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