Society of Toxicology Annual Meeting 13 M h 2012 13 March 2012 · 13 March 2012 Presentation...

Human iPS Cell Technology in Predictive and Mechanism-based Drug Discovery and Toxicity T ti U i Ph t t i B d A

Society of Toxicology Annual Meeting13 M h 2012

Testing Using Photometric-Based Assays

13 March 2012

Presentation Outline

iPS Cell Technology & Company Overview

iC ll P d t iCell Products iCell® Cardiomyocytes

iCell® Hepatocytes

Assessing Mechanisms of Toxicity through g y gMultiparametric Analysis of iCell Cellular Test Systems

iCell Hepatocyte VignettesCe epatocyte g ettes

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iPS Cell TechnologyUnlimited Potential

iPS cells are uniquely useful stem cellsf Derived from adult tissue via non-invasive methods

Can be expanded indefinitely Can be differentiated into any cell type in the body Fully pluripotent

iPS cells offer distinct advantages to ES cellsCan be created ia streamlined & non in asi e methods Can be created via streamlined & non-invasive methods

Eliminates political/social issues regarding tissue source Enables diversity of genotype and phenotype

Reprogram iPS cells multiplyDraw 1 small Differentiate iPS cells into

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Reprogram sample tissue into iPS cells

iPS cells multiply and expand in culture indefinitely

Draw 1 small sample from 1 person

any cell type in the body (unlimited numbers)

CDI Overview

Cellular Dynamics International (CDI) is the world’s largest producer of human iPS cells d iPS ll d i d ll tand iPS cell-derived cell types

Currently employs ~107 people in Madison, WI

Core competencies

Creation and culture of human iPS cells

Genetic engineering of iPS cells

Manufacture of human iPS cell-derived cell types Manufacture of human iPS cell derived cell types

iCell® Cardiomyocytes – first commercial product

iCell® Endothelial Cells – launched Q3 2011

iCell® Neurons - launched Q4 2011

iCell® Hepatocytes – pre-commercial evaluation

MyCell Custom Services

iPS cell reprogramming

iPS cell genetic engineering

iPS cell differentiation The Wall Street Journal

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iPS cell differentiation The Wall Street JournalGold Winner: CDITechnology Innovation Awards 2011

CDI OverviewQuality, Quantity, Purity

Quality Exhibit key cellular characteristics Recapitulate normal human biology Reproducible Target Cell (non proliferating) Reproducible Known and relevant genotype

Purit

y

Target Cell (non proliferating)

Cel

l

Days in Culture

Non-Target Cell (proliferating)

Quantity Purity

ays Cu tu e

Sufficient to support HTP drug screening and safety testing

Currently 1Bn iCell Cardiomyocytes/day

>95% pure Maintained over time

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y y y y Currently up to 1.2 Bn iCell Hepatocytes/week

(shipped >5 Bn to external partners in Q4 2011)

CDI OverviewPeer-Reviewed Publications

Booth #1507Workshop, Tues. Mar 13, 6-8:30PM , Intercontinental Hotel



iC ll P d t iCell Products iCell Cardiomyocytes

iCell Hepatocytes



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iCell CardiomyocytesProtein Characterization

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iCell CardiomyocytesGenomic Characterization

Gene ExpressioniCell Cardiomyocytes d28 and d120

Stem cellGene Category

Transition

Primary Heart culturesNovartis GNF expression atlas

Heart expression ≥ 10X median tissue expression

GO analysis confirms Cardiac-specific enrichment

Cardiomyocyte“More human than human”

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Data courtesy of Uli Certa and Josh Babiarz

iCell CardiomyocytesElectrophysiology Characterization

I ISpontaneous Action PotentialsIonic Currents

INa ICa-L

Ito IKr

Ifunny IK1 ElicitedAction Potentials

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iCell HepatocytesMorphology

Adherent monolayer

Round nucleus

Distinct nucleoli Distinct nucleoli

High cyto/nuclear ratio

Bi-nucleation (circles)

Bile canaliculi (arrows)

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iCell HepatocytesMorphology

Bile Canaliculi – 4 days post-plating

2 hours

4 days Hoechst1 day

2 d

CDFDA O l

2 days

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CDFDA Overlay

3 days

iCell HepatocytesLong-term culture vs. PHH

Day 1 Day 21Day 1 Day 21

Thank You

iCell Hepatocytes

Thank You

Primary Human Hepatocytes

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Poster #519

iCell HepatocytesProtein Characterization

Alpha-1-antitrypsin HNF4a

Albumin Hoechst - Occludin

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iCell HepatocytesIntrinsic Function

PAS

Oil Red BODIPY

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iCell Hepatocytes



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Cell ViabilityCellTiter-Glo®

CellTiter-Glo® Cell Viability Assay iCell Cardiomyocytes Luminescent, ATP-based assay Single reagent “add-mix-measure” format Amenable to automated HTS, cell

proliferation & cytotoxicity assaysproliferation & cytotoxicity assays

- 2- 4- 6- 8- 10- 12Log [Compound] M

iCell HepatocytesTroglitazone

TamoxifenTerfenadineTheophylline

iCell Hepatocytes

AcetaminophenChlorpromazine

17Log [Compound] M

Cell ViabilityCellTox-Green

iCell Hepatocytes

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Apoptosis/NecrosisApoTox-Glo

