Am J Physiol Cell Physiol 310: C520 –C541, 2016.

First published December 30, 2015; doi:10.1152/ajpcell.00166.2015.

CALL FOR PAPERS Stem Cell Biology

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3␤
signaling promote functional maturation of induced pluripotent stem
cell-derived neurons
Vsevolod Telezhkin,1 Christian Schnell,1 Polina Yarova,1 Sun Yung,1 Emma Cope,1 Alis Hughes,1
Belinda A. Thompson,1 Philip Sanders,2,3 Charlene Geater,1 Jane M. Hancock,4 Shona Joy,1
Luned Badder,1 Natalie Connor-Robson,1 Andrea Comella,2,3 Marco Straccia,2,3 Georgina Bombau,2,3
Jon T. Brown,5 Josep M. Canals,2,3 Andrew D. Randall,4,5 Nicholas D. Allen,1 and Paul J. Kemp1
1
School of Biosciences, Cardiff University, Cardiff, United Kingdom; 2Department of Cell Biology, Immunology and
Neuroscience, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; 3Centro de Investigación Biomédica
en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain; 4School of Physiology and Pharmacology,
University of Bristol, Bristol, United Kingdom; and 5Hatherly Laboratory, Institute of Biomedical and Clinical Sciences,
University of Exeter Medical School, Exeter, United Kingdom
Submitted 9 June 2015; accepted in final form 28 December 2015

