developing new
therapeutics. This study shows that ioMicroglia
display chemotaxis in response to C5a in a
dose-dependent manner, making them a
physiologically relevant model for assays
used in the development of new therapeutic
candidates targeting cell migration.
Chemotaxis assay workflow
3
3.4
106 105 104 103 -102.5 106 105 104 103 -102.5
C5aR-H
Isotype control
All cells
Singlets
Live cells
C5aR antibody
All cells
Singlets
Live cells
APC-H
2.4
C5aR+
0.28%
C5aR+
99.56%
1.6
0.8
0
3.4
2.4
1.6
0.8
0
Results
ioMicroglia show
homogenous expression
of the C5a receptor protein
C5a receptor (C5aR) expression is needed
to mediate a chemotaxis response to the
C5a chemoattractant in microglia. Expression
of C5aR in ioMicroglia was initially assessed
by flow cytometry, using a C5aR antibody
(Biolegend 344310). Over 99.5% of the
ioMicroglia population were observed to
express C5aR (Figure 1). This high expression
of the C5aR within the ioMicroglia population
indicates that the cells are equipped to show
a C5a-mediated chemotaxis response.
Figure 1: Flow cytometry analysis of
ioMicroglia shows expression of C5aR
(Biolegend 344310) with a purity of
above 99% versus the isotype control
(Biolegend 400219).
ioMicroglia display
C5a-mediated chemotaxis
in a dose-dependent manner
Rates of chemotaxis in response to varying
levels of a C5a chemoattractant were
assessed using the Incucyte® Chemotaxis
Analysis Software Module. This module
measures the change in the number of cells on
the top surface of a membrane from those on
the bottom surface of a membrane, in order
to quantify the movement of cells over time.
ioMicroglia migrated towards C5a via pores
in the insert into a reservoir containing the
chemoattractant in a dose-dependent manner
(Figure 2). Maximal migration was observed
with 10 nM of C5a with a 5-fold increase in
ioMicroglia present in the reservoir at 60
hours. This means that researchers can use
ioMicroglia to model chemotaxis in-vitro to
better understand neurodegenerative disease
mechanisms and guide drug development.
50 100
-800000
-600000
-400000
-200000
0
200000
Cells in insert
Control
1 nM
3 nM
10 nM
30 nM
100 nM
Control
1 nM
3 nM
10 nM
30 nM
100 nM
Time (hours)
Control 1 nM 3 nM 10 nM 30 nM 100 nM
0
1
2
3
4
