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Improved Human Relevance of Neurobiology: Study of ‘Omics in Mini-Brains
CAAT - Center for Alternatives to Animal TestingBloomberg School of Public Health, Johns Hopkins University, Baltimore, US
Dr. rer. hum. biol.
André Kleensang
OutlineI. Toxicology in the 21th century – A vision II. Organotypic ModelsIII. Ecosystem Omics & Systems ApproachesIV. If time left: Humans(Organs)-on-a-chip
I. Toxicology - $3 billion of testing to regulate $10 trillion of trade
Problems / Motivation•Throughput•Costs•Animal use•Mixtures•High-dose to low-doseextrapolation
•Applicability to newproducts/hazards (e.g. nano)
•Inter individual/speciesdifferences=> Low predictive capacity
=>Too precautionary
4
Examples: Reproducibility of animal tests
• Cancer bioassay: 57% (repeat or mouse vs. rat)• Reproductive Tox: 60% between species• Uterotrophic assay: 26% contradictory• Skin sensitization: 75% guinea pig vs. mouse• Severe eye irritation: 70% reproducible • Acute fish tox: up to 6 log orders different
Preclinical tox testing of Aspirin in animals would result in: •R 22 harmful if swallowed •(LD50 = 150-200mg/kg in rats)•R 36 irritant to eyes•R 37 respiratory irritant•R 38 irritant to skin•Not carcinogenic, but co-carcinogen (promotor)•Unclear mutagenicity•Embryonic malformations incat, dog, rat, mice, rabbit, monkey
=> Difficult to be brought tothe market today
Hartung (2009) ATLA, 37(2), 45 – 47
Too precautionary…
Dependent on use (chemicals, pesticides, drugs,…)and country (EU, USA,…) you may need in addition
• Repeated dose toxicity• Reproductive and developmental toxicity• Carcinogenicity• Genotoxicity• Immunotoxicity• Skin sensitization• Skin irritation/corrosion• Eye irritation• Phototoxicity• Toxico- and pharmacokinetics• …
Acute toxicity data is not enough …
• Replacement: Don’t use animals if a non-animal method exists that can answer the scientific question at hand.
• Reduction: If you must use animals, keep the number to the minimum necessary to answer the question.
• Refinement: If you must use animals, keep any pain or distress they experience to a minimum.
What are the 3Rs?
1959 20077
=> An atmosphere of departure in toxicology
=> New technologies from biotech and (bio)informatics revolution
NAS vision report Tox-21c
“We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments” F. Collins, NIH, 2008
“With an advanced field ofregulatory science, new tools, including functional genomics, proteomics, metabolomics, high-throughput screening, andsystems biology, we can
replace current toxicology assays with tests that incorporate the mechanistic underpinnings of disease and of underlying toxic side effects.” M.A. Hamburg, FDA 2011
In vitroIn silico
(rodent)Animal tests
Mode of action
PoT / Toxome
Deterministic RA(precautionary)
SystemsToxicology
ProbabilisticRA
Evidence-basedTox (EBT)
ITS &
validation
Quality AssuranceTechnology Use
de facto valid
Validation
Evidence-basedTox (EBT)
http://www.activegarage.com/wordpress/wp-content/uploads/past-future-sign.jpg
What we want to measure are the causal events
What we typically measure
PoT ALTEX. 2014;31(1):53-61.
