Discovery Sciences | Reagents & Assays
Mei DingIPSC&A (iPSCs, Primary cells, Stem Cells & Assays)
Discovery SciencesAstraZeneca
iPSC & iPSC derived cells-Values and Applications in drug discovery
Items
• Introduction-iPSC values and applications
• iCells Cardiomyocyte validation examples
• Future opportunities and challenges
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Drug discovery processes• Use of biologically relevant cells in physiological in vitro assays is very important in the early drug discovery processes• Multiple, biologically relevant targets and pathways are simultaneouslyinterrogated in biologically relevant cells
Biologically relevant cells• Human primary cells• Pluripotent stem cells derived cells
• ESC derived cells• iPSC derived cells
• Adult stem cells (ASC)• etc
Target Validation
Lead Generation
Lead Optimization
Pre-clinical Development
LGID CSIDLOID
Clinical Studies
FTIM
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Human primary cells • Difficult to obtain for certain cell types• Limited proliferative capacity• Challenge to maintain the in vivo phenotype in culture• Large batch variations
Human primary cells, ESCs & ASCsLimitations
hES cells • Limited access• Significant ethical and political debates
Human adult stem cells • Limited access• Often limited in their proliferative ability and can become only the cell type(s) of the organ where they come from
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Patients Patients
Screening
”Disease in a dish”
Differentiatedcells
iPS cell production
iPScells
Somaticcell
Cell expansion
iPSCcharacterisationDrugs
Reprogram-ming factors
• Gene expression • Epigenetics• DNA fingerfrinting• Karyotyping• Pluripotency
[Embryoid bodies(EB), Teratomas, directeddifferentiation]
Human iPSC & iPSC derived cells
iPSCgeneration
• Clone selection• Expansion • Banking
Reprogramming of human adult somatic cells to become hESC-like induced
pluripotent stem cells (iPSCs). hiPSCs have the ability to divide indefinitely
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Patients Patients
Screening
”Disease in a dish”
Differentiatedcells
iPS cell production
iPScells
Somaticcell
Cell expansion
iPSCcharacterisationDrugs
Reprogram-ming factors
• Gene expression • Epigenetics• DNA fingerfrinting• Karyotyping• Pluripotency
[Embryoid bodies(EB), Teratomas, directeddifferentiation]
iPSC
Human iPSC & iPSC derived cells
iPSCgeneration
In vitro differentiationto tissue specific cell
types
• Clone selection• Expansion • Banking
iPSCs largely resemble hESCs and can be differentiated in vitro to tissue
specific cell types
Discovery Sciences | Reagents & Assays
Human iPSC directed differentiation in vitro
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Patients
Screening
”Disease in a dish”
Differentiatedcells
iPScells
iPSCDrugs
In vitro differentiation to tissuespecific cell types
Assaydevelopment
iPS Cells
Endoderm
Liver
Intestine
Pancreas
Mesoderm
Lung
Heart
Fat
Ectoderm
Brain
Nervoussystem
Eye
• Possibilities
iPSCs have the potential to become all cell types of the body , can therefore
serve as a potential unlimited ex vivo source of human derivatives
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Human iPSCs & iPSC derived cells in Drug discovery
• Target ID / target validation• Disease in vitro models• Phenotypic screening• 2nd assays & MOA• Efficacy• Safety / Toxicity• DMPK• etc…
• Easy to handle• Scalable• Stable & Robust• hiPSC directed In vitro differentiation
to tissue specific cell types• Functional• Cost effective
hiPSC advantages hiPSC applications
Target Validation
Lead Generation
Lead Optimization
Pre-clinical Development
LGID CSIDLOID
Clinical Studies
FTIM
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iPSCgeneration
AZ iPSC capabilities (small-scale)
Scale up iPSCcharacterisation
Pluripotent QC of hiPSC
Grafting iPSCunder kidneycapsule in SCID mouse
Differentiatedcells
iPSC directeddifferentiation
in vitro
Accessing External World-leading Expertise in stem cell technology
Cellular Dynamics International (CDI) & AZ (CVMD, GSA, DMPK and DS)
• On-going collaboration with access to the iCELL products and other products for use in the safety, discovery and regenerative medicine programs
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Examples of AZ on-going iCells validation
• Use of human iPSCs in toxicity testing
• Use of human iPSCs in therapy areas - Cardiomyocyte proliferation assay
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In vitro safety tools
12 Joanne Bowes | April 2013 Innovative Medicines | Global Safety Assessment
Impact
• Biochemical and cellular assaysCell lines
• Recombinant human screens, cardiac ion channel screens,
• Transporter assays• Secondary Pharmacology
screens
Molecular
• Cardiomyocytes• Hepatocytes
Primary cells
• Isolated heart• Brain sliceTissue
Will human stem cell derived cells offer a scientific or technical advantage?
