modelling alzheimer’s disease - axolbio.com · to become pluripotent” induced pluripotent stem...
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Modelling Alzheimer’s Disease:
A High Throughput-Compatible Assay forDetecting Tau Aggregation Using iPSC-Derived Cortical Neurons
Alfredo Cabrera-SocorroNeuroscience R&DJanssen Pharmaceutica
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Outline
iPSC and related initiatives in Europe: EBiSC
Alzheimer’s Disease & tau
Modelling tau aggregation using iPSCs
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S. YamanakaJohn B. Gurdon
Nobel Prize in Physiology or Medicine 2012“for the discovery that mature cells can be reprogrammed
to become pluripotent”
Induced Pluripotent Stem Cells:iPSCs
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A virtually unlimited source ofhuman cells available for drug
discovery
Non-embryonic and non-tumoral
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European Bank forinduced pluripotent Stem Cells
Creating a self financing stem cellrepository for Europe
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What is the EBiSC project?
EBiSC Copyright © EBiSC Consortium
A €35 million, IMI funded project through which 26 leading European organisations
will establish a central facility for the collection, testing and distribution
of iPS cells to researchers.
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EBiSC Copyright © EBiSC Consortium
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Why create EBiSC?
8
With EBiSC: better use of research assets
provide samples of iPSClines to EBiSC
Creates distribution stocks & ensures quality
get iPSCs of known quality,faster & at less cost
Research projects creating iPSCs EBiSC Other researchers
EBiSC Copyright © EBiSC Consortium
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EBiSC : improving the research landscape in Europe
Research projects creating iPSCs EBiSC Other researchers
Create a catalogue of cell lines whichmeet user needs
Common standards for processing and
testing cell lines
Data management system which provides extensive data to users
but controls access
Consent forms &contracts which
meet needs of allstakeholders
Establish central facilities which use best cellculture technologies to operate at scale
EBiSC Copyright © EBiSC Consortium
What will EBiSC do?
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• By end 2016: validating working pipeline: reception, QC/banking, expansion,distribution and phenotyping
• Beyond 2016: expand catalogue to meet user demand leading to a selffinancing operation by 2019.
New EBiSC Commissioned iPSC lines
2014 2015 2016
Sanger HIPSCI linesHot Start lines from 7 iPSC centres
iPSC lines from other large scale global projects eg FP7, H2020, IMI—1, IMI-2
EBiS
C iP
SC c
atal
ogue
Foun
dato
nal
colle
cton
↑ First cells shipped to researchers from EBiSC
↑ Main central facility operational↑ Standard contracts
↑ Extended web portal operational↑ Standard Quality Control testing programme
↑ Harmonised production protocols v1
Copyright © EBiSC Consortium EBiSC
When will EBiSC deliver results?
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More about EBiSC…
Copyright © EBiSC Consortium
Contact us at: [email protected]
Visit our website at: www.ebisc.eu
EBiSC
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Outline
iPSC and related initiatives in Europe: EBiSC
Alzheimer’s Disease & tau
Modelling tau aggregation using iPSCs
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Dementia: one the most significant socialand health crisis of 21st century
Worldwide, nearly 44 million people have Alzheimer’s or a relateddementia: Denmark, Norway, Sweden, Belgium and The Netherlands(16.8M)
It cannot be prevented, cured or slowed.
Source: ADI
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Alzheimer’s Disease Pathophysiology
“Intraneuronal aggregates ofhyperphosphorylated and misfolded
tau”.
“Extracellular deposits of amyloid-b peptide abundant in the cortex of
AD patients”
Neurofibrillary tangles (NFTs) Amyloid plaques
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High correlation between tau lesionsand degree of dementia
• Braak stages: Clear correlation betweenthe spatiotemporal progression of NFTpathology and cognitive state.
• NFTs, neuron loss, and synaptic loss,parallel the progression of cognitivedecline
Asymptomatic(entorhinal)
Symptomatic(Hippocampal)
Severe (neocortical)
• Specific genetic variants of tau areassociated with familial forms offrontotemporal dementia (FTD)
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Tau – in normal and diseased state
• Tau is expressed in the CNS, primarily inaxons of neurons
• Tau binds to and stabilizes microtubulesthus ensuring proper transport of cargo tothe nerve endings
• In Alzheimer’s disease tau is phosphorylatedby multiple kinases, detaches frommicrotubules and aggregates which finallyleads to neuronal death
• It is hypothesized that toxic tau-species canspread from cell to cell thus “infecting”neighbouring neurons and spreading thedisease
Hanger, D., et al. Trends Mol Med 2009.
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Therapeutic approaches
Modified from Brunden et al., Nat Rev Drug Discov. 2009 Oct;8(10):783-93.
