DEVELOPMENT OF HIGH THROUGHPUT SYNAPTIC FUNCTIONAL ASSAYS IN NEURONS DERIVED FROM

HUMAN INDUCED PLURIPOTENT STEM CELLS AND CHARACTERIZATION OF PHARMACOLOGICAL RESPONSES

Pascal Laeng, Chris M. Hempel, James J. Mann, Jesse L. Mohn, Jeffrey R. Cottrell and David J. Gerber.

Galenea Corp, Wakefield MA 01880

MANTRAMultiwell Automated Neuronal Transmission Assay

Cell Culture. Neurons derived from human iPSCs (prepared from HIP™ iPS Cell-Derived Neural Stem Cells) were

obtained from GlobalStem (Rockville, MD). Frozen batches of differentiated neurons from HIP™ iPS Cell-Derived

Neural Stem Cells (HIP Neurons) were thawed, seeded in 96 well plates and maintained in serum free medium

provided by GlobalStem for up to 9 weeks. 96-well plates (Greiner) coated with poly-D-lysine and laminin were used

for these cultures and for primary neuronal cultures isolated from E18 rat embryos. HIP Neurons and rat neurons

were seeded in the same plates and tested in parallel. Rat neurons were maintained in Neurobasal medium

(Invitrogen) plus 2% B-27 Supplement (Invitrogen), 500 µM glutamine (Invitrogen), and 6.25 µM glutamate (Sigma).

Cultures were analyzed between 2 and 7 weeks in vitro on the MANTRA system or on a high resolution,

fluorescence microscope-based imaging and stimulation system. For both systems, fluorescence imaging was

performed in parallel with application of field stimulation trains. Immunofluorescence analysis was performed at

different time points to evaluate the expression and localization of presynaptic proteins and the sypHy reporter.

Reporter Viral Transduction. For analysis of presynaptic function, cultures were infected with an adeno-associated

virus (AAV) used to deliver a synaptophysin-pHluorin fusion fluorescent reporter construct (sypHy). The

synaptophysin-pHluorin reporter and the human synapsin promoter sequences were as previously described

(Hempel CM et al., 2011). The expression construct was generated by custom cDNA synthesis (Blue Heron Bio). A

recombinant adeno-associated virus of mixed serotype 1/2 (AAV1/2) was generated (GeneDetect). At 4 DIV or 7 DIV

respectively, rat and HIP neurons were infected with the hSyn-SypHy-AAV.

Evoked Ca+2 Transients. For analysis of ability of neurons to initiate action potentials following field stimulation,

neurons were incubated in assay buffer containing Fluo-4 for 1 hour and assayed on the MANTRA system as

described below.

MANTRA Assays. Plates containing neuronal cultures were placed on an Evolution P3 liquid handling robot (EP3;

Perkin Elmer) with which culture medium was replaced with assay buffer containing (in mM): NaCl 119, KCl 2.5,

dextrose 30, HEPES 25, MgCl2 2, CaCl2 2, D-AP5 0.05, and DNQX 0.02. Test compounds were added as part of

this wash step. Plates were transferred to a 30ºC incubator for one hour, transferred to the plate tray in the MANTRA

instrument, and subjected to a read/field stimulation protocol. Fluorescence readings were made using a 475/535

excitation/emission filter. Unless specified otherwise, field stimuli were 30V, 0.2 msec. The temperature of the

cabinet was set at 32ºC. Wells were imaged at 1 Hz with 300 msec exposures. Data files were processed using in-

house analysis routines (Igor Pro) and stored in a custom mySQL database.

High-resolution sypHy assays. To elicit action potentials 1 ms voltage pulses (4 or 6V) were passed using CX3

electrodes (Cellectricon) positioned manually inside individual wells of a 96-well plate. Stimulus patterns were

delivered by a stimulus isolation unit (Coulbourn Instruments) controlled by Igor Pro software (Wavemetrics) and a

DAQ system (National Instruments). Cultures were illuminated by a 475 nm LED (Cairn), filtered with a 470/525

emission/excitation filter cube (Zeiss), and imaged with a 1.3 NA 40x oil-immersion objective lens and an iXON

EMCCD camera (Andor) with 100 msec exposures at a frequency of 1 Hz. Fluorescence intensities were extracted

using ImageJ and analyzed with custom routines (Igor Pro).

