Functional Neuromuscular Junctions Formed by
Embryonic Stem Cell-Derived Motor Neurons
Joy A. Umbach1, Katrina L. Adams2,3, Cameron B. Gundersen1, Bennett G. Novitch2,3*
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of
America, 2Department of Neurobiology, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine, University of
California Los Angeles, Los Angeles, California, United States of America, 3Molecular Biology Interdisciplinary Graduate Program, University of California Los Angeles, Los
Angeles, California, United States of America
A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in
vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development
of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have
characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic
activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-
culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and
muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while
dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those
observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally
relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders
of synaptic dysfunction associated with MN disease.
Citation: Umbach JA, Adams KL, Gundersen CB, Novitch BG (2012) Functional Neuromuscular Junctions Formed by Embryonic Stem Cell-Derived Motor
Neurons. PLoS ONE 7(5): e36049. doi:10.1371/journal.pone.0036049
Editor: Marcel Daadi, Stanford University School of Medicine, United States of America
Received January 24, 2012; Accepted March 26, 2012; Published May , 2012
Copyright: ? 2012 Umbach et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Broad Center for Regenerative Medicine and Stem Cell Research at the University of California Los Angeles, and grants
to BGN from the California Institute for Regenerative Medicine (RB1-01367) and the Muscular Dystrophy Association (92901). KLA was also supported by the
University of California Los Angeles Cellular and Molecular Biology Training Program, Ruth L. Kirschstein National Research Service Award GM007185. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Allmotorfunctionsfromlocomotion torespiration dependonthe
communication between motor neurons (MNs) in the spinal cord
and muscle cells in different regions of the body. This vital activity is
susceptible to many neurodegenerative diseases, most notably
amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy
(SMA), resulting in MN dysfunction and ultimately death [1,2].
While progress has been made in identifying genes associated with
MN degeneration [3–5], the molecular and cellular processes
underlying disease onset and progression remain unclear.
Over the past decade, considerable attention has been focused
on using stem cell-derived MNs to model disease pathogenesis,
driven by demonstrations that mouse and human embryonic stem
cells (mESCs and hESCs) can be directed to form MNs in response
to developmental signals that promote MN formation in vivo [6–
9]. Recent studies have further shown that MNs can be similarly
produced from induced pluripotent stem cells (IPSC) including
those derived from ALS and SMA patients [10–12], and through
transcription factor-mediated reprogramming of fibroblasts .
A remaining challenge, however, is to establish methods to
evaluate the function of normal and diseased MNs obtained from
these sources in a physiologically relevant setting.
An important step towards this goal is the development of in
vitro assays to measure the synaptic activity of MNs at
neuromuscular junctions, as many studies have pointed to synaptic
dysfunction as an early readout and possibly an initiating event in
MN disease progression [14,15]. ESC and IPSC-derived MNs
have previously been shown to exhibit many molecular and
physiological properties associated with mature MNs [12,16,17].
Moreover, when transplanted into the embryonic chick spinal cord
[9,18,19] or peripheral nerve of mice , these neurons appear
to be capable of extending axons towards peripheral muscle
targets. Despite these successes, relatively little attention has been
placed on direct measurements of the communication between
stem cell-derived MNs and muscle cells. In part, this reflects the
inherent difficulties in isolating connected pairs of cells in mass
culture or transplantation settings.
In this study, we report the development of low-density culture
conditions that encourage the formation of neuromuscular
junctions between isolated ESC-derived MNs and muscle cells.
