www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100

Characterization of an Isogenic Disease Model of

Alzheimer’s Disease from Human iPSC-derived Neurons Coby Carlson, Jun Wang, Natsuyo Aoyama, Rachel Llanas, Kile Mangan,

Michael McLachlan, Tom Burke, Susan DeLaura, and Eugenia Jones

AD-model Generation

Amyloid Beta (1-40)

-11 -10 -9 -8 -7 -6 -5 -41000

1500

2000

2500

3000

3500

4000

Log [DAPT] (M)

Alp

ha

Co

un

ts

Genotype Phenotype iPSC line #

Control (WT) Healthy; normal 01279.107

APP A673T (homo) Isogenic AD model

01279.A27

01279.A32 APP A673V (het)

“Hot Topics” Introduction

The development of therapies for neurodegenerative diseases such as Alzheimer’s

disease (AD) has been hindered by limited availability of relevant human cells for

research and drug discovery. Using induced pluripotent stem (iPS) cell technology,

we have created an unlimited source of human neurons that incorporate specific

genetic profiles associated with AD pathology. Researchers are using these cells to

understand the fundamental mechanisms underlying AD and to identify novel drug

treatments for this disease.

AD is a progressive neurodegenerative disease that results in gradual memory loss

and impairment in the ability to learn or carry out daily tasks. Recent studies estimate

that AD may contribute to as many deaths as heart disease and cancer combined.

Development of therapies for the treatment of AD is lengthy, costly and has been

largely unsuccessful, with drug attrition rates of >90%. As a result, there are no cures

and few treatment options.

A hallmark of AD pathology is the development of toxic plaques in the brain that contain

beta amyloid (Aβ), which is produced from amyloid precursor protein (APP). To

build a cellular model that could help to better understand the mechanisms by which Aβ

is produced, we used iPSC technology and genetic engineering to create human

neurons that exhibit AD-specific profiles. Our studies with these human iPSC-derived

neurons highlights the “disease-in-a-dish” approach for AD drug development.

iPS Cells

Genetic

Engineering

Human Neurons

Cortical Neuron Production Electrical Activity in APP Variants Correlates

with Allele: A673T > WT > A673V

5 sec

A673T is the first variant in APP associated

with protection against amyloid pathology

and AD. A673T was identified in a whole

genome sequencing project of approx.

1,800 people from Iceland.

(Jonsson Nature 2012)

The A673V variant is also near the APP

beta-secretase cleavage site and the

mutation contributes to AD pathology not

only by increasing Aβ production, but also

by enhancing aggregation and toxicity.

(Di Fede Science 2009)

(B) Real-time qPCR

analysis of a focus gene

panel reveals modest but

significant differences in

gene expression; notably

protein kinase C delta

(PRKCD) – involved in

learning and memory –

and the AD-associated

protein alpha-2-macro-

globulin (A2M)

WT control

βIII-

Tu

bu

lin

A673T A673V

Nestin

(A) Characterization of neuron

purity by flow cytometry. Using

the expression markers of βIII-

Tubulin (TUJ1) and Nestin, we

observed >98% pure neurons.

WT control (98.6%), A673T

(99.6%), and A673V (98.9%).

Day 7

Day 14

Day 21

WT control A673T A673V

MAP2 / Synaptophysin / DAPI

(C) ICC staining of neurons in culture

A 40/42 Ratio

-11 -10 -9 -8 -7 -6 -5 -41

2

3

4

5

Log [DAPT] (M)

A

40

/42

Ra

tio

(A) Measurement of AD-relevant

biomarkers by AlphaLISA: Aβ (1-40),

Tau, and sAPPα in iPSC-derived

neurons produced from a healthy

donor.

(B) Modulation of APP processing with

the -secretase inhibitor (DAPT)

shows a change in the amount of Aβ

(1-40) and the Aβ 40/42 ratio.