Telezhkin V, Schnell C, Yarova P, Yung S, Cope E, Hughes A,
Thompson BA, Sanders P, Geater C, Hancock JM, Joy S, Badder
L, Connor-Robson N, Comella A, Straccia M, Bombau G, Brown
JT, Canals JM, Randall AD, Allen ND, Kemp PJ. Forced cell cycle
exit and modulation of GABAA, CREB, and GSK3␤ signaling pro-
mote functional maturation of induced pluripotent stem cell-derived
neurons. Am J Physiol Cell Physiol 310: C520 –C541, 2016. First
published December 30, 2015; doi:10.1152/ajpcell.00166.2015.—Al-
though numerous protocols have been developed for differentiation
of neurons from a variety of pluripotent stem cells, most have
concentrated on being able to specify effectively appropriate neu-
ronal subtypes and few have been designed to enhance or accel-
erate functional maturity. Of those that have, most employ time
courses of functional maturation that are rather protracted, and
none have fully characterized all aspects of neuronal function,
from spontaneous action potential generation through to postsyn-
aptic receptor maturation. Here, we describe a simple protocol that
employs the sequential addition of just two supplemented media
that have been formulated to separate the two key phases of neural
differentiation, the neurogenesis and synaptogenesis, each charac-
terized by different signaling requirements. Employing these me-
dia, this new protocol synchronized neurogenesis and enhanced the
rate of maturation of pluripotent stem cell-derived neural precur-
sors. Neurons differentiated using this protocol exhibited large cell
capacitance with relatively hyperpolarized resting membrane po-
tentials; moreover, they exhibited augmented: 1) spontaneous elec-
trical activity; 2) regenerative induced action potential train activ-
ity; 3) Na⫹ current availability, and 4) synaptic currents. This was
accomplished by rapid and uniform development of a mature,
inhibitory GABAA receptor phenotype that was demonstrated by
Ca2⫹ imaging and the ability of GABAA receptor blockers to
evoke seizurogenic network activity in multielectrode array re-
cordings. Furthermore, since this protocol can exploit expanded
and frozen prepatterned neural progenitors to deliver mature neu-
rons within 21 days, it is both scalable and transferable to high-
throughput platforms for the use in functional screens.
Address for reprint requests and other correspondence: N. D. Allen. School
of Biosciences, The Sir Martin Evans Bldg., Cardiff Univ., Museum Ave.,
Cardiff CF10 3AX, UK (e-mail: Allennd@cf.ac.uk).
C520 0363-6143/16 Copyright © 2016 the American Physiological Society
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neural differentiation; induced pluripotent stem cells; iPSCs; embry-
onic stem cells; EPSCs; patch clamp; GABAA; neuronal maturation
THE ABILITY TO DERIVE induced pluripotent stem cell (iPSC) lines
directly from human patients and healthy subjects has enor-
mous implications for biomedical research (37). Of particular
interest is the prospect of using patient-derived stem cell lines
to derive human in vitro models of disease, to use these to
research disease mechanisms in the context of human disease
genetics, and to develop assays with the highest disease and
human relevance for drug discovery (9). iPSC disease model-
ing has become especially prominent in the field of neurosci-
ence. First, patient-derived iPSCs have been used to model
neurodevelopmental disorders (including Down’s, Rett, and
Fragile X syndromes; Refs. 11, 15, 16), where the effects of
disease on the proper differentiation and maturation of neural
lineages have been studied. Second, there has been extensive
modeling of neurodegenerative disorders (including Alzhei-
mer’s disease, Parkinson’s disease, Huntington’s disease, and
amyloid lateral sclerosis) (5, 9, 21a, 23, 27, 33). Models have
been validated by phenotyping a spectrum of cell, molecular,
and metabolic features associated with neurodegeneration.
Most significantly for models of neurodegeneration, disease-
associated phenotypes have been seen to manifest in weeks in
the context of in vitro cultures, in spite of the fact that the
diseases may take decades to manifest in vivo. Third, there is
growing interest in iPSC models of neuropsychiatric disorders,
such as schizophrenia, bipolar, and autism spectrum disorders
(8, 22, 31, 36, 39), where there is even greater emphasis on
synaptic function and physiological endpoints.
While reprogramming technologies have enabled significant
resources of patient-derived iPSC lines to be derived and
banked, for effective disease modeling their utility is entirely
dependent on the quality of the protocols used to differentiate
them into the somatic cells of disease relevance. In the case of
neural differentiation, numerous protocols for neural induction
and neural progenitor patterning, or neural subtype specifica-
tion, have been developed and many succeed in generating
http://www.ajpcell.org
 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS
neurons with characteristic morphology and the appropriate
expression of multiple neuronal markers (13). However, in the
majority of cases, the extent of functional neuronal maturation
with respect to physiological endpoints is poorly described.
Most commonly, examples of induced action potential record-
ings are shown, and examples of spontaneous networked ac-
tivity are only reported from long-term cultures differentiated
and maintained for many weeks, typically involving coculture
of human pluripotent cell (hPSC)-derived neurons with mouse
astrocytes or astrocyte-conditioned medium (21, 35, 45, 47).
Moreover, problems associated with culture heterogeneity and
time-in-culture severely compromise the use of iPSC-derived
neuronal models for reproducible higher content and higher
throughput phenotyping (29, 42, 48, 49).
Here, we have addressed the shortcomings of current neu-
ronal differentiation protocols and developed new defined
media that generate highly mature neurons from iPSC-derived
neural progenitors, with high efficiency and reproducibility,
within only 21 days of plating and without the use of astrocyte
coculture or astrocyte conditioned medium. Critical pathways
that control neurogenesis and synaptogenesis include regulated
cell cycle exit and Notch pathway inhibition, with GABAA,
CREB, and WNT pathway activation. These have been ma-
nipulated using small molecules to produce two chemically
defined culture media, which, when applied sequentially, syn-
chronize progenitor cell neurogenesis to promote and acceler-
ate synaptogenesis and neuronal maturation within functional
networks.
MATERIALS AND METHODS
hiPSC Culture and Neural Differentiation
Studies were performed using two feeder-free human iPSC lines:
34D6, derived using integrating reprogramming vectors (gift from S.
Chandran, Edinburgh, UK; Ref. 6), and 33Qn1, derived using nonin-
tegrating reprogramming vectors (gift from C. N. Svendsen, Cedar
Sinai, Los Angeles, CA; Ref. 33). iPSCs were cultured on Matrigel-
coated plates (BD Biosciences, Oxford, UK) in mTeSR1 medium and
expanded by passaging using dispase following the manufacturer’s
protocols (Stem Cell Technologies, Cambridge, UK). For neural
induction, iPSC cultures were grown to 70% confluence and washed
three times with phosphate-buffered saline (PBS; Invitrogen Life
Technologies, Paisley, UK) and the medium was exchanged for the
neural induction SLI medium, which contained Advanced DMEM:
F-12 (with Glutamax), 1% penicillin/streptomycin (all from Life
Technologies), 10 ␮M SB431542 (Abcam, Cambridge, UK), 1 ␮M
LDN 193189 (Stemgent, Cambridge, MA), 1.5 ␮M IWR1 (Tocris
Bioscience, Abingdon, UK), and 2% NeuroBrew-21 without retinoic
acid (RA) (Miltenyi Biotec, Bisley, UK). On day (D)4 of differenti-
ation, confluent cultures were treated with 10 ␮M Y-27632 (Abcam)
for 1 h before cell dissociation with Accutase (GE Healthcare Life
Sciences, Little Chalfont, UK) and passaged onto fresh Matrigel-
coated plates, with a split ratio of 1:2. On D8, cultures were passaged
1:2 and cultured in LI medium, which contained Advanced DMEM:
F-12, 2 mM L-glutamine, 1% penicillin/streptomycin, 200 nM LDN
193189, 1.5 ␮M IWR1, and 2% NeuroBrew-21 without RA. Cultures
received daily medium changes. D16 iPSC-derived neural progenitor
cells (NPCs) were either used directly for neuronal differentiation (see
below), frozen in Cryostor CS10 (Stem Cell Technologies) for later
differentiations, or expanded in Matrigel-coated flasks in ADF-F
medium, which contained Advanced DMEM:F-12 (with Glutamax),
1% penicillin/streptomycin, 2% NeuroBrew-21 with RA, and 20
ng/ml FGF2 (Miltenyi Biotec).
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For neuronal differentiation, NPCs were dissociated using accutase
and plated at a density of 1 ⫻ 105 cells/13-mm glass coverslips.
Before use, coverslips were cleaned with 70% nitric acid, washed with
deionized water and then with absolute ethanol, oven sterilized, and
sequentially coated with 100 ␮g/ml poly-D-lysine (PDL in borate
buffer; Sigma-Aldrich, Poole, UK). They were then washed three
times in PBS before being coated with 1:100 Matrigel. D16 NPCs
were then plated onto the coated coverslips and were differentiated for
up to a further 35 days postplate-down (dpp), although the standard
time course lasted for only 21 dpp (i.e., a total of 37 days from iPSC
to functional neuron). The proliferation inhibitors N-[N-(3,5-difluoro-
phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT; Sigma-