This system enables the direct measurement of synaptic commu-
nication through dual patch clamp recordings. In this setting, MNs
form neuromuscular junctions containing functionally importan
synaptic proteins, and these synapses exhibit both spontaneous
and stimulus-evoked transmitter release. Together, these findings
constitute an important advance in validating the functional
identity of stem cell-derived MNs and providing a platform for
defining their synaptic properties under normal and diseased
PLoS ONE | www.plosone.org1 May 2012 | Volume 7 | Issue 5 | e36049
ESC-derived MNs form cholinergic synapses on muscle
cells under low-density co-culture conditions
To evaluate the synaptic activity of ESC-derived MNs, we first
developed culture conditions that were amenable to patch clamp
analysis of MN-muscle pairs. The initial step was to test whether
cells could form synaptic contacts when plated at low density
dish). We reasoned that such conditions might encourage the
non-synaptic contacts made when cells are plated at high densities.
Under these conditions, each culture dish yielded 1–4 isolated MN-
muscle cell pairs with Hb9::EGFP+axons projecting towards
spindle-shaped muscle cells (Fig. 1A, B). At the point of contact
between the axons and muscle cells there was a varicose
enlargement of the terminal bouton (Figs. 1 and 2). Bouton
diameter ranged from 3–11 mm in diameter with a mean diameter
of 6.962.0 mm (n=65) and was easily distinguished from motor
neuron soma, which were typically .20 mm in diameter. This
geometry of neuron-muscle pairing was sufficiently common that it
enabled the reliable identification of nerve and muscle cells that
were likely to have made functional synaptic contacts. The presence
of a-bungarotoxin (BTX) staining (Fig. 1C–E) further indicated that
nicotinic ACh receptors preferentially accumulated at these sites.
We next used immunofluorescence microscopy to investigate
whether other macromolecules characteristic of cholinergic
synapses were present at the nerve-muscle contacts. Proteins
associated with ACh metabolism including Slc18a3 (VAChT) and
the high affinity choline transporter Slc5a7 were detected at these
sites along with the SNARE proteins Snap25 and Syntaxin 1a
(Fig. 2A–L). Concomitantly, synaptic vesicle proteins such as
synaptophysin and SV2 were also present (Fig. 2M–R). These
results were representative of data obtained from at least two
separate culture dishes for each antibody. In each dish, at least 4
neuromuscular junctions were imaged and every Hb9::EGFP+
terminal showed immunoreactivity for these presynaptic proteins.
Collectively, these data indicate that sites of contact between ESC-
derived MNs and muscle cells contain components of the
molecular machinery associated with cholinergic synapses.
Neuromuscular synapses formed in vitro are functional
and trigger both spontaneous and evoked muscle
To determine whether the nerve-muscle contacts formed in
culture exhibit the functional properties of neuromuscular
junctions, we sealed patch clamp pipettes onto MN-muscle pairs.
Current injection into the ESC-derived MNs initially elicited
passive membrane responses in the MNs, but no electrical
response in muscle cells beyond the stimulus artifact (Fig. 3A).
However, once threshold was exceeded, the MNs fired an action
potential. With a brief delay (1–3 msec), MN action potentials
were followed by an excitatory post-synaptic current (EPC) in the
muscle cells (Fig. 3A). These EPCs are reminiscent of the classical
electrophysiological signature of synaptic communication between
MNs and muscle cells observed in en-bloc preparations .
From 60 dishes examined, 111 neuron-muscle pairs with
geometries similar to that shown in Fig. 1A were identified.
Successful patches (where the resting potential in both cells was
Figure 1. Morphology of neuromuscular junctions formed in
vitro by mESC-derived MNs. (A, B) Brightfield and fluorescence
images showing an Hb9::EGFP+ESC-derived MN extending an axon to
contact a muscle cell under low-density cell culture conditions. (C–E)
The adjacent trio of images shows a representative axonal varicosity
contacting a muscle cell stained for nicotinic ACh receptors using
fluorescent a-bungarotoxin (BTX) overlaid with Hb9::EGFP fluorescence
in the MN terminal bouton. Scale bars are 20 mm.
