Pascal Laeng, Chris M. Hempel, James J. Mann, Jeffrey R. Cottrell

MANTRA
Multiwell Automated Neuronal Transmission Assay
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
Introduction
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.
SypHy delivered by AAV transduction
a
SypHy responses measured on MANTRA: similar
frequency-dependence in rat and human neurons
a
Synaptophysin
H+
H+
H+
Effect of glia on MANTRA activity
5Hz
iCell Neurons – No glia
iCell Neurons – with glia
30Hz
H+
H+
H+
pHluorin
Rat Neurons
b
(3 weeks)
b
5Hz
Synapsin
30Hz
MAP2
Human
Neurons
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.
KO/transgenic
(6 weeks)
iCell Neurons grown in presence of glia show larger presynaptic responses at all frequencies.
(a) Evoked presynaptic waveforms at 5 and 30Hz from rat neuronal primary cultures
measured on MANTRA.
mNeurons
(b ) Human neurons show measurable presynaptic activity with lower magnitude but similar
overall waveform shapes compared to rat neurons.
hNeurons
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.
High resolution analysis of presynaptic
responses in rat and human neurons
Materials & Methods
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.
Ph. contrast
SypHy
Rat Neurons
Ph. Contrast/SypHy
(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).
Before Stimulation
After Stimulation
ROI Modulated
4000
(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.
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).
Evoked Ca+2 flux in human neurons
dF/F0 4V= 0.144
6V= 0.303
(Avg of 5 ROIs)
b
- Glia
Human Neurons
a
+ Rat Glia
hiPSC
Presence of glia increases neurite outgrowth and
synapsin expression in human neurons
Synapsin
GFP
Map2
- Glia
+ Glia
3800
dF/F0 4V = 0.189
(Avg of 5 ROIs)
3600
3400
n =1
0
20
40
60
80
100
120
140
Localization and quantification of synaptic response in rat and iCell neurons. Arrow heads and
circles show active presynaptic sites in rat and iCell 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.
(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.
Conclusions
Transduced cultures display show significant levels of evoked presynaptic activity after
5 weeks in culture (with signal ~1/6th of rat neuronal culture at 6 weeks).
Follow-up fluorescence microscopy analysis confirmed the synaptic localization of
sypHy signals in human neurons.
Preliminary results suggest that evoked presynaptic activity is larger in the presence of
glia.
a
•
•
•
•
•
MANTRA system instrumentation
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
Co-cultures neuron-glia
a
Future Directions
b
• 10 Hz for 1 second at indicated voltage
• Each voltage averaged over 5 trials
Rat Glia – No Neurons
b
- Glia
Rat Glia + Rat Neurons
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.
(a) Sustained
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.
+ iCell
Neurons
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.
+ Glia (Rat)
(a) Phase contrast – 5 DIV post seeding of neurons
on rat glia.
Ca2+
The high-throughput capacity of the MANTRA system provides a unique capability to test
multiple conditions in parallel to generate human iPSCs-derived neurons with optimal
synaptic functionality.
Rat Glia + hiCell Neurons
No
Neurons
+ Rat
Neurons
(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).
Acknowledgements
We thank members of Cellular Dynamics International, 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.