Materials & Methods

MANTRA System Instrumentation

The MANTRA instrumentation (left) consists of integrated 96-well parallel

imaging and field stimulation systems. Right, top shows the instrument deck

with its multiple technology components. Right, bottom shows the design of the

electrode tip module.

Introduction

Alterations in synaptic transmission are associated with a number of psychiatric

and neurological disorders, suggesting that an approach of directly targeting

synaptic function is an attractive strategy for CNS drug discovery. To this end,

Galenea developed a high-throughput screening technology, termed the

MANTRA™ (Multiwell Automated NeuroTRansmission Assay) system, for

identifying modulators of synaptic function in rodent primary neuronal cultures

(Hempel CM et al., 2011). We are employing the MANTRA system in an integrated

drug discovery platform that targets synaptic transmission at multiple levels.

The MANTRA system can be applied first to define synaptic functional alterations

in CNS disease model systems and then to perform screening campaigns to

identify compounds that restore normal synaptic function. In addition to neuronal

cultures from genetic mouse models, neurons derived from human induced

pluripotent stem cells (iPSCs) are a valuable cellular model system for measuring

neurotransmission abnormalities in a human disease-relevant context.

Use of human neurons for neurotransmission screening applications requires that

cultures achieve a sufficient degree of synaptic maturation to yield a measureable

proportion of synapses with pre- and post-synaptic functionality. Here, we show

that cultures of neurons derived from human iPSCs can be utilized in the MANTRA

system for high-throughput assays to measure evoked Ca+2 transients or evoked

pre-synaptic responses. Our results support feasibility of high-throughput

functional screening in human neurons.

hiPSC hNeurons

mNeuronsKO/transgenic

Acknowledgements

We thank members of GlobalStem (Rockville, MD), R. Josephson, R. Harvey,

and J.M. Auerbach for operational and technical support with HIP Neurons, and

members of Galenea, Marie Fitzpatrick and Rasheedah Malik for MANTRA

system operation and Qing Fei for cell culture assistance. This work was funded

in part by NIH grant 1RC4MH092889-01.

1. The high-throughput capacity of the MANTRA system provides a unique

capability to test multiple conditions in parallel to generate human iPSC-

derived neuronal cultures with optimal synaptic functionality.

2. Ultimately, the MANTRA system can be used to characterize synaptic

abnormalities in neurons derived from patients and to screen for

compounds that restore normal synaptic transmission.

Applications of MANTRA for New Functional

Phenotypic Assays in hiPSC-derived Neurons

The robust dynamic range observed with the evoked Ca+2 response on the

MANTRA system provides a quick and reliable assay to measure neuronal

excitability in cultures derived from hiPSC.

Transduced HIP neuronal cultures display measurable levels of evoked

presynaptic activity after 5-7 weeks in culture.

Enables application of human neurons in CNS drug discovery

Functional high content analyses performed with our high resolution imaging

system can be used as a secondary validation tool to confirm and extend

primary results derived from MANTRA screening.

Conclusions

High Resolution Analysis of Active

Presynaptic Sites in Human Neurons

Application to in vitro Pharmacology. Effects of

Anti-Epileptic Compounds in Human Neurons

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Ca++ Flux (Fluo-4) SV Release (SypHy)

Examples of pharmacological characterization in human and rat neurons

using the MANTRA system. Concentration-response curves for Ca+2 flux and

SypHy assays show acceptable dynamic range and reproducibility. Co102862

(state dependant Na+ channel inhibitor) and Lamotrigine show similar effects in

rat and human neurons in both assays.

Localization and quantification of functional presynaptic responses in

HIP neurons.

(a) Identification of active presynaptic sites was performed by subtracting

baseline SypHy signals from those evoked after stimulation.