Figure 2. mESC-derived MNs form cholinergic synapses with
muscle cells in vitro. Immunofluorescence analysis of proteins
expressed at nerve terminals of mESC-derived MNs. The three columns
show: (A, D, G, J, M, P) brightfield images of axon terminals contacting
muscle cells; (B, E, H, K, N, Q) green fluorescence associated with the
Hb9::EGFP MN reporter; (C, F, I, L, O, R) red fluorescence corresponding
to antibody staining for the indicated presynaptic proteins: Slc18a3
(VAChT), Slc5a7 (ChT1), Syntaxin 1a (Stx1a), Snap25, Sv2, and
Synaptophysin (Syp). Scale bar is 20 mm.
Functionality of Stem Cell-Derived Motor Neurons
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.230 mV) were obtained for ,37% of these pairs (41/111), of
which ,90% (37/41) showed functional synaptic responses as
illustrated in Fig. 3A. Overall, muscle resting membrane potentials
averaged 253.669.5 mV; S.D., while neuron resting potentials
were 40.9 mV69.4 mV; S.D. The records in Fig. 3A also
illustrate the variability in the synaptic delay, amplitude and time
course of EPCs. Scatter plots summarize the observed range of
synaptic delays, EPC amplitudes, EPC rise times, and EPC decays
(Fig. 4A–D). In particular, EPC amplitude varied from ,100 pA
at some synapses on day 3 to .1 nA on 4 day (Fig. 4A). EPCs
were abolished by the addition of the nicotinic ACh receptor
antagonist, d-tubocurarine (10 mM, n=3 trials; Fig. 3B), confirm-
ing the cholinergic nature of these EPCs. EPCs were similarly
eliminated by replacement of extracellular Ca2+with 10 mM
Mg2+(n=3 trials; data not shown). Moreover, in parallel
experiments using a patch pipette on the neuron alone, triggering
of neuronal action potentials led to visible muscle contractions in
,20% of the trials evaluated (3/15; data not shown). Collectively,
these data indicate that the neuromuscular junctions formed in
these cultures exhibit stimulus-evoked and Ca2+-dependent
neurotransmitter release capable of triggering muscle contraction.
An important advantage of the dual patch configuration is that
it enables one to conduct additional quantitative analyses of both
spontaneous and evoked synaptic events at these neuromuscular
junctions. We thus evaluated the profile of spontaneous miniature
(m) EPCs recorded in muscle cells for which evoked EPCs were
also obtained (Fig. 5A–D). The mEPC amplitude distribution
(Fig. 5B) is typical of the skewed Gaussian distribution observed at
70% of the nerve-muscle contacts in these cultures. Cumulatively,
mEPC frequencies ranged from 0.04–0.30 Hz, with mEPC
frequencies ,0.1 Hz characteristic of 3 d old cultures and
.0.1 Hz after 4 days in culture (Fig. 5C). Although we have not
yet undertaken a systematic evaluation of the quantal content of
the EPCs in this system, it is important to note that EPCs such as
that shown in Fig. 5A are very likely comprised of multiple quanta.
This conclusion derives from the fact that the amplitude of this
EPC is at least five times greater than the largest mEPC (Fig. 5A,
B, D). Based on this criterion, multi-quantal EPCs were observed
in ,95% (36/37) of the neuromuscular junctions from which
mEPC/EPC recordings were obtained. Taken together, these data
indicate that mESC-derived MNs are capable of coupling action
potentials to the synchronous release of multiple quanta at these
nerve-muscle contacts to elicit muscle contractions.
The definitive feature of MNs is their ability to form functional
neuromuscular junctions and thereby drive the contraction of
skeletal muscle cells. Our study provides critical evidence that
ESC-derived MNs can exhibit robust synaptic communication
with muscle cells under simplified in vitro culture conditions.
