OPTIMIZATION OF NEURONAL CULTURES DERIVED FROM HUMAN INDUCED PLURIPOTENT

STEM CELLS FOR HIGH THROUGHPUT ASSAYS OF SYNAPTIC FUNCTION

Pascal Laeng, Chris M. Hempel, James J. Mann, Jeffrey R. Cottrell and David J. Gerber, Galenea Corp, Cambridge MA 02139

MANTRAMultiwell Automated Neuronal Transmission Assay

SypHy Delivered by AAV Transduction

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

chosen as the 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) Characterization of iCell Neurons transduced by hSyn-sypHy-AAV.

Upper left: Expression of SypHy in iCell Neurons infected at 1DIV

and fixed at 2 weeks. MAP2 expressing cells represent

more than 95% of the cells in the culture and synapsin is

expressed in all neurons (presence of puncta). Arrows

show cells not stained for GFP.

Upper right: Co-expression of hSypHy and synapsin can be detected

(arrow heads, higher magnification).

Lower panel: Expression of SypHy in iCell Neurons 4 weeks post

transduction. Most neurons express hSypHy (70%) in cell

body and axons.

0 30 60 90 120 150 180

0.0

0.2

0.4

0.6

0.8

Time (sec)

Am

plitu

de

(

F/F

)

H+

H+

Synaptophysin

H+

H+

H+H+

1

2

3

4

pHluorin

a

SypHy

Synaptotagmin

Overlay

b

c d

(a) Sustained Ca2+ influx over the duration of the stimulus train indicates

reliable generation of action potentials by each stimulus pulse in iCell

Neurons.

(b) The EV50 of evoked Ca2+ transients in iCell neurons is similar to that

measured from rat forebrain neuronal cultures, indicating a similar

action potential threshold. Pretreatment with TTX fully blocked evoked

Ca2+ transients at all stimulus intensities (not shown).

Evoked Ca+2 Flux in Human Neurons

Ph. contrast Ph. Contrast/SypHy SypHy

Synapsin MAP2

b

• Fluo-4 imaging on MANTRA

• Response to 60 V, 10 Hz train

• 200 ms sampling interval

• Stimulus: 10 stimuli delivered

over 1 sec.

• Signal averaged over 5 wells

a

• 10 Hz for 1 second at indicated

voltage

• Each voltage averaged over 5

trials

b

Cell Culture. Post-mitotic human neurons derived from iPSCs (“iCell® Neurons”, Cellular Dynamics International,

USA) and primary neuronal cultures isolated from E18 rat embryos were seeded in 96-well plates (Greiner) coated

with poly-D-lysine with or without laminin. For some experiments, iCell Neurons or rat neurons were cultured with

rat or human astrocytes (Lonza) grown as a monolayer. iCell Neurons and rat neurons were seeded on the same

plates and tested in parallel. iCell Neurons grown in the absence of glia were maintained in serum free medium

provided by the manufacturer, while rat neurons were maintained in Neurobasal medium (Invitrogen) plus 2% B-27

Supplement (Invitrogen), 500 µM glutamine (Invitrogen), and 6.25 µM glutamate (Sigma). When neurons were co-

cultured with glia, medium consisted of Advanced DMEM/F12 plus 1% fetal calf serum. Cultures were analyzed

between 2 and 7 weeks in vitro on the MANTRA system or on a fluorescence microscope imaging system. For

both systems, fluorescence imaging was performed in parallel with 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 1 DIV or 7

DIV respectively, iCell and rat 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 platform 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 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).

Introduction

Materials & Methods

MANTRA System Instrumentation

Alterations in synaptic transmission are associated with a number of

psychiatric and neurological disorders, suggesting that approaches directly

targeting synaptic function represent an attractive strategy for CNS drug

discovery. We previously described the development of a high-throughput

screening technology, termed the MANTRA™ (Multiwell Automated

NeuroTRansmission Assay) system, for identifying modulators of synaptic

function (Hempel CM et al., 2011) in rodent primary neuronal cultures. 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 iPSC represent 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 human neurons derived from

induced pluripotent stem cells (iPSCs) can be utilized in the MANTRA

system for synaptic functional assays.

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.

hiPSC hNeurons

mNeurons KO/transgenic

iCell Neurons grown in presence of glia show larger presynaptic

responses at all frequencies.

Presence of Glia Increases Neurite Outgrowth

and Synapsin Expression in Human Neurons

Conclusions

Acknowledgements

We thank members of Cellular Dynamics International, Vanessa Ott,

George Kopitas, Susan DeLaura, Lucas Chase and Rachel Llanas for

operational and technical support with iCell Neurons, and members of

Galenea, Eva Xia and Marie Fitzpatrick for MANTRA system operation.

This work was funded in part by NIH grant 1RC4MH092889-01.

Transduced iCell neuronal cultures display measurable levels of

evoked presynaptic activity after 6 weeks in culture.

Enables application of human neurons to hit/lead (compound A)

validation in CNS drug discovery

Further optimization required for HTS in human neurons

Effect of Glia on MANTRA Activity

- G

lia

+ R

at

Gli

a

Synapsin GFP Map2 - Glia + Glia

a b

(a) Triple immunofluorescence of iCell neurons grown for 6 weeks in

absence or presence of rat glia. Similar results were obtained with

human glia (not shown).

(b) Synapsin immunofluorescence at higher magnification shows more

punctuate staining in axons and less staining in the cell body of

human neurons in presence of rat or human (not shown) glia.

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

unique capability to test multiple conditions in parallel to generate

human iPSC-derived neurons 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 to restore normal synaptic transmission.

Galenea is interested in developing these 2 approaches via

collaborations. If your group is interested please contact us at

plaeng@galenea.com.

Applications of MANTRA for New Functional

Phenotypic Assays in hiPSC-derived Neurons

iCell Neurons – No glia

iCell Neurons – with glia

(a) Phase contrast – 5 DIV post

seeding of neurons on rat glia.

(b) Analysis of expression of SypHy in

live cultures (not-fixed) at 6 weeks

post- transduction shows no

detectable signal in glia cultured

in the absence of seeded neurons

(negative control, low

magnification).

No

Neurons

+ Rat

Neurons

+ iCell

Neurons

+ Glia (Rat) - Glia b

SypHy Responses Measured on MANTRA: Similar

Frequency-dependence in Rat and Human Neurons

High Resolution Analysis of Presynaptic

Responses in Rat and Human Neurons

Co-cultures Neuron-Glia

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

magnitude but similar overall waveform shapes and frequency

dependence compared to rat neurons

(b) Compound A shows same effects on synaptic function in rat and human

iPSC-derived neurons measured on MANTRA

a

Localization and quantification of synaptic response in rat and iCell

neurons. Arrow heads and circles show active presynaptic sites in iCell and

rat neurons respectively. Preliminary data suggest that pre-synaptic

responses are similar in active synapses between rat and iCell neurons.

This suggests that the difference in MANTRA activity observed between rat

and human neurons might reflect a difference in the total number of mature

synapses/neurons present in the cultures.

Rat Glia – No Neurons Rat Glia + Rat Neurons Rat Glia + hiCell Neurons

a

b

b

Rat Neurons (3/5 weeks)

Human

Neurons (6 weeks)

(***p <0.001; **p < 0.01)

**

***

Rat Neurons Human Neurons

ROI Modulated

dF/F0 4V= 0.144 6V= 0.303 (Avg of 5 ROIs)

n =1

Human Neurons

Before Stimulation After Stimulation

Rat Neurons