development and characterization of scalable human induced ... · specifically midbrain...
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Abstract
www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100
Development and Characterization of Scalable Human Induced
Pluripotent Stem Cell-derived Midbrain Dopaminergic Neurons for
Drug Discovery and Disease Modeling
Hussey D, Chase L, McMahon C, Ma J, Meyer N, Chavez C, Mangan K, Carlson C, DeLaura S, Wang WB and Swanson B
Objective: Since the discovery of human induced pluripotent stem cells (iPSCs),
much excitement and interest has been created around this technology as a
platform for generating pluripotent stem cell lines from a range of specific genetic
backgrounds, both normal and diseased. We have developed highly consistent
and scalable differentiation protocol for making various types of human neurons,
specifically midbrain dopaminergic neurons. This protocol provides a consistent
platform to study various aspects of midbrain dopaminergic neuron biology,
including Parkinson’s disease.
Methods: Using an optimized episomally-derived human iPSC platform, we
developed a scalable method for the generation of differentiated, cryopreserved
human midbrain dopaminergic neurons (iCell® DopaNeurons). Gene expression
was analyzed by target-focused PCR arrays. Electrophysiological properties were
measured using whole-cell patch clamp and the network-level activity was
evaluated on multi-electrode array (MEA).
Results: Here, we present data characterizing gene expression for these floor
plate-derived midbrain dopaminergic neurons with proper regional and neural
subtype specifications. These cells displayed characteristic neuronal
electrophysiological properties, including ion channel activity, evoked and
spontaneous action potentials and excitatory post-synaptic currents. In addition,
results from the MEA showed characteristic excitatory phenotypes with responses
to known pharmacological agents and enhanced population bursts in an astrocyte
co-culture environment.
Conclusions: Robust and reproducible methods to generate functional iCell
DopaNeurons at high purity will enable the successful downstream production of
panels of disease-specific samples derived from donor iPS cells for the study of
neurodegenerative disorders such as Parkinson’s disease.
iCell DopaNeurons Characterization
iPSC technology grants access to
the CNS. The advent of induced
pluripotent stem cell (iPSC)
technology has enabled the use of
previously inaccessible human cells,
specifically neuronal cell types like
cortical or dopaminergic neurons.
Floor Plate-derived Midbrain Dopaminergic
Neuron Development
iPS Cell
Expansion
Midbrain
Specification
Floor Plate
Patterning
Cell
Cryopreservation
Day 0
Map2+/Nestin-
and
FoxA2+/TH+
Day 42
Neuron
Maturation
Midbrain DA
Neuron Induction
Schematic of midbrain dopaminergic neuron differentiation process
0
20
40
60
80
100
120
A B C D E F G
%
Po
sit
ive
[Molecule X]
FoxA2+Low High
[Molecule X]
Optimization of stages of midbrain
dopaminergic neuron differentiation
from human iPS cells using high
content imaging (HCI). Molecule X
titration during patterning identifies
optimal concentrations to achieve
high levels of co-expression of the
floor plate marker FoxA2 and the
roof plate marker Lmx1.
Differentiation protocol optimization
A) iCell DopaNeurons demonstrate
high viability and show extensive
neurite outgrowth within 2-3 days
post-thaw.
B) Identity and purity assessment by
flow cytometry and HCI. Flow
cytometry characterization of the
midbrain region specification was
performed by using expression of
FoxA2 and Lmx1 (96.9%) post-
thaw. HCI of FoxA2/LMX1
(midbrain) overlay, Map2 and
Nestin double stain, and TH and
FoxA2 double stain at 14 days
post-thaw. A TH+ purity of 83.5%
was quantified by flow cytometry
(data not shown).
C) Gene expression time course of
regional markers and neuronal
subtypes measured by qPCR
indicate that most genes are
expressed at very similar levels
over a 4-6 week period. An adult
human substantia nigra RNA was
included as a control for
comparison. Relative expression
versus GAPDH is depicted. Results
lower than 1x10-4 (gray shaded
box) are considered to be below
background or negative for
expression.
D) Spontaneous and evoked action
potentials recorded with a whole-
cell patch clamp show maturation
overtime (left). iCell DopaNeurons’
sodium and potassium channels
are inhibited by tetrodotoxin (TTX)
and tetraethylammonium (TEA),
respectively, as measured by
single-cell patch clamp (2-3 week
post-thaw).