Empirically, this is a significant observation, as the use of stem cell-
derived MNs for regenerative purposes or disease modeling
requires that the cells faithfully mimic their natural counterparts in
Figure 3. mESC-derived MNs trigger a post-synaptic response in muscle cells only when MNs fire action potentials. (A) In each pair of
recordings, the lower trace shows the MN response to depolarizing currents of increasing amplitude. For both MN1 and MN2, current was increased
in 0.5 nA steps from 2.5 nA on the left to 4.5 nA on the right. Stimuli were delivered at 30 s intervals. MN responses were initially passive, but upon
reaching threshold, MNs typically fired a single action potential with this stimulus duration. Each MN action potential elicited an EPC of variable
amplitude after a delay of 1–3 msec. (B) Addition of d-tubocurarine (10 mM) eliminated the stimulus-evoked EPC, but did not affect the MN action
Functionality of Stem Cell-Derived Motor Neurons
PLoS ONE | www.plosone.org3May 2012 | Volume 7 | Issue 5 | e36049
both molecular and functional properties. Our data show that
ESC-derived MNs express several proteins, including nicotinic
ACh receptors, Slc18a3 (VAChT), the high affinity choline
transporter Slc5a7, and SNARE proteins found at native
neuromuscular junctions, and exhibit both spontaneous and
action potential-dependent, multi-quantal secretion of ACh to
trigger post-synaptic potentials and muscle contraction. These
results further provide an important extension to previous studies
that have used bath application of glutamate to evoke post-
synaptic potentials and muscle contraction in high-density MN-
muscle cell cultures [13,16,22]. Moreover, the ability to quantify
the functional properties of individual nerve-muscle contacts offers
the opportunity to rigorously assess the impact of a variety of
experimental manipulations on these synaptic events.
To define the synaptic activity of MN-muscle pairs, our
investigation was intentionally restricted to the differentiated
progeny of mESCs and C2C12 muscle cells. Our data indicate
that MN-muscle synapses formed under these simplified condi-
tions recapitulate many features of neuromuscular communication
seen in vivo. For example, the frequency of spontaneous mEPCs
(0.04–0.3 Hz) is within the range reported for muscle fibers of late
embryonic and early post-natal rodents [23–26]. Similarly, the rise
and decay times of EPCs are within the range observed for
developing rat neuromuscular junctions . It is important to
note that functional MN-muscle synapses formed under these
conditions typically survived for a maximum of 8–9 days in
culture. This limitation might reflect a lack of support provided in
vivo by other cells including astrocytes and Schwann cells or
presynaptic inputs to the MNs from spinal interneurons. The low-
density culture conditions established in this study provide a
suitable platform for evaluating the influence of different cell types
in future work. Nevertheless, we have found that similar co-culture
of human ESC and IPSC-derived motor neurons with muscle cells
results in synaptic contacts that persist for several weeks (JAU,
KLA, and BGN, unpublished data), suggesting that at least some
aspects of synaptic stability are inherent to the MNs themselves
and highly variable between species.
During embryonic development, different classes of MNs
exhibit a high degree of selectivity in their choice of muscle
targets [27,28]. However, we infer from the present results that the
programs that dictate motor innervation patterns are sufficiently
malleable such that ESC-derived MNs can form functional
synapses on C2C12 cells. Although this observation is not
surprising given the promiscuity of mammalian MNs for forming
Figure 5. mESC-derived MNs exhibit both spontaneous and
evoked synaptic currents at neuromuscular junctions formed
in vitro. Patch pipettes were sealed onto MNs and muscle cells making
contact as shown in Fig. 1. (A, B) Representative records of spontaneous
mEPCs in a muscle cell and their amplitude distribution. (C) Scatter plot
of mEPC frequency over different times in culture. (D) Current injection
into the MN triggers an action potential that elicits a multi-quantal EPC
in the muscle cell.
Figure 4. Quantification of the properties of mESC-derived
MN-muscle synapses. (A–D) Scatter plots show the variability of the
following parameters as a function of days in co-culture: (A) EPC
amplitudes, where each point is the largest EPC recorded at each
synapse. Inset shows representative EPCs that correspond to the fuchsia
diamonds (time scale is 25 msec); (B) synaptic delay, measured from the
peak of the MN action potential to the start of the EPC; (C) EPC rise time;
(D) EPC decay time.
