Development of 96/384-well kinetic neurite outgrowth/stabilisation assays in
human iPSC-derived neurons using long term live cell imaging
S. L. Alcantara, J. Brown, P. Garay, L. McGillicuddy, T. O’Callaghan, T. Dale, D. Appledorn, O. McManus, V. Groppi & D.J. Trezise
Essen BioScience, Welwyn Garden City, UK & Ann Arbor, Michigan, 48108 USA
0.4
0.5
-6
300
66 h
284 h
IC50
52 µM
144 µM
-3
100
50
Vehicle
31M
250M
63M
500M
8
2.5
6
2.0
4
66-92 h
212-284 h
1.5
2. Treat cells with neuro-degenerative and/or neuro-protective agents.
•
200
300
-6
Phase image processing & quantification
0.4
300
200
0.5
-6
-3
3.0
100
50
-4
Log [Glut acid] (M)
IC50
10 µM
65 µM
66 h
284 h
-5
Vehicle
125M
4M
250M
31M
500M
63M
1000M
10
8
2.5
6
2.0
4
66-92 h
212-284 h
1.5
0
Fluorescence image processing & quantification
Tuj-1
100
200
300
Time (h)
Log [Kainic acid] (M)
-6
-5
-4
Early
Late
IC50
8 µM
2 µM
150
150
100
100
100
50
3.7 M
1.23 M
0.41 M
0.14 M
Vehicle (0.3% H2O)
230 M Kainic acid
33 M
11 M
0
50
50
0
0
-50
66-92 h
212-284 h
-50
0
100
200
Late phase neuroprotection (% IC80 KA)
Normalised Neurite length (% 66h)
150
300
-8
-7
-6
-5
-4
-3
Log [CNQX] (M)
Time (h)
0
0
100
200 100
0
100Time (h)
200
Time (h)
300
300
• Time-courses compare mean ± SEM neurite
2)
-100
length (mm/mm2) or branch point (1/mm
responses to increasing concentrations of
the
-200
growth factor BDNF (n=4) .
0
• Plate view shows the kinetics of neurite length
for human iCell Neurons in mono-culture.
Inter-well variability plot shows maximum neurite length ± SEM (mm/mm2) at several time points up to
14 days (CV= 12%, n=56 wells).
(mm/mm2)
www.essenbioscience.com
% Neuroprotection
length (% 66h)
Neurite
Normalised(%
IC80 KA)
100
50
0
0
100
200
300
150
100
50
0
48
96 144 192 264 312
Time (h)
Time (h)
150
50
300
78 M
312 M
1250 M
5000 M
Control
100
50
7 10 13 15 21
• Rat primary cortical neurons co-cultured with rat
primary astrocytes (Global Stem).
• End point staining (Green = Tuj1 primary antibody,
Red=mKate2 infection) indicates an infection efficiency
of 79 ± 3% at day 7 (n=8 wells, 9 images/well).
• Temporal profile of neurite length (mm/mm2) from
transiently infected red fluorescent protein.
• Variability plot of maximum red neurite length ± SEM
(mm/mm2) at several time points up to 13 days (CV=
5%, n=96 wells).
150
Kainic acid
150
100
50
15 M
63 M
250 M
Control
L-Glutamic acid
100
50
100
100
50
0
100
Vehicle (0.3% H2O
0
230 M Kainic acid
-100
33 M 200
Time (h)
-200
11 M
3.7 M
0
1.23 M
0.41 M
-100
0.14
M
-200
300
% neuroprotection calculated by normalising to the vehicle and kainic acid controls.
We thank Cellular Dynamics International for
technical support with iCell Neurons.
200
78 M
312 M
1250 M
5000 M
Control
150
100
100
11 M
3.7 M
1.23 M
0.41 M H1152
0.14 M
50
200
33 M 200
Time
Time(h)
(h)
300
300
• Treatment with CNQX, the growth factor
BDNF, or the RhoK0 inhibitor H1152 at 48
0
100
h followed by treatment on their own
Timeor
(h)
with an IC80 of kainic acid at 72 h.
• No effect on neurite formation exerted
by CNQX or H1152 in the absence of KA.
In contrast, BDNF augmented neurite
formation.
• Co-application with KA: CNQX and BDNF
inhibited and H1152 exacerbated KA
effects exemplifying different temporal
effects.