Aldrich) and PD0332991 (Selleckchem, Newmarket, UK) were tested
in neural differentiation medium that comprised Advanced DMEM:
F-12 (with Glutamax), 1% penicillin/streptomycin, 2% Neuro-
Brew-21 with RA (Miltenyi Biotec), 200 ␮M ascorbic acid (Sigma-
Aldrich), and 10 ng/ml brain-derived neurotrophic factor (BDNF;
MACS; Miltenyi Biotec). For differentiation studies, three protocols
were compared in this study: 1) SFDM4, 2) SCM-Base, and 3)
SCM1/2. In all cases, other than switching from SCM1 to SCM2,
half-medium changes were made every 2 days and cells were cultured
in a humidified incubator in 5% CO2-95% air at 37°C.
SFDM4 protocol. To the plated D16 NPCs, SFDM4 medium was
added, which contained Advanced DMEM:F-12 (with Glutamax), 1%
penicillin/streptomycin, 2% NeuroBrew-21 with RA (Miltenyi Bio-
tec), 200 ␮M ascorbic acid (Sigma-Aldrich), and 10 ng/ml BDNF
(Miltenyi Biotec). DAPT (10 ␮M; Sigma-Aldrich) was added for the
first 7 dpp.
SCM-Base protocol. To the plated D16 NPCs, SCM-Base medium
was added, which contained Advanced DMEM:F-12 (with Glutamax),
1% penicillin/streptomycin, 2% NeuroBrew21 with RA (Miltenyi Bio-
tec), 2 ␮M PD0332991 (Selleckchem), 10 ␮M DAPT(Sigma-Aldrich),
0.6 mM CaCl2 (to give 1.8 mM total CaCl2 in final complete medium;
Sigma-Aldrich), 200 mM ascorbic acid (Sigma-Aldrich), 10 ng/ml
BDNF, and 1 ␮M LM22A4 (Tocris Bioscience).
SCM1/2 protocol. To the plated D16 NPCs, SCM1 medium was
added for the first 7 dpp, which contained Advanced DMEM:F-12
(with Glutamax), 1% penicillin/streptomycin, 2% NeuroBrew21
(Miltenyi Biotec), 2 ␮M PD0332991 (Selleckchem), 10 ␮M DAPT
(Sigma-Aldrich), 0.6 mM CaCl2 (to give 1.8 mM total CaCl2 in final
complete medium; Sigma-Aldrich), 200 ␮M ascorbic acid (Sigma-
Aldrich), 10 ng/ml BDNF (Miltenyi Biotec), 1 ␮M LM22A4 (Tocris
Bioscience), 10 ␮M forskolin (FSK; Tocris Bioscience), 3 ␮M CHIR
99021 (Tocris Bioscience), and 300 ␮M GABA (Tocris Bioscience).
From 8 dpp, the NPCs were cultured in SCM2 medium, which
contained 1:1 Advanced DMEM/F-12 (with Glutamax):Neurobasal A
(Life Technologies), 1% penicillin/streptomycin (Life Technologies),
2% NeuroBrew21 with RA (Miltenyi Biotec), 2 ␮M PD0332991
(Selleckchem), 3 ␮M CHIR 99021 (Tocris Bioscience), 0.3 mM
CaCl2 (to give 1.8 mM total CaCl2 in final complete medium;
Sigma-Aldrich), 200 ␮M ascorbic acid (Sigma-Aldrich), 10 ng/ml
BDNF (Miltenyi Biotec), and 1 ␮M LM22A4 (Tocris Bioscience).
These media form the basis of an accelerated maturation protocol
(patent no. PCT/GB2014/053064, Neuronal Stem Cell Differentia-
tion; P. J. Kemp, N. D. Allen, and Cardiff University), available as the
SynaptoJuice kit via collaboration.
Western Blot Analysis
For CREB and ERK analysis, cells were rinsed with ice-cold PBS
and then harvested by scraping directly into RIPA buffer (Sigma-
Aldrich) containing complete EDTA-free protease inhibitors (Roche
Life Science, Welwyn Garden City, UK) and PhosSTOP phosphatase
inhibitors (Roche Life Science). Cell lysates were centrifuged at 120
g for 15 min at 4°C, and the supernatants were collected and
quantified by the Pierce BCA assay (Life Technologies). Twenty
micrograms of total protein were loaded per well into 4 –12% Bis-Tris
C522 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS
Bolt gels (Life Technologies) and electrophoresed at 165 V for ⬃1 h
in MOPS buffer. Proteins were transferred to nitrocellulose mem-
branes at 100 V for 1 h and membranes were blocked in 5%
milk-PBST (PBS plus 0.1% Tween-20; Sigma-Aldrich) for 1 h at
room temperature with agitation. Membranes were probed with the
appropriate primary antibodies at the dilutions and incubation time/
temperatures defined in Table 1. Secondary antibodies (donkey anti-
mouse IR800; Lorne Laboratories, Danehill, UK) and goat anti-rabbit
680 (Life Technologies) at 1/10,000 dilution were incubated for 1 h at
room temperature before visualization of blots on the Li-Cor Odyssey
system (Li-Cor Biotechnology, Cambridge, UK).
Immunocytochemistry
Cells were processed on 13-mm glass coverslips. They were fixed
with fresh, 4% (wt/vol) paraformaldehyde in PBS for 15 min at 37°C
and then washed three times with PBS. For all antibodies, other than
those raised against the NMDA receptors, the samples were blocked
and permeabilized using PBS containing 1% BSA (wt/vol), 0.1%
Tween-20 for 1 h at room temperature. For the NMDA receptor
antibodies, cells were blocked and permeabilized using 1% BSA
(Sigma-Aldrich), 0.3% Triton X-100 (Sigma-Aldrich), 0.03%
sodium-azide (Fluka BioChemike), and 5% normal goat serum (Vec-
tor Laboratories) for 45 min at room temperature before being incu-
bated overnight at 4°C in the appropriate primary antibodies at
dilutions defined in Table 1. After overnight incubation, primary
antibodies were removed and the cells were washed three times for 5
min in PBS before a 1- or 2-h incubation with fluorophore-conjugated
secondary antibody [Alexa Flour-488, goat anti-rabbit, Abcam; Cy3,
donkey anti-rabbit, Jackson Immuno Research (JIR); Alexa Fluor 488,
donkey anti-mouse, JIR no. 715-545-150; or Alexa Fluor 647, goat
anti-rabbit; Molecular Probes, Eugene, OR] at room temperature in
the dark. Nuclear staining employed Hoechst or DAPI stain at 1:5,000
in PBS. Coverslips were mounted in Vectashield Mounting Medium
(Vector Laboratories, Peterborough, UK) and imaged using Olympus
BX61 microscope with SIS F-view SSD camera and AnalySIS imag-
ing software (Olympus, Southend-on-Sea, UK); coregistration images
were obtained using a Leica TCS SP2 AOBS spectral or Leica SP5
TCS confocal microscopes.
Morphometric Analyses
Sholl analysis was performed on cells individually imaged for GFP
expression after low efficiency transfection of pmax-GFP using Li-
pofectamine 2000 (Invitrogen, Life Technologies). Neurons were then
fixed at 7 and 21 dpp with 4% (wt/vol) paraformaldehyde for 5 min at
room temperature and washed three times with PBS. Coverslips were
then mounted in Fluoromount (Sigma-Aldrich). Images of at least 50
neurons were taken for each condition and time point using an
Olympus BX61 microscope with SIS F-view SSD camera and Analy-
SIS imaging software (Olympus). When required, multiple images
were taken to capture the extent of single neuron processes and
images were stitched together using Microsoft Image Composite
Editor (Microsoft, Berkshire, UK). Images were converted to black and
white by splitting the RGB channels of each image, and the green channel
was selected in order for axons and neurites to be traced using the
simple neurite tracer plugin of Fiji software (http://fiji.sc/Simple
_Neurite_Tracer). With the use of this plugin, a line stack of the traces
was created for subsequent Sholl analysis. Sholl analysis was performed
using the Sholl analysis plugin (http://fiji.sc/Sholl_Analysis) with a start-
ing radius of 5 ␮m and final radius at the furthest point of the cell in
increments of 5 ␮m. Data relating to neurite length and intersection
were also obtained using the simple neurite tracer plugin. Data are
presented as scatterplots but also show mean ⫾ SE. Normalized
branching data (intersections per ␮m) are presented as bar graphs
showing mean ⫾ SE.
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Scanning Electronic Microscopy
Dendritic spines were visualized by scanning electron microscopy
(SEM). D16 NPCs (1 ⫻ 105) were plated on 13-mm plastic coverslips
(Thermanox plastic coverslips; Nunc), which were fixed and pro-
cessed for SEM imaging at 21 dpp. Samples were processed at the
Electron Microscopy Unit of the Scientific and Technological Centers
of the University of Barcelona, Spain. Images were taken using a
JOEL Field Emission Scanning Electron Microscope (JSM-7001F).
Electrophysiological Recordings and Analyses
Patch clamp. Voltage and current recordings were made using
conventional patch clamp in the whole cell configuration (20) em-
ploying either Axopatch 200B or Multiclamp 700A amplifiers inter-
faced to a computer running pClamp 9 using a Digidata 1322A A/D
interface (Molecular Devices, Sunnyvale, CA). All electrophysiolog-
ical studies were performed at a controlled room temperature of 22 ⫾
0.5°C. Recordings were digitized at 10 kHz and low-pass filtered at 2
or 5 kHz using an 8-pole Bessel filter. The standard bath solution
contained the following (in mM): 135 NaCl (Fisher Scientific, Lough-
borough, UK), 5 KCl (Fisher), 1.2 MgCl2 (Sigma-Aldrich), 1.25
CaCl2 (Sigma-Aldrich), 10 D-glucose (Fisher), 5 HEPES (VWR
International); pH was adjusted to 7.4 using 5 M NaOH. The standard
pipette solution contained the following (in mM): 117 KCl, 10 NaCl,
11 HEPES, 2 Na2-ATP (Sigma-Aldrich), 2 Na-GTP (Sigma-Aldrich),
1.2 Na2-phosphocreatine (Sigma-Aldrich), 2 MgCl2, 1 CaCl2, and 11
EGTA (Fisher); pH was adjusted to 7.2 with KOH.
Mean resting membrane potential (Vm) and spontaneous action
potential (sAP) characteristics of the neurons were determined during
120-s gap-free recording periods in current-clamp mode (I ⫽ 0 pA).
Neurons were then coded according to the type of sAP activity that
they demonstrated, defined as follows: 1) full spontaneous action
potentials (sAP-full, at least 1 excursion that overshoots 0 mV); 2)
attempted spontaneous action potentials (sAP-attempted, significant
excursions from resting Vm that did not reach 0 mV); or 3) quiescent
(sAP-none, no significant excursions from resting Vm). This then
allowed neurons to be categorized into the three separate groups
for further detailed analysis. Once Vm and sAP activity had been
