Download Toxicology: A Springboard for Stem Cell Scientists? - NAS

National Academy of Sciences Workshop
“Stem Cell Models for Environmental Health”
Washington, DC, June 3-4, 2010
Toxicology: A Springboard
for Stem Cell Scientists?
Roger A. Pedersen
Professor of Regenerative Medicine
Laboratory for Regenerative Medicine
University of Cambridge
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How can stem cells be used for treatment of diseases?
Transplantation
Stem cells
Differentiation
Tissue engineering
The major expected clinical use of stem cells is tissue
repair and replacement, i.e., regenerative medicine.
Jim Shapiro developed islet transplantation
Chris & Dana Reeve envisioned stem cell treatments for spinal cord injury
Achieving their vision will require
fundamental knowledge about how
stem cells can be induced to specialise
into therapeutic tissues, and whether
they are safe for transplantation.
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Derivation and scaleup of GMP-quality cells
Immune mis-match between donor and recipient
Epigenetic alterations may undermine stability
Other stem cell translational targets can
be more accessible than transplantation
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Disease modelling using pluripotent stem cellderived cells and tissues
Drug discovery using stem cell-derived cells
Use patient-specific cells (towards personalized
medicine)
Toxicity screening using stem cell-derived
cellular systems
Approaches to toxicology testing using
stem cell-derived cellular systems
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Modelling penetration of xenobiotics through
barrier cell populations (e.g., skin, blood vessels)
Activation/detoxication of xenobiotics by
primary metabolic cell types (e.g., liver, intestine)
Assessment of target cell damage and response
to xenobiotic/metabolite exposure
FP7 HEALTH.2010.4.2-9: Towards the replacement of
repeated dose systemic toxicity testing in human safety
assessment. (Funding: European Commission, 25M Euro +
COLIPA, 25M Euro, participant selection late 2010)
1: Optimisation of current methodologies and development of
novel methods to achieve functional differentiation of humanbased target cells in vitro.
a) Optimisation of stem cell technology as a source for humanbased target cells for toxicological purposes
b) Refinement of cell culture systems for long-term toxicity
testing
c) Exploitation of emerging mechanistically-driven methods
controlling cellular differentiation
FP7 HEALTH.2010.4.2-9: Towards the replacement of
repeated dose systemic toxicity testing in human safety
assessment.
2: Exploitation of organ-simulating cellular devices as alternatives
for long-term toxicity testing
a)Integration of target and metabolising cells to simulate multi-organrelated toxicity in vitro
b) Utilisation of scaffolds and microstructures to optimise the cellular
microenvironment
c) Development of novel cellular barrier models relevant to systemic
exposure
d) Optimisation of microsensors to monitor tissue responses in organsimulating devices
FP7 HEALTH.2010.4.2-9: Towards the replacement of
repeated dose systemic toxicity testing in human safety
assessment.
3: Establishment of endpoints and intermediate markers in human-based target
cells with relevance for repeated dose systemic toxicity testing.
a) Functional parameters as predictive signals of human long-term toxicity
b) Establishment of “-omics”-based markers as predictive signals of human longterm toxicity
c) Integration of markers for enhancement of human long-term predictive capacity
4: Computational modelling and estimation techniques.
5: Systems biology for the development of predictive causal
computer models.
6: Integrated data analysis and servicing.
d) Setting up a cell and tissue bank for in vitro toxicity testing
7: Coordination action.
How can stem cells be used to generate mechanismbased toxicology reporter systems?
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Induction of xenobiotic metabolizing networks
Cellular response to oxidative/nitrosative stress
Induction of apoptosis
Proliferation disturbances (cell cycle arrest or
promotion)
Strategies for reporter gene use in toxicology
• Constitutive expression (housekeeping gene
tagged)
• Direct activation (responsive gene tagged)
• Indirect activation (target of responsive gene
tagged)
• Conditional activation of tagged gene (e.g.,
by Cre or Tamoxifen)
• Permanently switch on or off (conditionally
target a constitutive gene locus)
Constitutive expression of GFP reporter gene
targeted to the human AAVS1 locus
Smith et al., Development 2008
Hockemeyer et al., Nature Biotech 2009
Constitutive expression of RFP reporter gene
targeted to the human ROSA26 locus
Irion et al., 2009
Generic Targeting Strategy
B1 FRT en2SA T2A H2B_Venus pA FRT loxP PGKp
neoR
pA loxP B2
ATG
exon
AUG
mRNAs
proteins
T2A H2B_Venus pA neoR pA
H2B_Venus neoR
or
RFP neoR
H2B_Venus will be expressed as a non‐fusion protein because of the T2A ribosome skip site (self‐cleaving peptide)
Targeting the human Brachyury gene
Targeting the human Brachyury gene
Targeting the human Brachyury gene
Potential targets for toxicology reporter genes
Oxidative/Nitrosative Stress
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Induction
of xenobiotic networks: CYP3A4
HMOX
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(HO)
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Response
to Oxidative stress: NRF2 target(s)
Induction of apoptosis: Caspases
Proliferation disturbances: Cell cycle genes
Hemeoxygenase-1
Conclusions
• Pluripotent stem cells can be induced to
differentiate into either xenobiotic
metabolizer or target cells
• Pluripotent stem cells can be genetically
modified to express reporter genes
• Reporter genes used can be assessed by
fluorescence, luminescence or combined
modalities
• Differentiated cells with reporter genes
can be interfaced with devices for high
content/high throughput screening
Acknowledgements
Gabrielle Brons
Zhenzhi Chng
Daniel Ortmann
Sasha Mendjan
Bowen Sun
Maria Ortiz
Andreia Bernardo
Lily Cho
Kate Quinlan
Tiago Faial
Thomas Moreau
Yifan Ng
Kathy Niakan
Smruthi Jayasundar
Cambridge Stem Cell Initiative
www.stemcells.cam.ac.uk
The Wellcome British Heart
Foundation
Trust
NIHR