Where we are now Where we need to be
Primary short-term objectiveRobust screens predictiveof human toxicity
Measured bySensitivitySpecificityPredictivityRobustness
Cost:benefit
Technology availability
Use of iCells in Safety Assessment
Innovative Medicines | Global Safety Assessment
Applied in three modes
1. Front loading of in vitro ‘black box’ and molecular assays prior to toxicology studies
• Routine screening within the design make test cycle
2. Bespoke target safety
3. Investigative toxicology• De-risk follow up compound series• Provide mechanistic understanding to observed in vivo toxicology
In vivo models
In vitro models
Lead Generation
Lead Optimization
Pre-clinical Development
LGID CSIDLOID
Clinical Studies
FTIM
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Potential use of iCell Products for Front loading
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Cardiotoxicity
1. Front loading - in vitro ‘black box’ and molecular assays in medium –high throughput format
Hepatotoxicity
Chronotropes Beat rate
Inotropes Beat rate/amplitudeCa2+ flux
ECG modulation APDIon channel inhibition
Pathology change High Content Biology (HCB)
Dysfunction Measure
Pathology change High Content Biology (HCB)
Innovative Medicines | Global Safety Assessment
Use of in vitro models in safety assessment
Applied in three modes
1. Front loading of in vitro ‘black box’ and molecular assays prior to toxicology studies
• Routine screening within the design make test cycle
2. Bespoke target safety
3. Investigative toxicology• De-risk follow up compound series• Provide mechanistic understanding to observed in vivo toxicology
In vivo models
In vitro models
Lead Generation
Lead Optimization
Pre-clinical Development
LGID CSIDLOID
Clinical Studies
FTIM
Innovative Medicines | Global Safety Assessment15
Target Safety and Investigate toxicologyPotential use of iCell in Impedance assay to explore target safety and human CV safety risks
• Using impedance to measure cell function (beating)
• Using siRNA and tool compounds to explore target versus off-target toxicity
• Data shown was from rat neonatal cardiomyocytes
• Moving to iCells: focus on human safety risks!
beat amplitude
beat rate
Time (sec)
Cel
l Ind
ex
RTCA Cardio impedance instrument quantifes cardiomyocyte beating
*
***
**
Target contributes to CV toxicity seen in vivo
A B
9 inhibitors
•Beat IC50 & kinase inhibition IC50’s not correlated•siRNA has negligible effect on beating
CV toxicity in vivo unlikely due to target Lamore & PetersAstraZeneca, Boston
Kinase-A
Kinase-B
CtrlsiRNA
K1 K2 K1/2
K1 K2 K3 K4
Ctr
lsiR
NA
•Kinase inhibitors ↓beat amplitude•Potencies correlate with kinase affinities•siRNA ↓ amplitude
K1-
4
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Proliferation assay for cardiac regeneration phenotypic screening
Background: Unmet medical need for post-MI and heart
failure patients
Fundamental problem: Loss of functional cells
Goal: Regenerate functional myocardium
Task: To identify noval targets or chemistry that can induce
cardiomyocyte proliferation for the treatment of post-MI
cardiac dysfunction and heart failure
Phenotypic screening approach
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• The phenotypic screening was the most successful approach for first-in-class drugs discovered between 1999 and 2008
• Idealy use native human cells (multiple, biologically relevant targets and pathways are simultaneously interrogated)
CDI iCell Cardiomyocyte
• Measure the desired biological effect (activity might be translated to human disease more effectively than that in target-based screens):
Cardiomyocyte proliferation
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Challenge 1: Mature CMsdon’t proliferate and iPSderived CMs proliferateslowly. Potential smallassay window
Total DNA
ATP
Edu DNA
synthesis
Ki67 Prolif. marker
Nuclearnumber
Cell number
Potential samll assaywindow due to slow CMproliferation
An ideal readout,but difficult for CMSensitive, an early
indication of cellproliferation
Edu High ContentImage assay
Assay options and challenges
Challenge 2: CMs areheterogenous (mono-, bi-,multi-nuclei). Can’t usenuclei to count cell number.Need a good membranemarker to do cellsegmentation.