recorded, current was injected to hyperpolarize Vm to approxi-
mately ⫺70 mV before 1-s current injection steps were imposed
from ⫺10 to ⫹180 pA in 10-pA increments to record induced
action potential (iAP) activity, which was coded as iAP-none (no
significant excursions from baseline during injection); iAP-at-
tempted single (significant excursions from baseline that do not
reach 0 mV); iAP-single (a single excursion that overshoots 0 mV);
iAP-attempted train (several excursions but only 1 overshoots 0
mV); and iAP-train (several excursions, at least 2 of that overshoot
0 mV). Where iAP trains were recorded, a spike frequency analysis
was performed. Input resistance was measured from the voltage
difference induced by the ⫺10-pA current step. Spike analysis was
performed on the first iAP using Clampfit 9; threshold was deter-
mined as the peak of the third differential of voltage with respect
to time during the upstroke of the action potential and all other
parameters are as defined extensively elsewhere (4).
Na⫹ currents were recorded using a standard voltage-step
protocol (holding potential of ⫺70 mV followed by 80-ms steps
from ⫺120 to ⫹80 mV in increments of 10 mV). For Na⫹ current
inactivation curves, cells were stepped for 200 ms to prepulse voltages
of between ⫺120 and ⫹80 mV in 5-mV increments before being
stepped for 200 ms to the test potential of 0 mV. Note that pharma-
cological dissection of the voltage-activated currents was not at-
tempted for logistical reasons but that cross contamination of maximal
inward Na⫹ current by outward K⫹ current was negligible at the
voltage at which they were measured (⫺30 and ⫺20 mV); similarly,
contamination of K⫹ current by Na⫹ current was negligible at the
time and voltage at which they were measured (⫹60 mV between 180
and 200 ms), since Na⫹ currents were already inactivated (see Figs. 7
 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS
and 8). Cell capacitance and series resistance were measured and
compensated; series resistance was compensated 60 –90%. Pipette
resistances were 8 –10 M⍀ when filled with the pipette solutions.
For activation and inactivation curves, conductance (G) was cal-
culated by dividing current by the appropriate driving force (Vc ⫺
ENa), where Vc ⫽ command potential and ENa ⫽ ⫹66.7 mV. G/Gmax
was plotted against voltage and fitted with a Boltzmann equation
using an iterative fitting routine in Microcal Origin:
G ⁄ Gmax ⫽ 1 ⁄ [1 ⫹ exp (V0.5 ⫺ Vc ⁄ k]
where Gmax is the extrapolated maximum conductance, V0.5 is the
voltage corresponding to half the maximum conductance, and k is the
slope factor.
Spontaneous miniature excitatory and inhibitory currents, and
postsynaptic GABA and NMDA currents, were recorded using
solutions modified form (17). The extracellular solution contained
the following (in mM): 127 NaCl, 20 CsCl, 5 BaCl2, 2 CaCl2, 12
glucose, 10 HEPES, 0.0005 tetrodotoxin (TTX), and 0.02 glycine
at pH 7.3 with NaOH. The intracellular solution contained the
following (in mM): 175 N-methyl-D-glucamine (NMDG), 40
HEPES, 2 MgCl2, 10 EGTA, 12 phosphocreatine, 2 Na2ATP, 0.2
Na3GTP, and 0.1 leupeptin at pH 7.3 with H2SO4. These solutions
set the Ecat and ECl at positions where at ⫺40-mV inward cationic
currents (NMDA) were detected as downward deflections and
inward chloride currents were detected as upward deflections.
Spontaneous miniature excitatory and inhibitory currents were
recorded for at least 2 min. Evoked postsynaptic currents were
induced by three 5-s pulses of either 100 ␮M GABA or 100 ␮M
NMDA at 90-s intervals delivered locally to neurons by a gravity-
driven rapid perfusion changer (RSC18; Biologic, Cedex, France).
Data were analyzed offline using MiniAnalysis (Synaptosoft),
Clampfit 10.3 (Molecular Devices), and GraphPad Prism 6 (Graph-
Pad Software).
Multielectrode arrays. D16 NPCs were plated at a density of
150 ⫻ 103 cells/well onto poly-D-lysine coated, 24-well multielec-
trode arrays (MEAs) and cultured sequentially using the three differ-
entiation protocols as described above. At 21 dpp, the medium was
switched for a HEPES-buffered physiological solution containing the
following (in mM): 130 NaCl, 3 KCl, 1 MgCl2 2 CaCl2, 10 glucose,
and 10 HEPES-NaOH at pH 7.4, and the MEAs were placed onto the
temperature-controlled (37°C) platform of a commercially available
MEA workstation (Multichannel Systems, Reutlingen, Germany).
Each well contained 12 electrodes, the activities of which were
recorded simultaneously before, during, and following manual addi-
tion of agonists, antagonists, and toxins, as defined in the text and
legends. Data were band-pass filtered between 100 and 2,000 Hz and
finally sampled at 20 KHz using the acquisition software provided
with the device. During recordings this software detected single unit
activity online and live displays of raw data, spikes per second
histograms, and detected spike waveforms were available. For anal-
ysis, raw data were converted to a HDF5 file format, which was
imported into the MatLab environment in which custom-written
analysis routines were coded. Spikes were redetected and sorted, and
time stamps were extracted allowing the various plots shown here to
be produced. Root mean square recording noise levels were ⬃1.7 ␮V.
Spikes were detected using a threshold method (usually 5 SD of the
mean) that detected either rapidly rising or falling events. Amplitudes
of clearly resolvable spikes uncontaminated by significant levels of
false detections varied between 10 and 100 ␮V. The major source of
this amplitude variation is likely to be the physical distance of each
detected neuron from the recording site. Typically, spontaneous spik-
ing activity could be detected on multiple electrodes within a single
well and in 24-well plates.
Calcium Imaging
The ratiometric Ca2⫹ sensitive dye fura-2 (Molecular Probes)
was used for Ca2⫹ imaging experiments. Neurons on glass cover-
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C523
slips were incubated in 4 ␮M fura-2-AM (dissolved in cell culture
medium) for 30 min at 37°C in a humidified incubator gassed with
5% CO2/95% air. Coverslips were placed in a perfusion chamber
mounted upon an Olympus IX71 inverted microscope equipped
with a Cairn monochromator-based epifluorescence system (Cairn
Instruments, Faversham, UK) and were continuously superfused
with standard extracellular solution (see above for composition).
Imaging was conducted at a controlled room temperature of 22 ⫾
0.5°C. Fura-2 was alternately excited with light of 340 and 380 nm
and images were captured at 510 nm using an Orca CCD camera
(Hamamatsu Photonics, Welwyn Garden City, UK). Solutions,
agonists, and antagonists were locally applied to the neurons using
a rapid solution changer. Following background subtraction of the
emission intensities evoked by each excitation wavelength, emis-
sion ratios (340/380) were calculated offline. A cell was deemed to
have responded to any stimulus if the emission ratio change was
more than 10 times the standard deviation of the fura-2 signal in its
preapplication period. Peak intensity changes from baseline in
response to applied agonists were calculated for each individual
cell. Averaged values obtained from independent experiments were
used to calculate the mean intensity.
Statistical Analyses
All summarized data are expressed as mean ⫾ SE. Statistical
comparisons of the means were performed using one-way or two-way
analysis of ANOVA with Tukey post hoc test, or Student’s t-tests, as
appropriate and as stated in text and figure legends; differences were
considered significant at P ⬍ 0.05.
RESULTS
In vitro differentiation and maturation protocols of human
neurons were developed using D16 NPCs derived from the
iPSC line 34D6 and further validated using the 33Qn1 line.
NPCs from each line were derived by culture in defined
medium with dual SMAD and WNT inhibition to enrich for
NPCs with forebrain fate potential (30). At D8, cultures were
characterized by high expression of SOX2, PAX6, and
NESTIN, and FOXG1 with a high incidence of neural rosette
formation identified by foci of zona occludens-1 (ZO-1) im-
munoreactivity at D16 (data not illustrated).
With the use of a standard protocol for neuronal differenti-
ation, D16-dissociated iPSC-derived NPCs plated on PDL/
Matrigel-coated coverslips and cultured in SFDM4 (25)
showed extensive neurite outgrowth and expression of ␤-III-
tubulin after 21 dpp. However, as commonly observed, after 21
dpp, cultures were seen to increase in complexity with signif-
icant clustering of cells, with apparent cell overgrowth, often
compromising cultures for use in developmental or electro-
physiological and imaging studies (data not illustrated).
Inhibitors of Cell Cycle and Notch Signaling Force Cell
Cycle Exit and Synchronize Neurogenesis
To synchronize neuronal differentiation we first determined
whether blockade of Notch signaling and the cell cycle check-
point cyclin-dependent kinases CDK4 and CDK6 would pro-
mote cell cycle exit. After D16 34D6 NPCs were plated,
blockade of Notch signaling with the ␥-secretase inhibitor
DAPT (10 ␮M) significantly reduced the proportion of cells
that stained for MKI67, a marker of cell proliferation, from
21.8 ⫾ 1.5 to 12.9 ⫾ 1.8% at 3 dpp (P ⬍ 0.05), and from
47.2 ⫾ 3.6 to 25.7 ⫾ 10% at 7 dpp (P ⬍ 0.001; Fig. 1, A and
B). Treatment with 2 ␮M PD0332991, a selective CDK4/6
C524 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

