A novel human embryonic stem cell derived cell model for use in

high content functional assays of neurodegenerative disorders

Stebbeds W1, Nancolas B1, Smith, G1, Gowers I1, Jones P3, Roth B3, Hamby M3, Cook K1, Anton J1,Iovino M1, Gardner C1, Lachize

S2, Lo K2, Cheung M2, Mitchell P3, Lazari O1, McAllister G1, Ray, J3

1 Discovery from Charles River, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL 2 Discovery from Charles River, Darwinweg 24, 2333 CR Leiden, Netherlands 3 Neurodegeneration Consortium, MD Anderson Cancer Center, Institute for Applied Cancer Science, Houston, Texas, USA

1 Introduction

As average life expectancies increase, the prevalence of neurodegenerative disorders is also increasing. Despite

decades of research and numerous therapeutics entering clinical trials, treatment options for neurodegenerative

diseases remain extremely limited and current therapies are only available for symptom management.

The use of disease relevant cells in the search for greater understanding and novel therapeutic approaches to complex

neurodegenerative disorders is imperative to achieve clinically relevant data. Similarly, the use of high content

approaches using multi-parametric analyses to investigate pathophysiology as well as on-target and off-target effects of

potential therapeutics on phenotype has gained traction in recent years. This is due to the increased productivity of

these methods at generating first in class drugs when compared to conventional target based screening(1).

We describe herein a novel cell model using embryonic stem cells differentiated into medium spiny neurons based on

the modulation of SMAD, GABA, CREB and GSK3β signalling(2-3). These cells present a neuronal phenotype after 23

days of differentiation with a complex neurite network and further differentiation produces a phenotype presenting

multiple markers of mature medium spiny neurons.

Using this system we have been able to use these cells for numerous assays looking at a wide range of

neurodegenerative disorders. The data we present here demonstrates the expression levels of relevant proteins

associated with neuronal differentiation. These cells have then been used to assess the neuroprotective effect of a

number of compounds, identified from a separate phenotypic screen performed in an immortalized cell line by assessing

neurite length and cell viability as a surrogate for neurotoxicity.

2 Materials & Methods

3 Results

4 Conclusions

Charles River Discovery have produced a disease relevant cell model for the investigation of neurodegenerative disorders using high

content phenotypic assays to develop more clinically relevant therapies. The principal advantages of this cell model are:

1) Rapid differentiation of neurons, with a complex network of MAP2 and β-III tubulin positive neurites observed by day 23;

2) Amenable to high content assays for simultaneous assessment of target abundance, morphology and viability;

3) Consistent and predictable expression pattern over the course of differentiation: enables the tailored use of cells with the

desired characteristics with low inter-experimental variability;

4) Short differentiation time course amenable to genetic modulation: cell model has been used to study the effect of

knockdown and overexpression of number of different disease relevant genes.

These cells have now been used in a number of different assays to study the effect of putative therapeutic agents, yielding disease

relevant data in a cost-effective and time-efficient manner

Nestin Type VI intermediate filament protein Transient expression during early development

Pax6 Transcription factor Specifies a neuroectoderme fate

βIII-tubulin Microtubule highly expressed in neuronal cells

MAP2 Microtubule associated protein 2 Marks neuronal cell body and dendrites

GABA/GAD GABA - inhibitory neurotransmitter Glutamate decarboxylase – required for GABA production

CTIP2 Transcription factor Labels MSN within striatal cell population

FOXP1 Transcription factor Co-expresses with CTIP2 and DARPP32 in mature MSNs

Oct4 Transcription factor Highly expressed

Nanog Transcription factor Loss promotes differentiation

DRD1 DRD2 Dopamine receptors

DARPP32 Marker of MSN Inhibitor of PP1 or PKA

hESC maintenance and expansion

mTeSR or CM+F

CN03 induced neurite retraction assay

CN

03 tr

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N03

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mpd

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Inhibition of CN03 induced retraction

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CN03 induced neurite retraction at 4 hours

