Target

Identification

Target

Validation

Compound

Screening

Lead

Optimization

Preclinical

Trials

Clinical

Trials

www.cellulardynamics.com Madison, WI USA (608) 310-5100

Introduction

Here we demonstrate the differentiation of midbrain dopaminergic neurons from

human iPS cells. Cell type-specific characterization data, as well as various

application data are also presented. Robust and reproducible methods to

generate DA neurons at high purity, coupled with the development of functional

cell-based assays with “normal” cells, will enable the successful downstream

production of panels of disease-specific samples derived from patient iPS cells for

the study of neurological disorders such as Parkinson’s Disease.

Summary

Cell Type Characterization

Measuring Pharmaco-Influences

Functional Application Data

Human Donor Terminally

differentiated

cell types

Induced

Pluripotent Stem

(iPS) Cells

Based upon work published in the literature (Kriks et al.

2011), coupled with additional differentiation and workflow

improvements, an optimized midbrain dopaminergic neuron

differentiation protocol was established. This resulted in the

ability to produce large-scale cryopreserved DA neurons.

Human iPSC-derived DA neurons were plated

in 96-well plates coated with PLO/laminin and

cultured for 1-2 weeks with regular media

changes. On the day of the assay, cells were

loaded with Calcium 6 dye (Molecular

Devices), stimulated with various ligands, and

increases in fluorescence were detected on a

FLIPR Tetra system. Representative data

presented here shows a titration of kainic acid.

Characterization and Functional Applications of Human

iPS Cell-derived Midbrain Dopaminergic Neurons

Coby Carlson1, Lucas Chase1, Kile P. Mangan1, Arne Thompson1, Susan DeLaura1, Jayne Hesley2,

Oksana Sirenko2, Evan Cromwell2, Brad Swanson1, and Vanessa Ott1

1 Cellular Dynamics International, Inc., Madison, WI USA 2 Molecular Devices, LLC., Sunnyvale, CA USA

Relevant midbrain dopaminergic markers are expressed in DA Neurons

post-thaw. High Content Imaging (HCI) of FoxA2 (left; 94% positive), LMX1

(center; 96% positive), and FoxA2/LMX1 overlay (right; 91% co-positive)

demonstrate a highly pure population (>90%) of midbrain neurons. The

high degree of purity was also confirmed by flow cytometry analysis.

FoxA2 / Hoechst LMX1 / Hoechst FoxA2 / LMX1

Dopaminergic (DA) Neuron:

producer of dopamine; found in

different regions in the CNS with

the highest concentration in the

midbrain.

Dopamine: role in voluntary

movement and a broad array of

behavioral processes such as

mood, reward, addiction, and

stress.

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.

Differentiation Protocol

Development

iPS Cell

Expansion

Midbrain

Specification

Floor Plate

Patterning

Midbrain DA

Neuron Induction

Cell

Cryopreservation

Day 0

FoxA2+ FoxA2+ / Lmx1+ Map2+/Nestin- and

FoxA2+/TH+

Map2+/Nestin- and

FoxA2+/TH+

Day 42

Neuron Maturation

0

20

40

60

80

100

120

A B C D E F G

%

Po

sit

ive

[Molecule X]

FoxA2+ Lmx1+ FoxA2+/Lmx1+

Low High [Molecule X]

High content imaging (HCI)

serves as a useful tool for

optimizing critical stages of

midbrain dopaminergic neuron

differentiation from human iPS

cells. Data shown here (left)

shows the titration of a critical

component (Molecule X) used

during patterning to achieve

high levels of co-expression of

the floor-plate marker FoxA2

and the roof-plate marker Lmx1.

Midbrain DA Neurons: located

in the substantia nigra compacta

(SNc) and the ventral tegmental

area (VTA); send fibers to other

regions in both sides of the

brain.

Parkinson's Disease (PD):

caused by selective degenera-

tion of the SNc DA neurons.

What is a Dopaminergic Neuron?

0

20

40

60

80

100

% V

iab

ilit

y

Lot 1

Thaw Viability

Day 3 Post-Thaw Day 7 Post-Thaw Day 14 Post-Thaw

iCell DopaNeurons are highly viable, have significant

neurite outgrowth within 2-3 days post-thaw, and

maintain high purity for extended time in culture. Lot 2 Lot 3

Leveraging the power of iPSC technology,

CDI is building out a Disease & Diversity

Panel, which includes terminally differentiated

cell types from donors of diverse ethnic and

disease-specific populations. Not only will

there be more “controls”, but there is also a

heavy focus on neurological diseases,

including Parkinson’s Disease-related targets

such as LRRK2 and alpha-synuclein.

Patient-Derived iPS Cells:

Models for Parkinson’s

In collaboration

with NINDS, CDI is

making iPSC from

monogenic PD

donors: available

at the end of 2014.

Ca2+ Flux Assay

Multi-Electrode Array

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

Rela

tive E

xp

res

sio

n (

vs.

GA

PD

H)

Day 7 PT

day 14 PT

Day 21 PT

Day 28 PT

Day 42 PT

Human SN

Regional

Specification

Dopaminergic

Identification

Neuronal

Subtypes

Gene expression time course as measured by qPCR indicates 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.

EC50 = 8.6 M

24 h

Apomorphine (APO; 7.5 M)

50 sec

Fre

qu

en

cy (

Hz)

Fre

qu

en

cy (

Hz)

24 h

Vehicle Control

APO + D1 antagonist (SCH 23390; 4 µM)

Fre

qu

en

cy (

Hz)

24 h

DA neurons were cultured on 48-well MEA plates coated with PEI/laminin and their

electrical activity was analyzed at Day 8 following treatment w/ different compounds.

In this example, velocity graphs depicting instantaneous mean firing rate levels over

time (binned at 500 msec) were generated with the iCell NeuroAnalyzer before and

after drug addition. Stimulation w/ apomorphine resulted in increased excitatory and

connectivity levels (ie. # of bursts and bursting peak heights) after another day in

culture. Co-application of a D1 antagonist abolished the induced response of APO,

suggesting homeostatic receptor modulation following dopamine receptor

stimulation. Control cultures displayed no change over time.

High Content Imaging

Map2 / Nestin / Hoechst TH / FoxA2 / Hoechst

Fo

xA

2–

AF

64

7

Lmx1–AF488

High Viability and Expected Morphology

High Purity Determined by Multiple Methods

Gene Expression

Optimized Protocol

Phenotype – Day 14

DA neurons can be cultured for

extended period of time. After two

weeks, there is a significant degree

of neurite outgrowth and the

sophisticated network formed at this

point is reminiscent of a classical

neuronal phenotype.

DA Neurons are >80% TH-positive.

500

400

300

200

100

0

-100

-200

-300

-400

-500

Vo

lta

ge

(

V)

IC50 values:

Rapa = 1.3 M

Rot = 4.3 M

Sal = 3.6 M

Untreated DA neurons stained with MitoTracker®

Orange (left), while treatment with CCCP (0.4 M)

demonstrates mitochondrial toxicity (right). Images

acquired at 20X. Nuclei are stained blue with DAPI.

MitoTracker Red / Cyto-ID

Cells treated with

chloroquine (30 M)

In collaboration with Molecular

Devices, we are developing

HTS-compatible assays for

autophagy (left), mitochondrial

toxicity (above), and neurite

outgrowth (data not shown).

Additionally, software algorithms

for custom analysis of pheno-

typic assays are also being

developed (right).