Table 1. List of primary antibodies and dilutions used for either Western blot analysis or immunocytochemistry and supplier
details
Target Host Dilution Application Supplier

␤-III tubulin Rabbit 1/500 ICC Sigma-Aldrich

CREB Rabbit 1/1,000 WB Cell Signaling
ERK1/2 Mouse 1/2,000 WB Cell Signaling
GABA Rabbit 1/100 ICC Sigma-Aldrich A2052
GAPDH Mouse 1/5,000 WB Protein Tech Europe 60004-1-Ig
MKI67 Mouse 1/100 ICC Vector Labs MM1
MAP2 Mouse 1/600 ICC Sigma-Aldrich AP20
MAP2ab Mouse 1/500 ICC BD Transduction 610460
NESTIN Mouse 1/500 ICC Millipore MAB5326
NR1 Rabbit 1/300 ICC Alomone Labs AGC-001
NR2A Rabbit 1/300 ICC Alomone Labs AGC-003
NR2B Rabbit 1/300 ICC Alomone Labs AGC-002
Phospho-CREB Rabbit 1/1,000 WB Cell Signaling (S133) 87G3
Phospho-ERK1/2 Mouse 1/2,000 WB Cell Signaling (T202/Y204) E10
PSD95 Rabbit 1/100 ICC Abcam ab18258
Synaptophysin Mouse 1/200 ICC Abcam ab8049
All antibodies were used at 4°C with overnight incubations. WB, Western blot analysis; ICC, immunocytochemistry.