Time course experiments

4 hour end-point assessment of compound A CRC β-3 tubulin β-3 tubulin

CN03 CN03 + 15 µM Cmpd A

Figure 7: Differentiated neurons were used in a phenotypic assay to observe the neuroprotective effect of putative therapeutic compounds. Compounds were tested in a CN03 induced neurite retraction assay. This assay is based on the

principle that CN03 activates the Rho pathway, leading to microtubule disassembly and neurite retraction (B)(4). Compounds with the ability to inhibit this retraction are considered to be neuroprotective. In the first instance, human embryonic

stem cells differentiated to a neuronal phenotype were exposed to CN03 with or without the lead compounds for 24 hours, with neurite length measured by live cell imaging every 20 minutes (A). The images were then analysed using the

InCell Developer Toolbox from GE, separately segmenting nuclei, soma and neurites (C). A time course indicated that a four hour incubation yielded the optimum neurite retraction window, showing stable retraction and no decrease in

neurite length when cells were exposed to CN03 and a control compound. Subsequently, the effect of 1.5 nM to 50 µM of the lead compounds on inhibition on neurite retraction were assessed at 4 hours (D).

Figure 1: Schematic overview of the neuronal differentiation process utilized at Charles River Discovery. Pluripotent human ES cells are

cultured in selection and maturation media over 40 days, based on the methods used by Telezhkin and co-workers (2016)(3). To validate

the differentiation, the expression levels of key differentiation markers are assessed at pre-determined time points.

Nes

tin

CTI

P2

β-III Tubulin Expression Day 16 Day 23 Day 30 Day 37

Figure 6: β-III tubulin staining by ICC, quantification of the percentage of positive cells and neurite area over the course of neuronal differentiation. The expected β-III tubulin expression profile was achieved, with the vast majority of cells

expressing at day 23 and maintaining expression thereafter. As this staining was so consistent and perfectly stained the neurites, it was used to calculate neurite area over time, showing that neurite area increases rapidly from day 18

through to day 23, and maintains a similar neurite length throughout differentiation thereafter.

MA

P2

Q48 area neurites/cell count

day 1

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day 1

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Day 16 Day 23 Day 30 Day 37

Day 16 Day 23 Day 30 Day 37 Day 16

CTI

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DAP

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Figure 2: Nestin staining by ICC and quantification of the percentage of positive cells over the course of neuronal differentiation. The expected Nestin

expression profile was achieved with the majority of cells expressing Nestin at day 16 (early development) but losing expression during neuronal maturation.

Figure 3: CTIP2 staining by ICC and quantification of the percentage of positive cells over the course of neuronal differentiation. The expected CTIP2

expression profile was achieved and the number of positive cells increasing throughout the differentiation and maturation process, with a maximum achieved

at differentiation day 37.

Figure 4: MAP2 staining by ICC and quantification of the percentage of positive cells over the course of neuronal differentiation. The expected MAP2

expression pattern was achieved with no expression prior to differentiation and an increase to approximately 100% positive cells by day 23, which was

maintained up to day 44 and beyond.

Figure 5: mRNA data for 6 of the markers used to assess neuronal differentiation and maturation: Nanog, Nestin, DRD1 and 2, MAP2 and β3-tubulin

(BCL11B). These data closely match the protein expression data shown elsewhere on this poster, with expression increasing and decreasing at the

expected differentiation time points.

5 References

1) Swinney D. C. & Anthony J. (2011) How were new medicines discovered? Nat Rev Drug Discov.

10(7):507-19.

2) Chambers SM, et al. (2009) Highly efficient neural conversion of human ES and iPS cells by dual

inhibition of SMAD signaling. Nat Biotechnol. 27(3):275-280

3) Telezhkin V, et al. (2016) Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling

promote functional maturation of induced pluripotent stem cell-derived neurons. Am J Physiol Cell

Physiol. 310(7):C520-41.

4) Kranenburg O, Poland M, van Horck FPG, Drechsel D, Hall A & Moolenaar, WH. (1999) Activation of

RhoA by Lysophosphatidic Acid and Ga12/13 Subunits in Neuronal Cells: Induction of Neurite

Retraction. Mol. Biol. Cell. 10(6):1851-1857.

The Neurodegeneration Consortium

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