ApoTox-Glo Triplex Assay Viability, Cytotoxicity, Apoptosis

Step 1: Live cell measurement of viability & cytotoxicity

Step 2: Measurement of apoptosis

y, y y, p p

Utilizes two distinct fluorogenic substrates Cell permeable live-cell protease substrate Cell impermeable dead-cell protease substrate

Utilizes a luminogenic caspase-3/7 substrate

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Apoptosis/NecrosisApoTox-Glo

iCell Cardiomyocytes iCell HepatocytesiCell Cardiomyocytes iCell Hepatocytes

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Cell Type SpecificityProteasome Inhibitors

Bortezomib (PS-341; marketed as Velcade): first FDA-approved proteasome

iCell Cardiomyocytes*K562

inhibitor for use in the treatment of certain myeloma cancers

*Bortezomib (and other proteasome inhibitors) are known to partially

On-target potency(cytotoxicity by apoptosis)

Off-target safety

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( ) yinhibit the viability assay protease biomarker at concentrations >1μM; >50 fold the EC50 for K562 cells.

Cell Type SpecificityHDAC Inhibitors

U937 iCell Cardiomyocytes

Log [Apicidin] M Log [Apicidin] M

K562 iCell Cardiomyocytes

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On-target potency(cytotoxicity by apoptosis)

Off-target safety

Mitochondrial ToxicityMitochondrial ToxGlo Assay

Mitochondrial Tox-Glo AssayStep 1: iCell CardiomyocytesStep 1: Live cell measurement of cytotoxicity Utilizes a cell-impermeable dead-cell protease

fluorogenic substrateSt 2Step 2: Utilizes a luminogenic measure of ATP

K562

No MitoTox

MitoTox

• Oligomycin: phosphorylation inhibitor• Antimycin: respiratory chain inhibitor• Na Azide: electron transport chain inhibitor

CCCP: uncoupling agent (respiratory & phosphorylation)

MitoTox

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• CCCP: uncoupling agent (respiratory & phosphorylation)




iCell Hepatocytes



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Hepatotoxicity: AflatoxinHepatotoxicity: Aflatoxin

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iCell HepatocytesHepatotoxicity: Aflatoxin

Aflatoxins: • Naturally occurring mycotoxins produced by the fungus Aspergillus

120150

• Naturally occurring mycotoxins produced by the fungus Aspergillus• Metabolized by the liver to a reactive intermediate

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80

100

120

+ Keto + ANF- Keto + ANF

100

150HepG2Hum HepiPS Hep

lity

(RLU

)

HepG2PHHiCell Hepatocytes

00

20

40

10 20 30 40 50 60 70 80 90 1000 10 20 30 40 500

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% V

iabi

0 10 20 30 40 50 60 70 80 90 1000 10 20 30 40 50 Aflatoxin-B1 (M)

Conclusions:Conclusions: iCell Hepatoyctes exhibit a hepatotoxic response to Aflatoxin-B1,

similar to primary human hepatoyctes

Aflatoxin-B induced hepatotoxicity is inhibited by ketoconazole

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Aflatoxin-B1 induced hepatotoxicity is inhibited by ketoconazoleand alpha-naphthoflavone, inhibitors of P450 enzymes

iCell HepatocytesHepatotoxicity: Aflatoxin

ApoTox-GloCellTiter-Glo®

GSH/GSSG-Glo Luminescent Glutathione Assay

SH/G

SSG

SH/G

SSG

Glutathione Assay

PHHiCell Hepatocytes

Rat

io G

S

Rat

io G

S

PHHiCell Hepatocytes

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Aflatoxin (μM) Menadione (μM)

Hepatotoxicity: Rheumatoid ArthritisHepatotoxicity: Rheumatoid Arthritis

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iCell HepatocytesHepatotoxicity: Rheumatoid Arthritis

Conclusions:Conclusions: No apoptosis or cytotoxicity is observed in RO-1 treated cells

Both RO-2 and RO-3 cause rapid increase in caspase 3/7 within 5 h resulting in cell death within 24 h

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resulting in cell death within 24 hPoster #519

iCell HepatocytesHepatotoxicity: Rheumatoid Arthritis

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Poster #519

Summary

• iPS cell technology provides unprecedented access to human biology.

• CDI has developed a novel commercial platform for reprogramming and differentiating iPS cells and products.

• Promega has developed an extensive portfolio of simple, predictive assays for determining mechanisms of toxicityassays for determining mechanisms of toxicity.

• When used together, CDI’s iCell products & Promega’s photometric-based assays provide a relevant & accessible platform for assessing mechanisms of toxicity.

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Thank YouThank You

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Proteasome-Glo Cell-Based Assays Luminescent, real-time assays to measure:

Chymotrypsin-like proteasome activity Trypsin-like proteasome activity Caspase-like proteasome activity

“Add-mix-measure” format amenable to automated HTS

“The proteasome: a suitable antineoplastic target”Julian Adams (2004) Nature Reviews Cancer. 4:349-360( )

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Society of Toxicology Annual Meeting 13 M h 2012 13 March 2012 · 13 March 2012 Presentation...

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