inhibitor (50), resulted in a greater loss of MKI67 immunopo-
sitive cells at 3 dpp (to 0.9 ⫾ 0.6%; P ⬍ 0.001) and 7 dpp (to
7.9 ⫾ 4.7%; P ⬍ 0.0001, Fig. 1, A and B). In combination,
␥-secretase and CDK4/6 inhibition resulted in a significant and
synchronous sustained cell cycle exit at 7 dpp (to 4.2 ⫾ 0.5%;
P ⬍ 0.0001; Fig. 1, A and B). Treatment with DAPT and
PD0332991 also enhanced neural differentiation, as evidenced
by an increase in the proportion of cells expressing the neuro-
filament protein ␤-III-tubulin at 7 dpp (control: 64.3 ⫾ 1.6%;
DAPT/PD0332991: 84.6 ⫾ 3.6%; P ⬍ 0.001) and a marked
reduction in the level of expression of the neural progenitor
marker NESTIN (control: 92.9 ⫾ 1.9%; DAPT/PD0332991:
32.1 ⫾ 3.7%; P ⬍ 0.0001), compared with NPCs cultured in
the absence of DAPT and PD0332991 (Fig. 1C). Thus SCM1
medium was formulated to include these two small molecules.
SCM1 Enhances Neurogenesis
In search of the cellular basis for the well-documented
benefit of astrocyte coculture to neuronal differentiation (19,
26, 40, 51), we have previously identified excitatory GABAA-
dependent depolarization and consequent voltage-activated
Ca2⫹ entry as critical to the process (45). GABAA receptor
activation leads to Ca2⫹-dependent CREB and ERK phosphor-
ylation, which in turn plays a central role in regulating neuro-

A Control DAPT PD0332991 PD0332991/DAPT

3 dpp
MKI67

Fig. 1. ␥-Secretase- and cyclin-dependent kinase 4/6 inhibi-

tion promote synchronous cell cycle exit and differentiation of
34D6-derived neural progenitors (NPC). A: MKI67 immuno-
reactivity (green) and cell nuclear staining with DAPI (blue)
7 dpp

in NPC cultures at 3 days postplate-down (dpp) (top) and 7

dpp (bottom) after plating in either control medium or me-
dium supplemented with 2 ␮M PD0332991 or 10 ␮M DAPT
or both. Scale bars ⫽ 100 ␮m, as indicated. B: mean percent-
age of MKI67-positive cells at 3 and 7 dpp, analyzed by
one-way ANOVA with Tukey’s post hoc test: *P ⬍ 0.05,
***P ⬍ 0.001, ****P ⬍ 0.0001 (N ⫽ 3). C: NESTIN (top) B 3 dpp 7 dpp C Control PD0332991/DAPT
and ␤-III-tubulin (bottom) immunoreactivity of induced plu-
ripotent stem cells (iPSC)-derived neural progenitors differ- ***
NESTIN

60 60
MKI67 positive (%)

MKI67 positive (%)

entiated for 7 dpp in either control medium or medium ***

*
supplemented with 2 ␮M PD0332991 and 10 ␮M DAPT. 50 50
Scale bars ⫽ 100 ␮m. Percent cell counts for NESTIN were
control: 92.9 ⫾ 1.9% and DAPT/PD0332991: 32.1 ⫾ 3.7%; 40
****
40
Student’s t-test: P ⬍ 0.0001 (N ⫽ 3). Percent cell counts for 30
30
III-TUBULIN

***
␤-III-tubulin were control: 64.3 ⫾ 1.6% and DAPT/
PD0332991: 84.6 ⫾ 3.6%; Student’s t-test: P ⬍ 0.001 20 20
(N ⫽ 3).
10 10

0 0
/D D

/D D
PD P T
D rol

PD P T
D rol
T

T
AP

AP
AP

AP
t

t
on

on
C

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS C525
genic gene expression (34, 52). Functional expression of neu- Consistent with developmental regulation of CREB during
ronal voltage-gated Na⫹ and K⫹ channels during differentia- in vivo neural development (34), Western blot analysis of
tion and maturation can also be regulated by agents such as the 34D6 cultures differentiated for 7 days showed high levels of
adenylyl cyclase inhibitor FSK that directly elevate cAMP (1, CREB expression in NPCs that decreased with differentiation
43). Thus SCM1 was formulated to include a higher total in all media (P ⬍ 0.01; Fig. 2, A and B). Differentiation was
extracellular concentration of Ca2⫹ ([Ca2⫹]o) of 1.8 mM and associated with CREB phosphorylation, and significantly neu-
300 ␮M GABA to drive this GABAA-dependent signaling rons differentiated in SCM1 showed the greatest increase in the
pathway and 10 ␮M FSK to augment tonically the intracellular phosphorylation of available CREB (phosphoCREB:CREB:
cAMP concentration. P ⬍ 0.05; Fig. 2, A and C). Differentiation did not affect total

A se F I
4 - Ba /2
PC FD
M
CM M1 MKI67 NESTIN DAPI MERGE Neurite length
N S S SC ns

Neurite length ( m)
2000 ***

SCM1/2 SCM-Base SFDM4

1500
%
CREB 43kDa
4
1000

44kDa
4 500
ERK1/2 42kDa
4
0
SCM-Base SCM1/2 SCM-Base SCM1/2
7 dpp 7 dpp 21 dpp 21 dpp
P-CREB 43kDa
4
J Intersections
ns
800 **

Total interesections
P-ERK1/2 4
42kDa
600
G GABA MAP2 DAPI MERGE
43kDa
4 400
GAPDH
SCM1/2 SCM-Base SFDM4

200

B C 0
SCM-Base SCM1/2 SCM-Base SCM1/2
normalized to GAPDH

normalized to GAPDH

* 7 dpp 7 dpp 21 dpp 21 dpp

1.5 ** 80 ***
Relative CREB

P-CREB:CREB

60
** K Sholl 7 dpp Sholl 21 dpp
**

Interesections
Interesections
1.0 12 SCM-Base 12 SCM-Base
SCM1/2 SCM1/2
40 10 10
ns
0.5 8 8
20
6 6

0.0 0 4 4
M e
M 4
SC ase
M 4

2
SC as

2
PC

2
PC

SC DM
2
SC DM

H
1/
1/

-B
-B

N
N

Week1 (7 dpp) Week3 (21 dpp)

