Welcome to the SLAS 2015 Tutorial - Cisbio Bioassays · Welcome to the SLAS 2015 Tutorial presented by February 10th, 2015 ... HTRF/iPS cells tutorial SLAS 2015. 16 20 February 2015

1

Welcome to the

SLAS 2015 Tutorial presented by

February 10th, 2015

Coby Carlson Francois DegorceCarl Peters

CLARIOstar Highlights:

• Linear Variable Filter Monochromator The only adjustable bandpass monochromator

• Linear Varible Dichroic The only reader with an adjustable dichroic

• Monochromator, Spectrometer, Filters Three key technologies in one reader

• Superb luminescence dynamic range

• Two reagent injectors

CLARIOstar

Wavelength

Tra

nsm

itta

nce

Adjustable bandwidth

Adjustable peak wavelength

Wavelength

LVSWP LVLWP

Tra

nsm

itta

nce

*Angle of the arrow representing the light beam relative to the LVFs

is not Indicative of propagation angle or polarization of the light beam

LVF1LVF2

BMG‘s Linear Variable Monochromator is Different: Continuosuly Adjustable Bandpasses up to 100nm

Dichroics prevent excitation and unwanted light from reaching the

detection stage of the instrument. They are very important for optimal

sensitivity, but traditional dichroics have a fixed cutoff wavelength.

Without an adjustable dichroic a reader has to rely on the user having

the proper dichroic installed, or in worst case a 50/50 beam splitter

which reduces sensitivity.

BMG‘s Linear Variable DichroicThe Only Reader with an Adjustable Dichroic

ExcitationSource

DetectionSystem

Well

505LP Dichroic485nm

520nm

CLARIOstar

Technical features for performing HTRF

Optics:

Enclosed free air optical light path• 0.1 mm focus precision

• All assays top or bottom reading

CLARIOstar

CLARIOstar Technical Features

Top and Bottom Reading, Focus & Optical FlexibilityIf an assay can be read from the top, it can be read from the bottom on the CLARIOstar

and can focus with 0.1 mm precision which is key for detecting monolayers of cells. This

includes polarization, time-resolved fluorescence, luminescence.

High-resolution well scanning of adherent cells Z Axis

Adjustment

Liquid

level

CLARIOstar


Filters:

Ex / Em & dichroic slides• Easy to Change

• Identified using filter scan

Exictation Source:

High performance Xenon Flash Lamp

Detection System:

Photo Multiplier Tube (PMT)

CLARIOstar


TR-FRET

HTRF• TR-FRET measurement based on Cisbio chemistry

• Cisbio set some instrument quality limits which the CLARIOstar achieves

Delta F

Low 3 h

Delta F

high 3 h

Delta F

low 21 h

Delta F

high 21 h

PHERAstar

mean of 622 965 25 1100

CLARIOstar

(gain 2600)20 751 30 880

Omega

(gain 2600)Not meas. Not meas. 29 713

Cisbio Limit > 10 > 500 > 15 > 600

Please note that the CLARIOstar is HTRF certified for black and white plates.

Special Thanks to:

Team of German Engineers

BMG LABTECH Application Scientists

CDI and Cisbio

COMBINING HTRF DETECTION

TOOLS TO IPS CELL MODELS

MOVING TOWARDS MORE

BIOLOGICAL RELEVANCY

SLAS Washinghton D.C., US

Februray, 2015

1320 February

2015

Supporting all drug discovery steps with advanced assays

Cell signaling research

Drug discovery at all stages and all therapeutic areas

Standardized HTSComplex biological approaches

Unlimited applications

Biotherapeutics

Small molecules

AD & HTS

Hit-to-lead

TA

HTRF/iPS cells tutorial SLAS 2015

1420 February

2015

How Cisbio pioneered and refined cell-based TR-FRET

approaches


Cell and

biology

Add&read

Process

1520 February

2015

Efficient assay readout require optimal detection devices

Highly performing excitation sources (high energy flashlamp, laser)

Very selective signal collection and filtration (PMTs)

Time-resolved fluorescence mode


1620 February

2015

Custom assay

development services

An ever growing portfolio of cell-based tools

Ready-to use kits

Phospho-

protein

assays

Cytokine

assays


1720 February

2015

Creating efficient detection tools – enabling more

biological relevancy


Artificial Physiological

BiochemicalCell-based

(over-expressed)

Cell-based

(endogenous

primary, 3D, iPS)