(C) Comparison of Aβ (1-40) levels in

neurons produced from a healthy

donor (WT, gray), an isogenic APP

A673T variant (blue), and an

isogenic APP A673V variant (red)

shows differences in the amounts

detected (HTRF assay). Treatment

with small-molecule inhibitors of

‒secretase (ie. BMS 299897 and

DAPT) resulted in decreased assay

signal (AlphaLISA) in all three APP

backgrounds.

AD Biomarker Levels Differ in APP Mutant and

Normal iPSC-derived Neurons

Amyloid Beta (1-40) Levels

To introduce these mutations in human iPSC-derived neurons, we genetically

engineered a “control” iPS cell line (01279.107) from an apparently healthy normal

Caucasian male donor (with no family history of neurological disorders) to engineer

either the A673T or A673V alleles.

;

• The A673T and A673V mutations were introduced in human iPSCs using TALENS.

• The APP A673T variant is homozygous, whereas the APP A673V variant is

heterozygous. Both are clonal.

• Cortical neurons were differentiated from these three unique isogenic iPSC lines

using CDI’s proprietary differentiation protocol outlined in the next panel.

These lines can be purchased from CDI.

β-secretase -secretase Aβ APP

Ala→Thr = protective and Ala→Val = causative A673

Day 6 Day 7 Day 15 Day 8

Bu

rsti

ng

Ra

te (

BP

M)

Fir

ing

Ra

te (

Hz)

WT A673T A673V WT A673T A673V WT A673T A673V WT A673T A673V

(A) Real-time heat map of

activity electrical activity

recorded on the 48-well

Maestro MEA system from

Axion BioSystems.

(B) Display of action potentials

captured from a 4x4 grid of

electrodes in a single well

(top) and the raster plot

depicting action potential

ticks across time for all 16

electrodes (bottom). Blue

tick marks indicate a

“Poisson surprise”-defined

burst.

(C) Mean firing rate (Hz; red)

and bursting rate (BPM;

blue) for WT, A673T, and

A673V neurons (n≥12) over

different DIV. Notice that

as the cultures mature,

bursting behaviors are

enhanced for A673T and

decreased for A673V.

Summary

Human iPS cells were genome-edited to create isogenic

cell lines for modeling Alzheimer disease (AD) .

Cortical neurons can be derived from human iPS cells

with different genetic backgrounds at high purity.

Wild-type APP, A673T, and A673V neurons express Tau,

APP, and amyloid beta isoforms. Treatment with drugs

modulated Aβ levels in the expected manner.

Protocols for handling iPS cell-derived neurons from CDI, as well as

performing various applications (ie. ICC, MEA) are available online.

Please contact CDI with any questions at: support@cellulardynamics.com

Future Directions

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B

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Schematic of cortical neuron differentiation process

Post-thaw

MyCell Neurons iPS Cell

Expansion

Neuron

Maturation

Day 0 Day 28-30

βIII-Tubulin-(+)

Nestin-(-)

Cell

Cryopreservation

Neuronal

Differentiation A B

C

Bursting electrical activity in neurons carrying the

Icelandic protective APP variant (A673T) is higher than

WT neurons, which are more active than AD-associated

A673V variant.

These data here are consistent

with previously reported AD

models of transfected HEK

293 cells and primary rodent

neurons (Jonsson Nature 2012

& Benilova J Biol Chem 2014).

The advantage of using Multi-Electrode Array (MEA) technology to assess neuronal

cultures is that it is a non-invasive, label-free method that enables the direct sensing

of voltage to characterize network-level phenotypes.

Recently published work from Genentech using these

neurons purchased from CDI shows similar differences

in APP processing (Maloney J Biol Chem 2014).

MyCell Neurons

(WT control)

Leveraging the power of iPSC technology, CDI is building out a

panel of MyCell Disease & Diversity Products, which includes

human neurons from donors of diverse ethnic and disease-

specific populations. Not only will there be more “controls”, but

also other innate disease models for ALS, Rett Syndrome,

Parkinson’s Disease, and epilepsy are being generated.

Abstract Control

Number: 1983

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