Development and Characterization of Scalable Human nduced ... · Abstract Madison, WI USA +1 (608)...
1
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 Chase L, McMahon C, Ma J, Grinager J, Meyer N, Chavez C, Meline B, Liu J, Carlson C, Mangan K, Wang WB and Swanson B 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. Using an optimized episomally-derived human iPSC platform, we have developed highly consistent and scalable differentiation protocols for making various types of human neurons. Based upon previously published work (Kriks et al. 2011), we developed a scalable method for the generation of differentiated, cryopreserved human midbrain dopaminergic neurons (iCell ® DopaNeurons). This protocol provides a consistent platform to study various aspects of midbrain dopaminergic neuron biology, including Parkinson’s disease. Phenotypically, iCell DopaNeurons are floor plate-derived midbrain dopaminergic neurons. During differentiation, these cultures go through a highly pure midbrain progenitor phase as shown by high level expression of forkhead box A2 (FoxA2) and the LIM homeobox transcription factor 1a/b (Lmx1). Upon further differentiation, midbrain dopaminergic neurons are generated as identified by expression of microtubule- associated protein 2 (Map2), FoxA2, Lmx1 and tyrosine hydroxylase (TH). Utilizing a genetic-based selection strategy, the differentiating dopaminergic neuron cultures are purified to >90% neurons as determined by expression of Map2 and the absence of the progenitor marker nestin. iCell DopaNeurons quickly acquire a branched neuronal morphology and display midbrain dopaminergic markers when thawed and maintain a high level of dopaminergic neuron purity for extended culture post-thaw. Functionally, these cells display characteristic neuronal electrophysiological properties, including proper ion channel activity, fire both evoked and spontaneous action potentials and show excitatory post-synaptic currents. In addition, these cells display characteristic excitatory phenotypes with responses to known pharmacological agents. Finally, as a proof-of- concept for the ability to scale out a neuronal differentiation process, episomally-derived human iPSCs from various donors were created and differentiated into a highly purified neuronal population, exemplifying the potential application of this technology to panels of donors as a means to study larger human populations. iCell DopaNeurons Characterization Summary and Conclusions 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 % Positive [Molecule X] FoxA2+ Lmx1+ 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 FoxA2 / LMX1 Day 3 Post-Thaw Day 7 Post-Thaw Day 14 Post-Thaw 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 FOXG1 OTX2 EN1 FOXA2 LMX1A NURR1 TH AADC GIRK2 VMAT2 DRD2 DBH SNCA SYN1 SYP VGLUT1 VGLUT2 VGAT CHAT OLIG2 Relative Expression (vs. GAPDH) Day 7 PT day 14 PT Day 21 PT Day 28 PT Day 42 PT Human SN Regional Specification Dopaminergic Identification Neuronal Subtypes Map2 / Nestin / Hoechst TH / FoxA2 / Hoechst (A) Morphology (B) Identity and Purity (C) Gene Expression FoxA2 Lmx1 Evoked Action Potentials Spontaneous 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. iCell DopaNeurons co-cultured with iCell Astroyctes. (C) iCell DopaNeurons’ neuronal activity is modulated by co-culture with iCell Astroyctes. Example velocity graphs of iCell DopaNeuron activity levels show population bursts are tuned and enhanced with the addition of increasing amounts of iCell Astroyctes (10K, 25K, 50K and 100K) at 32 DIVs. (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 DIVs. Note the ‘synchronous’ action potentials on different electrodes. Ctrl .9 1.9 3.8 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 50 100 200 7.5 Apomorphine APO {15μM} + D1 Antagonist D2 Antagonist D1 Agonist Firing Rate (Hz) Bursting Rate (BPM) APO (μM) SCH23390 (μM) SKF83822 (μM) L-741,626 (μM) (B) 60 sec Electrode # Firing Rate (Hz) Apomorphine {7.5 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 DIC, 72 hr post transfection) Late Transfection (21 DIC, 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 Neuron – 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 are achieved independent of the source and genotype of the donor sample. Morphology on plating confirms neural characteristics. 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 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.
Transcript of Development and Characterization of Scalable Human nduced ... · Abstract Madison, WI USA +1 (608)...
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
Chase L, McMahon C, Ma J, Grinager J, Meyer N, Chavez C, Meline B, Liu J, Carlson C, Mangan K, Wang WB and Swanson B
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. Using an optimized episomally-derived human iPSC platform, we have
developed highly consistent and scalable differentiation protocols for making various
types of human neurons. Based upon previously published work (Kriks et al. 2011), we
developed a scalable method for the generation of differentiated, cryopreserved human
midbrain dopaminergic neurons (iCell® DopaNeurons). This protocol provides a consistent
platform to study various aspects of midbrain dopaminergic neuron biology, including
Parkinson’s disease. Phenotypically, iCell DopaNeurons are floor plate-derived midbrain
dopaminergic neurons. During differentiation, these cultures go through a highly pure
midbrain progenitor phase as shown by high level expression of forkhead box A2 (FoxA2)
and the LIM homeobox transcription factor 1a/b (Lmx1). Upon further differentiation,
midbrain dopaminergic neurons are generated as identified by expression of microtubule-
associated protein 2 (Map2), FoxA2, Lmx1 and tyrosine hydroxylase (TH). Utilizing a
genetic-based selection strategy, the differentiating dopaminergic neuron cultures are
purified to >90% neurons as determined by expression of Map2 and the absence of the
progenitor marker nestin. iCell DopaNeurons quickly acquire a branched neuronal
morphology and display midbrain dopaminergic markers when thawed and maintain a
high level of dopaminergic neuron purity for extended culture post-thaw. Functionally,
these cells display characteristic neuronal electrophysiological properties, including
proper ion channel activity, fire both evoked and spontaneous action potentials and show
excitatory post-synaptic currents. In addition, these cells display characteristic excitatory
phenotypes with responses to known pharmacological agents. Finally, as a proof-of-
concept for the ability to scale out a neuronal differentiation process, episomally-derived
human iPSCs from various donors were created and differentiated into a highly purified
neuronal population, exemplifying the potential application of this technology to panels of
donors as a means to study larger human populations.
iCell DopaNeurons Characterization
Summary and Conclusions
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+Lmx1+
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
FoxA2 / LMX1
Day 3 Post-Thaw Day 7 Post-Thaw Day 14 Post-Thaw
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
DopaminergicIdentification
NeuronalSubtypes
Map2 / Nestin / Hoechst TH / FoxA2 / Hoechst
Fo
xA
2–A
F6
47
Lmx1–AF488
(A) Morphology
(B) Identity and Purity
(C) Gene Expression
Fo
xA
2
Lmx1
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.
iCell DopaNeurons co-cultured with iCell Astroyctes.(C) iCell DopaNeurons’ neuronal activity is modulated by co-culture with iCell Astroyctes. Example velocity graphs of iCell DopaNeuron
activity levels show population bursts are tuned and enhanced with the addition of increasing amounts of iCell Astroyctes (10K, 25K, 50K
and 100K) at 32 DIVs. (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 DIVs. 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 AntagonistD2 AntagonistD1 Agonist
Fir
ing
Rate
(H
z)
Bu
rsti
ng
Rate
(B
PM
)
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{7
.5 µ
M}
(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 DIC, 72 hr post transfection)
Late Transfection (21 DIC, 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 Neuron – 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 are achieved
independent of the source and
genotype of the donor sample.
Morphology on plating confirms neural
characteristics. 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
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.