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Presentation Outline

• Introduction to platform technologies / providers

• Brief overview of iPSC technology and CDI

• HTRF assays with iPSC-derived cell types• Focus on therapeutic areas:

• Diabetes and Alzheimer’s Disease

• Summary and Final Thoughts

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iPS Cell TechnologyRevolutionary Access to Human Biology

Differentiate into

all 208 cell types in

the human body

208 C

ell

Typ

es

Represent

any individual

genotype

6 Billion People

Reprogramming

Dif

fere

nti

ati

on

Mastery of all three

technology platforms

is evolutionary,

and promises to

reinvigorate drug

discovery

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iPS Cell TechnologyOvercoming Current Limitations

Primary Human Cells Transformed Cell Lines Animal Models

• Limited availability

• Variable

• Unstable

• Limited characterization

• Poor recapitulation

• Limited key functional

characteristics

• Cannot represent human

diversity

• Represents animal biology,

not human biology

• Resource intensive

• Large cmpd requirements

• Animal welfare issues

Induced pluripotent stem cell (iPSC) technology overcomes limitations of

existing cell models – Availability, Functionality, Reproducibility, Translatability

21

CDI Overview and Product Portfolio

2009 2010 2011 2012 2013 2014

iCell Cardiomyocytes iCell

Endothelial Cells

MyCell Products

iCell

Hepatocytes

iCell Astrocytes

iCell

Hematopoietic

Progenitor

Cells

iCell Skeletal

Myoblasts

iCell Cardiac

Progenitor CellsiCell

DopaNeurons

iCell Neurons

CDI is the world’s largest producer of human

iPSC-derived cell types

Headquartered in Madison, WI

Currently employs ~138 total staff

~650 yrs human stem cell experience

Core competencies

Creation and culture of human iPS cells

Genetic engineering of iPS cells

Development of new differentiation protocols

Manufacture of human iPS cell-derived cell types

iCell Products = normal healthy donor

MyCell Products = represent patient-

derived or a disease-specific donor iPS

cell line

iCell

Macrophages

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iCell Products and HTP Screening?

Quality

Quantity Purity

Drawnel, et al. “Disease modeling and phenotypic

drug screening for diabetic cardiomyopathy

using human induced pluripotent stem cells”

Cell Reports. 2014; 9: 1–11.

Xu, et al. “Prevention of β-amyloid induced toxicity

in human iPS cell-derived neurons by inhibition of

cyclin-dependent kinases and associated cell cycle

events” Stem Cell Research; 2013;10: 213–227.

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• HTRF assays with iPSC-derived cell types• Focus on therapeutic areas



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ELISA vs. HTRF Comparison“Real-world” CDI example

iCell Astrocytes

Liang et al. (2004) J. Neurochem.

What can they do?

LXR agonist (T0901317)

stimulates ApoE expressionLiterature reference?

WB-to-ELISA conversionELISA-to-HTRF conversion

Standard Curve ELISA data

The minimum detectable dose (ie. the “sensitivity”)

of Apolipoprotein E is typically ~0.03 ug/mL

HTRF Assay Data for ApoE releasein human iPSC-derived Astrocytes

-10 -9 -8 -7 -6 -50

5

10

15

20

25

30

Log [T0901317] (M)

[Ap

oE

] (n

g/m

L)

Develop an application for ApoE secretion

Standard Curve

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HTS-Compatible Format = Lots of Data“Bang for your buck”

iCell Macrophages

• M1 or M2 activation?

• Cytokine release assays

• Matrix experiments to define

IL-6 HTRF assay data

Several kits have been tested:

• TNF-alpha

• IL-1b

• IL-6

• IL-10

• IL-12

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Cardioprotection

iCell Cardiomyocytes

Activation of AKT signalingin iCell Cardiomyocytes

(HTRF pAKT assays)

-13 -12 -11 -10 -9 -8 -7 -60

500

1000

1500

2000

2500

3000

-100

0

100

200

300

400

Thr308

Ser473

Log [IGF-1] M

Delt

a F

%

Delta

F%

Normoxia Hypoxia0

10

20

30

40

50

*

% A

po

pto

tic C

ells

(TU

NE

L/D

AP

I)

AB C

D

EDAPI

TUNEL

Normoxia Hypoxia

Control IGF-1 Insulin0

25

50

75

100

125

*****

Casp

ase-3

/7 A

ctiv

ity

F

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Diabetes and Metabolic DiseasesControlling Blood Glucose