Edu HCS assay
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Load cells
with Edu
Alexa Flour azide
CuSO4
Fix
cells
Staining of nuclei
& other markersImage
Edu staining
Assay parameters optimized
• Different assay media
• Coating: Gelatin, Collagen, CellBind surface
• Edu loading conc
• Cell density
• With or without serum starvation
• Compound treatment period
Plate cells (3.5k c/w)
384-w
Exchange to CDI M M
2-3 days
Add cpds & Edu in
Assay M
Fix cells
2 days 2 days
Staining& Image
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Medium supplement effect on proliferationMedium-BMedium-A
Edu staining
CTnT staining
10x
• A basal CM proliferation in medium-A, 6-7% of nuclei were Edu+ with 2-d Edu loading
• Medium-B enhanced CM proliferation significantly
• bFGF in medium-A gave slightly less effect than medium-B alone
0.0
5.0
10.0
15.0
20.0
25.0
Medium-A Medium-B bFGF in medium-A
% E
du +
nuc
lei
Tool compound effect on CM proliferation
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BIO (GSK3beta inhibitor) bFGF
EC50 = 160 nM EC50 = 120 pg/ml
BIO
Concentration (µM))0.1 1
%Ed
u+
nucl
ei(n
orm
aliz
edda
ta)
-20
0
20
40
60
80
100
0.03 0.1 0.3 1 Concentration (µM)% E
du +
nuc
lei(
norm
aliz
edda
ta)
bFGF
Concentration (ng/ml)0.1 10
%E
du+
nucl
ei(n
orm
aliz
edda
ta)
-20
0
20
40
60
80
100
120
0.1 1 10 100Concentration (ng/ml)% E
du +
nuc
lei(
norm
aliz
edda
ta)
• GSK3beta inhibitor BIO and bFGF induced dose dependent increase in the number
of Edu+ nuclei, an early indication of CM proliferation
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Future opportunities• “Disease in a dish” - Disease modeling
• Patient-derived iPSCc• Genome editing to introduce disease variant
• “Patient in a dish” - Personalised medicine • “Clinical trials in a dish” - Patient variation • Opportunity to increase understanding of clinical trial out-come by
generating iPS cells from stored blood samples• Regenerative therapy
Human iPSC & iPSC derived cells
Challenges• Control differentiation to a fully mature cell (ex. CM)• Reach a highly pure, homogeneous and mature population• Reach full ‘adult’ maturation when cultured in vitro.
Acknowledgements
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This is cross-function activity
Discovery SciencesHenrik Andersson, Gabriella Brolén, Elke Ericson, Philip Worthington, Sam Peel, Anna Jonebring, Anders Lundin, Anette Persson-Kry, Björn Löwenadler
CVMDLauren Drawley, Qing-Dong Wang, Britt-Marie Kihlberg, Alan Sarbish, Bruno Becker
GSAHelen Garside, Joanne Bowes, Amy Pointon, Clay Scott, Matt Peters, Sara Lamore
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