100
50
0
0
300
200
150
100
50
0
200
0 Time (h)
0
300
150
300
100
200
Time (h)
150
Red neurite length (mm/mm2)
Media
Time
CNQX(h)
100 0
Red neurite length (mm/mm2)
100
Co-culture
Red neurite length (mm/mm2)
230 M Kainic acid
300
100
0
-200
50
(100 µM)
200
0
300
EC80 KA
100 H O
Vehicle (0.3%
2
(50 ng/ml)
200
0
50
(33 µM)
200
-100
100
H1152
300
0
BDNF
BDNF
+ EC80 KA
- KA
100
150
Late phase
0
6
Differential excitotoxic effects in mono- vs co-culture
100
0
Early phase
50
150
0
EC80 KA
200
5
days post-coculture
+ EC80 kainic acid
150
100
CNQX
Late phase
100
50
3
0
0
300
300
Early phase
Time (h)
300
300
Early
Late
IC50
8 µM
2 µM
200
33 M 200
Time(h)
(h)
Time
Vehicle
200
150
100
100
100
Late phase
Time (h)
100
150
200
Early
Late
00
Early phase
12 24 48 72 120 168 336
0
0
250
EC80 KA
6
0
50
Late phase
0
50
Vehicle (0.3% H2O
230 M Kainic acid
0
0
200
11 M
3.7 M
1.23 M
0.41 M
0.14 M
50
Early phase
0
100
100
Early phase
20
150
300
0.14 ug/ml
0.01 ug/ml
Vehicle
Media
H1152
CNQX
EC80 KA
40
200
100 ug/ml
11 ug/ml
1.23 ug/ml
150
100
Late phase
60
250
(1/mm2)
points
branch
Normalised
(% 66h)
length
Neurite
Normalised
Normalised Neurite length (mm/mm 2)
Neurite length (mm/mm2)
Maximise data per well for high value cell types
150
200
IC50
8 µM
2 µM
100
BDNF
Vehicle
• Phase-contrast images and analysis masks (Yellow = neurite, Blue = cell body) show the kinetics of
neurite outgrowth in human iCell Neurons in mono- and co-culture.
• Masks quantify neurite length (mm/mm2), cell body clusters (1/mm2), and branch points (1/mm2) for
human iCell Neurons over the experimental time-course.
No kainic acid
Late phase
Neurite length 55.7 mm/mm2
Cell body cluster 463 /mm2
Branch points 727 /mm2
Early phase
Neurite length 47.6 mm/mm2
Cell body cluster 491 /mm2
Branch points 557 /mm2
Early
Late
Vehicle
Neurite length 32.0mm/mm2
Cell body cluster 493 /mm2
Branch points 346 /mm2
Normalised Neurite length (% 66h)
Neurite length 4.2 mm/mm2
Cell body cluster 495 /mm2
Branch points 31 /mm2
% Neuroprotection
Normalised Neurite length (% 66h)
150
200
150
0
Differential neuroprotective kinetics
IC50
8 µM
2 µM
combined
Log [Glutamic acid] (M)
• Excitotoxitity induced by an IC80
concentration of KA (230 µM) to
iCells in mono-culture at 72 h. Timecourse data normalised to 66 h
neurite length (mean ± SEM, n=4).
• CNQX,
the
kainate
receptor
antagonist, inhibited
both the initial
150
and late phases of kainic acidinduced neurite disruption.
-100
-9
mKate2
-3
CNQX reversal of KA-induced excitotoxicity
Day 14
Neurite length 137.8 mm/mm2
Branch points 2861 /mm2
5. Monitor in the IncuCyte ZOOM live-cell imaging
system: capture and analyse phase and fluorescent
images every 6-12 h for the assay length. (Red =
mKate2, Blue = segmentation mask).
2
0
-3
Neurite length 65.5 mm/mm2
Branch points 900 /mm2
Red neurite length (mm/mm 2)
Day 3
0-66 h
212-284 h
4. Prepare the co-culture by removing the vector from
neurones and seeding astrocytes. Replace media ±
treatments every 2-3 days.
Mono-culture
Day 1
-4
Early phase neuroprotection (% IC80 KA)
Day 0
-5
1.0
Normalised AUC
3. Monitor in the IncuCyte ZOOM live-cell imaging system: capture and analyse phase and fluorescent
images every 2-6 h for 3 to >12 days.
150
2
100
0.6
3. Infect the cells with the membrane targeted-RFP
vector for 20 h.
KA or GA (1 µM – 1 mM) were applied 1 h or 72 h post-seeding of human iCell Neurons in mono-culture.
Time-course data shows neurite length (mm/mm2) (treatment at day 0) or normalised (to 66 h) neurite
length (treatment at day 3). The AUC values were determined from the time-courses (mean ± SEM, n=4).