II. Organotypic cultures
Organotypic cultures implies a multi-cellular structurerepresenting at least some of the characteristics and
functions of the tissue it models
Conclusion
“All models are wrong, but some are useful”
George EP Box (2005): Statistics for Experimenters (2nd ed.) p 440
Your scientific question should determine which model to use
Limitations and Challenges3D in vitroLower reproducibility
Endpoints generally need optimization from 2D
Challenge to study on a single cell level
Limited perfusion
2D in vitroLack organ function, structure and complexity
Cancer or immortalized cells
Organ-on-chipCostly and complex
Low availability
Demands engineering
skills
Not suitable for high-throughput screeningOrgan specific biomarkers
Organ structure
Consist of numerous different cell types
– Many subtypes of neurons– Astrocytes– Oligodendrocytes– Microglia
Challenges
• Most complex organ in the human body
• Blood brain barrier
• Cerebrospinal fluid in the brain difficult to mimic
• Different extracellular matrix than commonly found in other organs
Organ function
• Sensitive organ to xenobiotic perturbations
• Action potential
• Calcium waves in neurons and astrocytes
• Myelination
• Cell-cell interactions
Re-aggregating cultures – Example brain
Preparation of rat primary aggregating brain cell cultures
Oligodendrocyte/neuron SynapsesAggregate (26 days)
Astrocyte Myelinated axon20µm 1µm 1µm
1µm 1µm
Trapp et al., 1979
Inner ½ outer ½
Neur. 32% 13% Astro. 57% 71% Oligo. 9% 3%Others 2% 13%
1979-2017
Trapp et al., 1979
iPSCs Neural Precursor Cells Gyratory Shaking 3D brain culture
Human iPSC-derived 3D neural model
Hogberg et al., (2013) Stem Cell Res Ther. 4(Suppl 1:S4)Pamies et al., (2014), Exp Biol Med 239:1096-107Pamies et al., (2017), Altex 34(3):362-376
Re-aggregating cultures – Example brainOrgan function
Micro-electrode arrays to measure spontaneous electrical activity
Intracellular calcium response to
neurotransmitters
Myelination of axons by oligodendrocytes
Pamies et al. (2017) ALTEX 34(3):362-376
III. Ecosystem: ‘Omics’
We need as well• (Epi)Genomics
(Methylation)• Proteomics
(Phosphorylation)• Metabolomics (flux)• Visualization, interpretation, text mining• Systems/network approaches
Cave: differential transcriptomics gives only part of the picture!
To understand
Andersen et al. (2013) Arch Toxicol 87:7-11
Why Systems Toxicology / Network Analysis?
• Systems approaches offer a dimensionality reduction that is helpful to interpret the data
• Allows for examining biology as a system rather than at a part-by-part basis
• Differential/ANOVA/heat map statistics are not adequate to understand a complex system
• Enrichment (KEGG, BioCyc etc.) and Gene Ontology (GO) depended on using existing annotations/knowledge
Group KEGG, WikiPathways, Reactome p-value
IV. Human-on-chip• Started as a program for
countermeasures to bioterrorism• Multi millionaire programs
– DARPA (Defense Advanced Research Agency), NIH and FDA
– DTRA (Defense Advanced Research Agency)
– EU project (Body-on-chip)
http://en.wikipedia.org/wiki/Organ-on-a-chip
For more information:http://www.ncats.nih.gov/tissuechip
http://www.extremetech.com/extreme/167748-us-army-develops-human-on-a-chip-tech-for-chemical-testing-fleshy-robot-soldiers
http://en.wikipedia.org/wiki/Organ-on-a-chip
http://en.wikipedia.org/wiki/Organ-on-a-chiphttp://www.ncats.nih.gov/tissuechiphttp://www.extremetech.com/extreme/167748-us-army-develops-human-on-a-chip-tech-for-chemical-testing-fleshy-robot-soldiershttp://en.wikipedia.org/wiki/Organ-on-a-chip
3-tissue-representative organ-on-a-chipsystem
Skardal et al. (2017) Nature’ Scientific Reports 7:8837
Example: two organ-on-a-chip interaction
Skardal et al. (2017) Nature’ Scientific Reports 7:8837
Regulatory acceptance…
There is one thing stronger than all the
armies in the world, and that is an idea whose time
has come. Victor Hugo
On résiste à l'invasion des armées;On ne résiste pas à l'invasion des idées.
The time will come soon to replace current animal toxicology assays with tests that incorporate the mechanistic
underpinnings of disease and of underlying toxic side effects !
http://en.wikipedia.org/wiki/File:Agriculture_in_Volgograd_Oblast_002.JPGhttp://www.fotofinder.com/preview/8EA06F4613DBAF78/?usrtrck=searchresult_link_icon_preview&searchresultid=747b1090af0ba3fc2d5b280371bf1593&start=5&text=Metallpresse
Improved Human Relevance of Neurobiology: Study of ‘Omics in Mini-BrainsOutlineI. Toxicology - $3 billion of testing to regulate $10 trillion of tradeSlide Number 4Slide Number 5Slide Number 6What are the 3Rs?Slide Number 8Slide Number 9Slide Number 10Slide Number 11II. Organotypic culturesConclusionLimitations and ChallengesRe-aggregating cultures – Example brainPreparation of rat primary aggregating brain cell culturesSlide Number 17Slide Number 18Re-aggregating cultures – Example brainSlide Number 20Why Systems Toxicology / Network Analysis?Slide Number 22IV. Human-on-chip3-tissue-representative organ-on-a-chip�systemSlide Number 25Slide Number 26Slide Number 27