SF

0 0
SF

0 20 40 60 80 100 0 20 40 60 80 100
D E Distance from Distance from
SCM-Base

L
normalized to GAPDH

normalized to GAPDH

soma ( m) soma ( m)
2.0 2.0
*
Relative ERK1/2

**** Intersection per micron

per m
P-ERK:ERK

1.5
1.5 1.5 **** **

1.0 1.0 1.0

Interesections
SCM1/2

ecti

0.5 0.5 0.5

0.0 0.0
M se

-B 4

0.0
M 4

PC

SC ase
PC

M M

2
SC DM

e e
2
SC -Ba

2 as 2
as 1/ 1/
1/
SC FD
1/

M pp
N

M pp -B pp
M

-B p
SF

C M dp SC 7 d CM 1 d SC21 d
S 7 S 2

Fig. 2. SCM1/2 media enhance neural differentiation of iPSC lines. A: Western blot analysis of CREB, ERK1/2, phospho-CREB, and phospho-ERK1/2 in
34D6-derived NPCs and at 7 dpp following differentiation in SFDM4, SCM-Base, and SCM1 media. B: quantitation of CREB normalized to GAPDH. Total
CREB levels were downregulated compared with the levels in NPCs in all conditions (**P ⬍ 0.001, N ⫽ 3). Differences in levels between SCM1 and SCM-Base
or SFDM4 cultures were nonsignificant (ns). C: phospho-CREB:CREB ratio increases with differentiation, with SCM1 inducing a greater phospho-CREB:CREB
increase compared with SFDM4 and SCM-Base conditions (one-way ANOVA with Tukey’s post hoc test: *P ⬍ 0.05, **P ⬍ 0.001, ****P ⬍ 0.0001, N ⫽ 3).
D: quantification of ERK1/2 normalized to GAPDH. No significant changes in ERK1/2 levels were seen between NPCs and SFDM, SCM-Base, or SCM1
differentiated cultures by one-way ANOVA with Tukey’s post hoc test. E: phospho-ERK2:ERK2 ratio was increased in cultures differentiated with SCM-Base
and SCM1 compared with SFDM4 (one-way ANOVA with Tukey’s post hoc test: ****P ⬍ 0.0001), and greater ERK2 phosphorylation was seen in SCM1
compared with SCM-Base (*P ⬍ 0.05). F: MKI67 and NESTIN staining in 34D6 21 dpp SFDM, SCM-Base, and SCM1/2 differentiated cultures. SCM1/2
cultures expressed significantly less MKI67 and NESTIN (one-way ANOVA with Tukey’s post hoc test: P ⬍ 0.0001). Percent cell counts for MKI67 staining
were SFDM4: 12 ⫾ 1.4 and SCM-Base and SCM1/2: ⬍0.5% (N ⫽ 3). Percent cell counts for NESTIN were SFDM4: 64 ⫾ 7%, SCM-Base: 70.5 ⫾ 10.1%,
and SCM1/2: ⬍1% (N ⫽ 3). G: MAP2 and GABA staining in 34D6 21 dpp SFDM, SCM-Base, and SCM1/2 differentiated cultures. Percent cell counts for MAP2
were SFDM4: 32.0 ⫾ 3.7%, SCM-Base: 84.5 ⫾ 2.7%, and SCM1/2: 98.5 ⫾ 1.1%. Both SCM-Base and SCM1/2 cultures expressed significantly more MAP2
(P ⬍ 0.01, P ⬍ 0.001 respectively) and GABA (P ⬍ 0.01, P ⬍ 0.0001, respectively) than SFDM4 cultures (N ⫽ 3). H: exemplar image traces of individual
GFP labeled neurons differentiated in SCM-Base or SCM1/2 media for 7 and 21 dpp taken from n ⫽ 48 –51 neurons in each group from N ⫽ 3 platedowns.
Morphometric analysis of all neurons differentiated in SCM-Base or SCM1/2 media for 7 and 21 dpp. I: scatterplots show individual, mean, and SE of neurite
length (***P ⬍ 0.001). Morphometric analysis of all neurons differentiated in SCM-Base or SCM1/2 media for 7 and 21 dpp. J: scatterplots show total
intersections derived from Sholl analysis (**P ⬍ 0.01). K: distance vs. intersection mean plots to highlight the differences in the first 100 ␮m from the soma.
L: total neuronal length data shown as normalized intersections per ␮m (**P ⬍ 0.01, ****P ⬍ 0.0001).

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C526 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

ERK1/2 levels (Fig. 2D); however, greater levels of sustained
and selective phosphorylation of ERK2 were also seen in
SCM1 cultures (P ⬍ 0.05; Fig. 2, A and E). Together, these
data show a greater activation of CREB and ERK2 signaling in
neurons differentiated with SCM1.
SCM1 and SCM2 Support Ongoing Neuronal Maturation
As GABA stimulation, leading to sustained CREB phos-
phorylation, has a transient developmental role in neurogenesis
(24), GABA and FSK were removed from the second medium,
SCM2, to allow the progression of neuronal maturation during
the second phase of the protocol. Since the proneurogenic
effects of Notch inhibition are required only for the early phase
of neurogenesis and continued ␥-secretase inhibition could
impair neuronal function (46), DAPT was also removed from
SCM2.
To identify further supplements, we performed a directed
screen of growth factors and small molecules for their ability to
enhance neuronal excitability as early as 14 dpp, measuring the
proportion of cells demonstrating each type of induced action
potential (iAP) activity (see Electrophysiology Recordings and
Analysis). Surprisingly, little effect on iAP activity was seen in
response to treatments with BDNF, FGF2, IGF1, or Activin A.
However, the screen showed that addition of 3 ␮M of the
GSK3␤ antagonist CHIR99021 increased the percentage of
neurons firing single iAPs from 60 to 100% and increased those
firing attempted iAP trains from 0 to 30% (data not illustrated).
Therefore, 3 ␮M CHIR99021 was incorporated into the SCM1
and SCM2 media for further analysis.
Following differentiation for 21 dpp, SFDM4 cultures showed
characteristic cell clumping and neural progenitor overgrowth that
contained 64 ⫾ 7% NESTIN⫹ cells and retained a significant 12

A B SCM1/2 sAP C SCM1/2 SCM-Base SFDM4

0

Voltage (mV)
0.5 s
Voltage (mV)

SCM1/2 sAP coding at 21 dpp 30

20 -10
10
28 dpp 0 -20
-10
-20 -30
-30 -40
-40 ns
-50 *
-50 ns
30 100 ms -60
Voltage (mV)