GLUCOSE

MUSCLE

LIVER

ADIPOCYTES

β-cells α-cells

PANCREAS

INTESTINE

High blood sugar

= insulin released

= glucose taken into cells

Low blood sugar

= glucagon released

= stimulate gluconeogenesis

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Diabetes and Metabolic DiseasesUse of iPSC-derived cell types to study disease

iCell Hepatocytes

iCell Skeletal Myoblasts

iCell Cardiomyocytes

iCell Neurons

iCell Endothelial Cells

GLUCOSE

MUSCLE

LIVER

ADIPOCYTES

β-cells α-cells

PANCREAS

INTESTINE

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Insulin-stimulated Pathway Analysis

iCell Hepatocytes

Inhibition of AKT signalingin iCell Hepatocytes

(HTRF pAKT Ser473 assay)

-10 -9 -8 -7 -6 -5 -40.0

0.4

0.8

1.2

1.6

IC50 = 254 nM

Log [PI-103] (M)

HT

RF

Rati

o

(665 n

m /

620 n

m)

Activation of AKT signalingin iCell Hepatocytes

(HTRF pAKT Ser473 assay)

-11 -10 -9 -8 -7 -6 -50.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

Log [Insulin] (M)

HT

RF

Rati

o

(665 n

m /

620 n

m)

High blood sugar = insulin released = glucose taken into cells

Low blood sugar = glucagon released = stimulates gluconeogenesis

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Glucagon-mediated Cell Signaling Events

iCell Hepatocytes

High blood sugar = insulin released = glucose taken into cells

Low blood sugar = glucagon released = stimulates gluconeogenesis

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Modulating PI3K/AKT Signaling in iCell SkM

iCell Skeletal Myoblasts

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Hot Target in Metabolism – PCSK9

iCell Hepatocytes

0.001

0.010

0.100

1.000

GAPDH HNF4A LDLR PCSK9

Re

lative

Ge

ne

E

xp

ressio

n

• PCSK9 regulates cholesterol levels in the blood

• One of the key drivers of high LDL cholesterol

• PCSK9 binds to LDL receptor, targets it for destruction

• If PCSK9 does not bind, LDLR can remove cholesterol

• Drugs that block PCSK9 can lower cholesterol

http://www.broadinstitute.org/

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iPSC-based Alzheimer’s Disease Model

Genotype Phenotype Catalog #

Control (WT) Healthy / normal 01279.107

APP A673T (homo) Isogenic

AD model

01279.A27

APP A673V (het) 01279.A32

iCell Neurons MyCell

Disease and

Diversity

Products (DDP)

iPS Cells

Genetic Engineering

Human Neurons

β-secretase -secretaseAβAPP

Ala→Thr = protective and Ala→Val = causativeA673

A673T• 1st variant associated with protection

• Identified in a whole genome seq.

project of ~1,800 people from Iceland

• Jonsson et al. (2012) Nature

A673V• Also near BACE1 cleavage site

• Mutation contributes to AD pathology

• Increased Aβ production, and

enhanced aggregation and toxicity

• Di Fede et al. (2009) Science

MyCell Products

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Genotype Impacts PhenotypeBiomarker Levels Differ From WT

DIV 20 4

Thaw and

Plate Cells

1

Change

Media

3

Run HTRF

Assay

Collect

Media

Factors that impact biomarker detection:

• Cell density

• Media composition

• Time in culture

• Dilution factor

Maloney, et al. “Molecular mechanisms of

Alzheimer’s disease protection by the A673T allele

of APP” J. Biol. Chem. 2014; 289: 30990-31000.

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HTRF technology can address relevant biology

HTRF is a well-established assay technology in the drug discovery community

CLARIOstar has been validated for use with HTRF assays

iPSC technology is changing the way we study human disease

CDI has Applications and Support team to help ensure success

Development of HTS-compatible assays demonstrates possibilities for screening

with relevant cell types (secondary screens in TA most likely)

Quality of the data matters – good cells, good assays, good instrument for

detection are all a must!!

Develop applications with normal cells – and we are then poised to compare

assay performance with disease-specific or patient-derived cells

Phrases like “Human brain in a dish” and “in vitro clinical trials” are real

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Thank you !!

CDI

Please visit us at our Booth:

BMG Labtech #929

Cisbio #923

CDI #1304

Documents

Welcome to the SLAS 2015 Tutorial - Cisbio Bioassays · Welcome to the SLAS 2015 Tutorial presented by February 10th, 2015 ... HTRF/iPS cells tutorial SLAS 2015. 16 20 February 2015