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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. PedersenProfessor of Regenerative Medicine

Laboratory for Regenerative MedicineUniversity of Cambridge

Differentiation

How can stem cells be used for  treatment of diseases?

Stem cells

Tissue engineering

Transplantation

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.

• 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

• Disease modelling using pluripotent stem cell- derived 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

• 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 human- based target cells in vitro.

a) Optimisation of stem cell technology as a source for human- based 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 alternativesfor long-term toxicity testing

a)Integration of target and metabolising cells to simulate multi-organ- related 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 organ- simulating devices

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 toxicityb) Establishment of “-omics”-based markers as predictive signals of human long- term toxicityc) 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 causalcomputer models.

6: Integrated data analysis and servicing.d) Setting up a cell and tissue bank for in vitro toxicity testing

7: Coordination action.

FP7 HEALTH.2010.4.2-9: Towards the replacement of repeated dose systemic toxicity testing in human safety assessment.

How can stem cells be used to generate mechanism- based toxicology reporter systems?

• 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

T2AT2A H2B_VenusH2B_Venus pApA neoneoRR pApA

AUG

Generic Targeting Strategy

exon

H2B_Venus will be expressed as a non‐fusion protein because of the T2A ribosome skip site (self‐cleaving peptide)

mRNAs

proteins

ATG

H2B_VenusH2B_Venus neoneoRR

B1B1 B2B2en2SAen2SA T2AT2A H2B_VenusH2B_Venus neoneoRRloxPloxP PGKpPGKppApA pApA loxPloxPFRTFRTFRTFRT

RFPRFP neoneoRRor

Targeting the human Brachyury gene

Targeting the human Brachyury gene

Targeting the human Brachyury gene

Oxidative/Nitrosative Stress

Potential targets for toxicology reporter genes

• Induction of xenobiotic networks: CYP3A4

• Response to Oxidative stress: NRF2 target(s)

• Induction of apoptosis: Caspases

• Proliferation disturbances: Cell cycle genes

Hemeoxygenase-1

HMOX 1(HO) 1

• 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

Conclusions

AcknowledgementsAcknowledgements

The Wellcome Trust

British Heart Foundation

Gabrielle Gabrielle BronsBrons ZhenzhiZhenzhi ChngChng Daniel Daniel OrtmannOrtmann

Sasha Sasha MendjanMendjan Bowen SunBowen Sun Maria OrtizMaria Ortiz

AndreiaAndreia BernardoBernardo Lily ChoLily Cho

Kate QuinlanKate Quinlan Tiago FaialTiago Faial

Thomas MoreauThomas Moreau YifanYifan NgNg

Kathy Kathy NiakanNiakan SmruthiSmruthi JayasundarJayasundar

Cambridge Stem Cell Initiativewww.stemcells.cam.ac.uk

NIHR