Functionality of Stem Cell-Derived Motor Neurons
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neuromuscular junctions in vitro , precise matching of MN
and muscle subtypes might nevertheless be crucial for ensuring full
synaptic activity and stability . Progress has recently been
made in understanding the mechanisms underlying MN fate
selection [27,31,32], and it should be fruitful to determine whether
this information can be harnessed to bias the differentiation of
mESC-derived MNs to favor the innervation of specific classes of
muscle cells both in vitro and in vivo.
Another important use for this co-culture system will be for
modeling neuromuscular disorders. There is abundant evidence
for an early and profound impairment of neuromuscular
transmission in amyotrophic lateral sclerosis , and we showed
previously that mutant forms of superoxide dismutase 1 (SOD1)
alter the morphology and survival of hESC-derived MNs in vitro
. Consequently, conditional expression of mutant SOD1 in
MN-muscle co-cultures is likely to provide an informative system
for clarifying the impact of SOD1 mutant alleles on nerve-muscle
communication. Similarly, recent data suggest that proprioceptive
circuits may be particularly vulnerable in spinal muscular atrophy
. The in vitro system developed here might accordingly be
expanded to assess the underlying cellular and molecular
mechanisms that contribute to this decline in synaptic input to
MNs. Thus, in addition to their utility for helping to answer
fundamental biological questions, these co-cultures have clear
applications in addressing problems of medical significance.
Materials and Methods
Differentiation of mESCs
Hb9::EGFP mESCs  were maintained and differentiated into
MNs as previously described [9,36]. Briefly, mESCs were plated
on 60 mm bacterial petri dishes in core MN medium to elicit
embryoid body (EB) formation. Core MN medium consisted of a
1:1 mixture of Dulbecco’s Modified Eagle’s Medium/F12
(DMEM/F12) and Neurobasal Medium supplemented with
Knockout Serum Replacement, Glutamax, and 2-mercaptoetha-
nol (560 nM), Penicillin/Streptomycin, and Primocin (50 mg/ml;
Invivogen). Except as noted, media components were obtained
from Invitrogen. After 1 d in culture, EBs were pipetted through a
100 mm strainer to remove large aggregates. The next day, EB
culture media was replaced with MN differentiation medium [core
MN medium containing N2 supplement (16), Retinoic Acid
(1 mM; Sigma) and Purmorphamine (1.5 mM; EMD Biosciences)].
After 5 d of differentiation, EBs were dissociated using papain
(0.5 U/ml; Worthington) in HBSS for 20 min at 37uC with gentle
trituration. Cells were collected by centrifugation and washed with
MN differentiation medium prior to plating with muscle cells.
Co-culture of MNs and C2C12 muscle cells
C2C12 cells (CRL-1772) were obtained from the American
Type Culture Collection and cultured in myoblast growth medium
[DMEM supplemented with 15% fetal bovine serum (FBS), L-
glutamine (1 mM) and antibiotics as above]. When the cells
reached 60–70% confluence, they were washed with PBS and
transferred to muscle differentiation medium [DMEM with 0.5%
FBS, insulin (10 mg/ml)-transferrin (5.5 mg/ml)-selenium (39 nM),
L-glutamine (1 mM) and antibiotics]. After 2 d, the medium was
supplemented with cytosine arabinoside (Ara-C; 10 mM), and cells
were cultured for another 2 d to eliminate dividing cells.
Differentiated myotube cultures were dissociated using trypsin
(0.05%) and plated at low density on Matrigel-coated 35 mm
culture dishes (1.26104cells/dish) in differentiation medium
containing 1 mM Ara-C. 1–2 d after muscle cells were plated,
1.26105mESC-derived cells, of which at least 10% were
Hb9::GFP+MNs, were added to each dish and the medium was
changed to core MN medium supplemented with Brain-Derived
Neurotrophic factor (10 ng/ml), Glia-Derived Neurotrophic
Factor (10 ng/ml) and Ciliary Neurotrophic Factor (20 ng/ml);
neurotrophic factors were obtained from Prospec. Within 1–2 d,
motor axons made contact with muscle cells, and functional nerve-
muscle contacts were observed for at least 6–7 d.