1.E-06
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
FO
XG
1
OT
X2
EN
1
FO
XA
2
LM
X1
A
NU
RR
1
TH
AA
DC
GIR
K2
VM
AT
2
DR
D2
DB
H
SN
CA
SY
N1
SY
P
VG
LU
T1
VG
LU
T2
VG
AT
CH
AT
OL
IG2
Re
lati
ve
Ex
pre
ss
ion
(vs
. G
AP
DH
)
Day 7 PT
day 14 PT
Day 21 PT
Day 28 PT
Day 42 PT
Human SN
Regional
Specification
Dopaminergic
Identification
Neuronal
Subtypes
(A) Morphology
(B) Identity and Purity
(C) Gene Expression
FoxA2 / LMX1 Map2 / Nestin / Hoechst TH / FoxA2 / HoechstF
oxA
2–
AF
64
7
Lmx1–AF488Lmx1
Fo
xA
2
Evoked Action PotentialsSpontaneous Action
Potentials
Transiently Transfected Midbrain
Dopaminergic Neurons
A) Cultured iCell DopaNeurons reveal spontaneous and consistent
activity at 8 days in culture. Velocity graphs (top) and raster plots
(bottom) of activity measured on an Axion multi-electrode plate
shows neuronal activity over ~4 minute recording. Raster plots
mark action potentials (ticks) on individual electrodes over time
while velocity graphs depict the instantaneous mean firing rate of
the wells entire neuronal population for each 500 msec. Red circles
on the velocity graphs indicate an instantaneous (burst) increase in
population mean firing rate ≥16 Hz. The addition of apomorphine
(15 µM), a potent D1- & D2-receptor agonist, noticeability increases
activity of neuronal cultures 24 hr following treatment and wash.
B) Mean firing rates (Hz) (top: red) and ‘Poisson-surprise’ bursts per
minute (BPM) (green: bottom) are shown for cultures 24+ hr after
being treated with various D1- & D2-receptor ligands for 1 hr and
then washed. Note the increased bursting rates are selectively
responsive to D1-receptor activation.
C) iCell DopaNeurons’ neuronal activity is modulated by co-culture with
iCell Astrocytes. Example velocity graphs of iCell DopaNeurons
activity levels show population bursts are tuned and enhanced with
the addition of increasing amounts of iCell Astrocytes (10K, 25K,
50K and 100K) at 32 DIV.
D) Example raster plot of all 8 electrodes from a single well for a 10
minute recording of iCell DopaNeurons co-cultured with 100K iCell
Astrocytes at 32 DIV. Note the ‘synchronous’ action potentials on
different electrodes.
Ctrl .9 1.9 3.8 7.5 15 30 Ctrl .01 .2 .7 2 6 18 Ctrl .5 .8 1.2 1.8 2.7 4 Ctrl 6.25 12.5 25 50 100 2007.5
ApomorphineAPO [15µM]
+ D1 Antagonist D2 AntagonistD1 Agonist
Fir
ing
Ra
te (
Hz)
Bu
rsti
ng
Ra
te (
BP
M)
APO (µM) SCH23390 (µM) SKF83822 (µM) L-741,626 (µM)
(B)60 sec
Ele
ctr
od
e #
Fir
ing
Rate
(H
z)
Ap
om
orp
hin
e [
0.5
µM
]
(A)
+ 24 Hours
(C)
60 sec
20
Hz
10K 25K 50K 100K
(D)
Electrical Activity: Bursting Plasticity Modulated via Dopamine
Receptor Activation
(D) Electrophysiology
Early Transfection (4 DIV, 72 hr post transfection)
Late Transfection (21 DIV, 72 hr post transfection)
iCell DopaNeurons are
efficiently transfected with a
GFP fluorescent reporter
using ViaFect Transfection
Reagent (Promega).
Quantification of the
transfection efficiency reveal
the optimal culture time prior
to transfection is 4 days
verses 21 days. Transfection
efficiency was quantified
using flow cytometry.
iPSC-derived Neuron Panels: Process and Quality
Isotype Control
Nestin
MyCell Neurons – Day
28
Nestin
5 ml Pellet =
~4 Billion Neurons
A robust cortical neuron
differentiation process is
demonstrated across
episomally reprogrammed
iPS cells from multiple donors
and starting materials. These
data show that >90% pure
neurons with neural
morphological characteristics
are achieved independent of
the source and genotype of
the donor sample. This
process can be used to
generate large quantities of
neurons from a single batch.Day 28
Morphology & Purity
Blood 1-5 from PBMC;
Blood 6-11 from LCL
Day 3 Post-Thaw Day 14 Post-ThawDay 7 Post-Thaw
Summary and Conclusions
Human iPS cells were used to produce floor plate-derived midbrain dopaminergic neurons at high purities with proper regional and neural
subtype specifications.
These dopaminergic neurons efficiently express a fluorescent reporter after transient transfection.
Bursting electrical activity in midbrain dopaminergic neurons can be selectively modulated with a D1-receptor drug.
Co-culture with human iPS cell-derived astrocytes and these pure neurons enhance population bursts and show ‘synchronous’ action
potentials.
Neuron differentiation can be scaled out to expand genetic background offerings and scaled up to produce large quantities.
Robust and reproducible methods to generate functional iCell DopaNeurons at high purity will enable the successful downstream production
of panels of disease-specific samples derived from donor iPS cells for the study of neurological disorders such as Parkinson’s disease.
iCell DopaNeurons co-cultured with iCell Astroyctes