(b) Specific evoked SypHy signals from active presynaptic sites were

quantified and averaged from 16 ROIs. Arrowheads show active

presynaptic sites.

Preliminary data suggest that presynaptic responses in active synapses

are similar between rat (not shown) and HIP neurons (dF/F0 = 0.27).

Before After

a

Before After AfterBefore

b

Time (s)

SypHy Delivered by AAV Transduction

b

(a) The pH-sensitive GFP, pHluorin, tagged to synaptophysin (sypHy) was applied

as a presynaptic functional reporter for the MANTRA system. An adeno-

associated virus (AAV) of mixed 1/2 serotype was used to deliver sypHy to

neuronal cultures. SypHy expression was driven by the human synapsin

promoter (hSyn-sypHy-AAV).

(b) Immunocharacterization of HIP Neurons transduced by hSyn-sypHy-AAV.

Upper: Expression of SypHy in HIP Neurons infected at 7 DIV

and fixed at 7 weeks. MAP2 (blue) expressing cells

represent more than 90% of the cells in the culture.

GFP (green) signal reveals robust expression of hSypHy

reporter (low magnification).

Lower left: Higher magnification pictures show co-expression in

the same field of view of the presynaptic protein,

synapsin (SYN, red), in MAP2 (blue) expressing

neurons.

Lower right: Higher magnification pictures show co-expression of

SypHy reporter (GFP, green) and synapsin (SYN, red)

in the majority of labeled presynaptic sites.

1

1 2 3

2

3

SynaptopHluorin Waveform

a

MAP2GFP

GFP SYNSYN/MAP2SYN SYN/GFP

Evoked Ca+2 Flux: Measures of Excitability

of Human and Rat Neurons in MANTRA

Estimation of excitability of human neurons in the MANTRA System:

reproducibility and comparison with rat neurons.

Upper: Evoked Ca+2 transient waveforms were elicited by increasing stimulus

pulse intensities over time (average of 4-6 wells). Profiles from 3

independent experiments are compared.

Lower: Comparison of concentration-response curves (amplitudes/volts) for

three independent experiments. Calculated EV50s were within close

range in the three experiments. HIP Neurons grown for 7 weeks had

similar EV50s to those measured from rat forebrain neuronal cultures (3

weeks), indicating a similar action potential threshold.

HIP Neurons

EV50

(volts)

dF/F0

(Max)

Exp.1 17.1 1.0

Exp.2 14.7 0.9

Exp.3 17.1 1.20.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

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(a) Human neurons show measurable pre-synaptic activity with lower magnitude

but similar overall waveform shapes and frequency dependence compared to

rat neurons.

(b) PMA and an adenosine agonist show same modulatory effects on presynaptic

activity in human iPSC-derived and rat neurons (Hempel et al., 2011).

a

b

Evoked SypHy Responses in Human and

Rat Neurons on MANTRA

High Resolution Analysis of Ca+2

Responses in Human Neurons

Frequency Dependent Evoked Ca+2 Flux and Ca+2

Channel Pharmacology on MANTRA

b

a

(a)Example of stimulation protocol: single action potential (SAP) stimulus, 1 Hz,

10 Hz and 30 Hz stimulus trains.

(b) Ca+2 and Na+ channels pharmacology: evoked Ca+2 influx is blocked by TTX

and partially blocked with a mixture (Cono-Aga-SNX):

- Cav2.1 inhibitor (P-type): w-agatoxin IVA

- Cav2.2 inhibitor (N-type): w- conotoxin GVIA

- Cav2.3 inhibitor (R-type): SNX-482

Rat Neurons

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Comparison between Rat and Human Neurons

Human Neurons Human Neurons

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Validation with Modulators of Presynaptic Activity

(a) Examples of Ca+2 images recorded before and after electrical stimulation

show robust Ca+2 responses in the cell bodies and across the entire network

of neuronal processes.

(b) Quantification of Ca+2 responses at different stimulation frequencies

(c) Waveforms recorded with our high resolution microscope imaging analysis

system show similar profiles to those recorded on the MANTRA system.

a

c

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Time (s) Time (s) Time (s)

415.27/D17