Fluorescence microscopy and immunostaining
Cultures were fixed in 3% paraformaldehyde in PBS for
15 min, washed twice with PBS, permeabilized with 0.1% Triton
X-100 in PBS for 15 min, and blocked in 10% normal goat serum
in PBS for 15 min. Primary antibodies (0.5–2 mg/ml) were added
and specimens were incubated for 12–16 h at 4uC. After extensive
PBS washes specimens were incubated with secondary antibodies
(Alexa 594 goat anti-mouse or goat anti-rabbit IgG; Invitrogen) for
1 h followed by PBS washes. The following primary antibodies
were used: Slc18a3 (VAChT, Millipore; AB1588); Slc5a7 (choline
transporter 1 (ChT1), Millipore; AB5966)); Sv2 (Developmental
Studies Hybridoma Bank); Snap25 (Stressgen; VAP-SV0002);
synaptophysin (Syp, Sigma; S-5768); syntaxin 1a (Stx1a, Stressgen;
VAM-SV013). Nicotinic acetylcholine (ACh) receptors were
detected through bath application of Alexa 594-a-bungarotoxin
(Invitrogen) to the cultures prior to fixation. Epifluorescence
images were obtained using an Olympus IX70 inverted micro-
scope equipped with a Sensicam cooled CCD camera (PCO) and a
Lambda 10 shutter (Sutter Instruments) controlled by Axon
Instruments Imaging Workbench. Images were processed using
Adobe Photoshop and CorelDRAW software.
Cultures were screened for isolated MN-muscle cell pairs where
the axon branched minimally and the axon terminal formed a
visible contact with a muscle cell that was ,0.1 mm from the cell
body. Patch pipettes were sealed sequentially onto both cells using
methods described in . Pipette solutions were in mM, muscle:
K-gluconate (140), Hepes (10), CaCl2(1), MgCl2(1), EGTA (11),
QX-314 (5); neuron: K-gluconate (140), Hepes (10), EGTA (1),
Mg-ATP (4), Na-GTP (0.3). The bath solution for recording was in
mM: NaCl (120), KCl (1.9), KH2PO4(1.2), Na-bicarbonate (20),
CaCl2(2.2), MgCl2(1.4), Hepes (7.5). In all cases, pH was adjusted
to 7.2. Cells with resting potentials ,230 mV were discarded.
After an initial period to record at least 25 spontaneous miniature
excitatory post synaptic currents (mEPCs) in the muscle cell
(voltage clamped at 280 mV), MNs (maintained in current clamp
mode at approximately 270 mV) were stimulated by 0.5 msec
current injections of increasing amplitude from +0.5 to +6 nA.
Data were collected using Axopatch 2B patch clamp amplifiers
with 4-pole Bessel filtering at 5 kHz. Signals were digitized and
stored using pClamp and Axotape software (Axon Instruments)
and analyzed using pClamp and miniAnalysis (Synaptosoft).
We thank Hynek Wichterle for the generous gift of Hb9::EGFP mESCs;
Amy Hurwitz and Destaye Moore for technical assistance, Lou Ignarro for
facilities, and Samantha Butler, Harley Kornblum, and Felix Schweizer for
helpful discussions and comments on the manuscript.
Conceived and designed the experiments: JAU KLA CBG BGN.
Performed the experiments: JAU KLA. Analyzed the data: JAU CBG
BGN. Wrote the paper: JAU CBG BGN.
Functionality of Stem Cell-Derived Motor Neurons
PLoS ONE | www.plosone.org5 May 2012 | Volume 7 | Issue 5 | e36049
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PLoS ONE | www.plosone.org6 May 2012 | Volume 7 | Issue 5 | e36049