Spreading inhibitor
Active / passiveimmunization
Proteasomeactivator
Tau kinases &
phosphatases
Tau assemblyinhibitors
Microtubulestabilizers
Autophagyenhancers
HSP90inhibitors
• Blocking aggregation
• Stop spreading
• Promoting clearance
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Tau aggregation: seeding model
Seeding
• Aggregated tau induces misfolding of normal tau (seeding hypothesis).• Evidence for seeding in vitro and in vivo
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Outline
iPSC and related initiatives in Europe: EBiSC
Alzheimer’s Disease & tau
Modelling tau aggregation using iPSCs
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Need for a physiologically relevant/robustmodel to study TAU aggregation
• Human/Neuronal specific
• High reproducibility (≠primary cultures)
• Sensitive
• Scalable (high throughput)
Verheyen et al., PLoS One, 2015Medda et al., J Biomol Screen (under review)
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Robust differentiation protocol
Commercially available iPSC lines Generation of NPCs (Axol biosciences):
•Dual SMAD inhibition 12 days•Freezing stocks
Final differentiation: BDNF, GDNF, cAMP
384w format (HCI and HTS)
NPCs NPCsiPSCs Neurons
0 12 30 70 (40)0 12 30 70 (40)
30-40 days
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Robust culture conditions to allowstandardization:
Neurospheres/Organoids– Intrinsic capacity to form in vitro a “cortical like” structure.
Scaffolding biomaterials:– Matrigel ®
Homogeneous cell distribution (HCI) Long term stability/adherence (synapse strength) Reduced shearing stress due to media change/plate
handling Scalability/automation (avoid pre-coating)
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TAU aggregation in human iPSC derivedneurons cultured in matrigel/384WP
Matrigel coating/embedding (10 days) Polyornithin/Laminin Coating (10 days)
Matrigel scaffold
• Use of matrigel:•Homogeneous cell distribution.•Synaptic stability/strength •Reduces shearing stress•Compatible with imaging
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Matrigel scaffold
• Low density matrigel:
- Dilution 1:15 (MG:N2B27)
- Cells sediment before MG gels
- Cells are “coated” with matrigel
- Thickness: 50um (approx.)
- For TAU aggregation assays
Day 7
7 Days in vitro
• High density matrigel:
- Dilution 1:1 (MG:N2B27)
- No sedimentation, fast gelling (37C)
- Cells are “embedded” in matrigel
- Thickness: 200um (approx.)
- For electrophysiology
7 weeks in vitro
Choi et al, Nature 2014
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Neurons 30 DIV
•96 well plate, 30K cells /well•384 well plate, 10K cells/well
Dynamic culture format tomaintain healthy neurons
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Neurons 30 DIV
CellTiter-Glo®
Dynamic culture format tomaintain healthy neurons
• IncuCyte ®
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Differentiated neurons are transducible
30 DIV
7 DIV
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Adenoviral-mediated TAU aggregationusing iPSC derived neurons
• Experimental setup:•10k Cells/well•AAV P301L•384WP: 75uL media, 20uL lysate
• Conditions:•No treated cells (C-)•No treated cells + K18 Fibrils (K18)•AAV P301L•AAV P301L + K18 (P301L +K18)
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TAU aggregates in human neurons
*n=3, One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison testversus non seeded control).
HA AT8 DAPI
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alphaLISA is the non-wash ELISA assay alternative alpha = amplified luminescent proximity homogeneous assay 3-4 hours assay
The higher the emission, the more TAU aggregation
How to measure TAU aggregation?alphaLISA - TAU
Counts
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Defining baseline valuesand sampling time
*n=3, One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison testversus non seeded control).
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K18 Dose response(Z’=0,52)
n=6 technical replicates
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Shortening protocol: 22 days
*n=3, One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison testversus non seeded control).
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Assay reproducibility:across cell lines and users
*n=3, One-way Anova. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Dunnett’s multiple comparison testversus non seeded control).
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New assay to study TAU aggregation
• Human/Neuronal specific
• High reproducibility (≠primary cultures)
• Sensitive
• Scalable (high throughput)
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Towards the standarization of iPSC technologyfor drug development in neuroscience:
Perspectives and challenges
1. To tackle different aspects of tau pathology: seeding,aggregation, clearance?
2. Test available mutant cell lines and isogenic controls: lessartificial
3. Optimizing maturation process: mature neurons in shortertime, co-culture (astrocytes)
4. Testing chemically defined scaffolding materials: eg.hyaluronic acid
0 7 10 30
AAV K18 Sampling? ? ?
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Thanks! • Xavier Medda• Annick Diels• An Verheyen• Ines Royaux• Andreas Ebneth
Paul Janssen Research Center, Beerse (Belgium)