Treatment at day 0: KA attenuated both neurite formation (0-66h) and maintenance (212-284h) in a
concentration dependence manner. In contrast, GA only inhibited neurite maintenance, with little or no
effect on neurite formation.
Treatment at day 3: KA yielded an early (66-92 h) and late (212-284 h) phase inhibition. GA treatment
produced a small early phase inhibition, whilst exhibiting a pronounced late phase effect.
•
1. Seed selected neuronal cell type in the microplate (96 or 384-well) and allow cells to adhere for 1 h.
100
1.5
Mean red neurite length (mm/mm 2)
Treatment
0
1000M
Time (h)
•
•
1000M
0
10
0
0
125M
0.8
Time (h)
3.0
125M
+
-4
500M
Log [Kainic acid] (M)
Time (h)
150
-5
63M
2.0
Infection efficiency
(% mKate2/Tuj-1)
200
250M
1.0
2. Thaw and seed selected neuronal cell type in microtitre plate 1 day prior to co-culture preparation and
allow adherence for 4 h.
Phase neurite length (mm/mm2)
100
Vehicle
2.5
1. Thaw and bulk up rat primary astrocytes 3-4 days
ahead of co-culture preparation.
Day 10
Red neurite length (mm/mm2)
0
0-66 h
66-284 h
10
1.2
Phase neurite length (mm/mm2)
1000M
1.0
Neurite length formation AUC x 10 3 (mm/mm 2)
125M
0.6
Neurite length (mm/mm2)
Neurite length formation AUC x 10 3 (mm/mm 2)
Neurite length (mm/mm2)
500M
1.5
Early phase AUC x 10 3 (66-92h)
14 d
63M
0.8
20
Red neurite length (mm/mm2)
0d
250M
2.0
30
Normalised neurite length (% 66h)
iCell Neurons
iPSC
1d
3d
Vehicle
1.0
IC50
489 µM
184 µM
66 h
284 h
Day 3
Late phase AUC x10 3 (212-284h)
IncuCyte ZOOM
live-cell imaging
system
10
2.5
Late phase AUC x103 (212-284h)
96 & 384-well NeuroTrack assay
20
L-Glutamic acid
1.2
Early phase AUC x 103 (66-92h)
• Application of the excitotoxic agents kainic acid
(KA) and L-glutamic acid (GA) disrupted iCell
neurite formation.
Interestingly, differential
IC50
239 µM
125 µM
0
Normalided Neurite length (% 66h)
• These data demonstrate that combining kinetic
measurements with complex biological monoand co-culture systems may lead to improved
models/understanding of neurological disease.
Treatment at day 0
• In rat primary cells, striking differences were
observed between mono- and co-culture. KAinduced excitotoxicity was greater in mono- vs coculture, as opposed to GA, where neurite
disruption was enhanced in co-culture.
66 h
284 h
30
Treatment at day 3
• Here we describe novel in-vitro assays for
quantifying
neurite
parameters
(length,
branching) kinetically in human iPSC-derived
(iCell Neurons, CDI) neurons in mono-culture and
primary cryopreserved (Global Stem) neurons
and astrocytes in co-culture.
Kainic acid
Neurite length maintenance AUC x 10 3 (mm/mm 2)
effects were observed. Whilst KA affected both
neurite formation and maintenance, GA had little
effect in formation and greater effect in
maintenance.
Neurite length maintenance AUC x 10 3 (mm/mm 2)
• Measurements of neurite dynamics are central to
our current understanding of neuropathology,
neuronal injury, regeneration and embryonic
development.
Cells
Primary co-culture NeuroTrack assays
iPSC mono-culture: Excitotoxic effects of KA & GA
Red neurite length (mm/mm2)
Summary & Impact
100
50
100
200
300
200
300
Time (h)
15 M
63 M
250 M
Control
100
50
0
100 0
200
0Time (h)
300
100
200
0
300
Time (h)
100
Time (h)
• Treatment with KA or GA in mono- and co-culture at 72, 144, 216
and 288 h (blue arrows). Data is presented as mean ± SEM (n=8).
• KA treatment yielded substantially greater neurite disruption on
neurones grown in mono-culture, compared to those co-cultured
with astrocytes.
• Conversely, GA induced minimal effects on the neurite outgrowth
of neurons grown in mono-culture, however substantial
Vehicle
KA (5 mM)
GA (250 M)
disruption was observed when co-cultured with astrocytes,
% excitotoxicity calculated by normalising to vehicle.
particularly at later time-points.
M/C
C/C
We thank GlobalStem for technical support with
primary frozen neurons.