Development and Characterization of Scalable Human Induced ... · specifically midbrain...

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