20
-70
SCM1/2 sAP
10 sAP-None sAP-Attempted sAP-Full
sAP-Full 0
-10 0

Voltage (mV)
sAP-Attempted -20 ****
sAP-None -30 -10 ns ****
-40 -20
-50
-30
SCM-Base sAP -40
SCM-Base sAP coding at 21 dpp 40
Voltage (mV) Voltage (mV)

30
20 0.5 s -50
28 dpp 10 -60
0
-10 -70
-20 SCM-Base sAP
-30 sAP-None sAP-Attempted sAP-Full
-40 0
Voltage (mV)

40
30 -10
20 100 ms
10 -20
sAP-Full 0
-10 -30
sAP-Attempted -20 -40
sAP-None -30
-40 -50
SFDM4 sAP -60
Voltage (mV) Voltage (mV)

40
SFDM4 sAP coding at 21 dpp 30 -70 SFDM4 sAP
20 0.5 s
10 sAP-None sAP-Attempted sAP-Full
0 0
-10
Voltage (mV)

-20 *
-30 -10
-40 -20
40 -30
30
20 100 ms -40
10
sAP-Attempted 0 -50
-10
sAP-None -20 -60
-30
-40 -70
Fig. 3. SCM1/2 protocol enhances the generation of 34D6-derived neurons exhibiting spontaneous action potentials. A: pie charts showing the proportion of
neurons that exhibit spontaneous action potentials (sAP-full, green), attempted spontaneous action potentials (sAP-attempted, orange), and no spontaneous action
potentials (sAP-none, red) in 34D6-derived neuronal precursors differentiated into neurons for 21 dpp using SCM1/2 (top, n ⫽ 73 from N ⫽ 5), SCM-Base
(middle, n ⫽ 19 from N ⫽ 2), or SFDM4 (bottom, n ⫽ 19 from N ⫽ 2) protocols. Insets: proportions at 28 dpp for precursors differentiated using SCM1/2
(n ⫽ 17) and SCM-Base (n ⫽ 6) protocols. B: current-clamp recordings (injected current ⫽ 0 pA), exemplifying the model activity for 34D6-derived
neuroprecursors differentiated into neurons for 21 dpp using SCM1/2 (top), SCM-Base (middle), or SFDM4 (bottom) protocol; expanded time-base sections of
traces are shown below each main trace. C: plots of resting membrane potential (Vm) values of means ⫾ SE (top) and individual neurons derived from 34D6
neuronal precursors and differentiated for 21 dpp grouped according to the type of spontaneous activity that they each exhibited; horizontal lines show
means ⫾ SD. One-way ANOVA, Tukey’s post hoc test: *P ⬍ 0.05, ****P ⬍ 0.0001, number of observations and platedowns are as defined in A.

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS
⫾ 1.4% population of MIK67⫹ proliferative cells, with just 32 ⫾
3.7% showing expression of the postmitotic neurofilament MAP2.
In contrast, cultures differentiated in SCM-Base or SCM1/2 me-
dia maintained a more monodispersed neuronal morphology, with
very few MKI67⫹ cells (⬍0.5%) indicating robust cell cycle exit,
and the majority of cells expressed MAP2 (⬎95%) and the
neurotransmiter GABA (Fig. 2, F and G). Interestingly, residual
NESTIN expression was nevertheless seen at 21 dpp in SCM-
Base-differentiated neurons (70.5 ⫾ 10.1%) and was localized
more to the soma than to neurites suggesting an immature neu-
ronal phenotype. NESTIN staining was rarely seen in the SCM1/2
cultures suggesting greater developmental maturation using the
SCM1/2 protocol.
To characterize further the morphology of neurons synchro-
nized in development by differentiation in the SCM-Base and
SCM1/2 conditions, individual cells were marked for analysis
C527
by low-efficiency GFP transfection and expression (Fig. 2D).
At 7 dpp, neurons differentiating in SCM-Base (which lacked
FSK, GABA, and CHIR99021 but retained BDNF/LM22A for
neurotrophic support) showed extensive neurite outgrowth
characteristic of early primary neuritogenesis (3). In contrast,
even at 7 dpp, neurons differentiated in SCM1 already exhib-
ited consolidation of primary neurites for axonal elongation,
demonstrating an accelerated developmental program in this
medium. Specifically, neurons differentiated in SCM1 for 7
dpp exhibited significantly longer neurite outgrowth (P ⬍
0.001) with lower number of intersections (P ⬍ 0.01), reduced
primary neurite complexity close to the soma (Sholl plot of first
100 ␮m), and consequently lower normalized intersections per
micrometer across the entire neuron (P ⬍ 0.0001). By 21 dpp,
neurons differentiated in SCM-Base had also acquired the
more mature phenotype (Fig. 2E).

A B SCM1/2 sAP C
0.5 s SCM1/2 SCM-Base SFDM4
40
Voltage (mV) Voltage (mV)

SCM1/2 sAP coding at 21 dpp 0

20

Voltage (mV)
0 -10
-20 -20
-40 -30
-60 -40
40 -50 **** ***
100 ms
20 -60 ****
sAP-Full 0 -70
sAP-Attempted -20 -80 SCM1/2 sAP
sAP-None -40 sAP-None sAP-Attempted sAP-Full
-60 0
****

Voltage (mV)
-10 ns **
SCM-Base sAP -20
-30
40 0.5 s
Voltage (mV) Voltage (mV)

SCM-Base sAP coding at 21 dpp 20 -40

0 -50
-20 -60
-40 -70
-60 -80 SCM-Base sAP
40 sAP-None sAP-Attempted sAP-Full
100 ms 0
20
0 -10 **
Voltage (mV)

sAP-Full -20 -20

sAP-Attempted -40 -30
sAP-None -60 -40
-50
SFDM4 sAP -60
40
Voltage (mV) Voltage (mV)

0.5 s -70
SFDM4 sAP coding at 21 dpp 20 SFDM4 sAP
-80
0 sAP-None sAP-Attempted sAP-Full
-20 0
-40
Voltage (mV)

-10
-60
-20
40
100 ms -30
20
0 -40
-20 -50
sAP-Attempted
-40 -60
sAP-None
-60 -70
-80
Fig. 4. SCM1/2 protocol enhances the generation of 33Qn1-derived neurons exhibiting spontaneous action potentials. A: pie charts showing the proportion of
neurons that exhibit spontaneous action potentials (sAP-full, green), attempted spontaneous action potentials (sAP-attempted, orange), and no spontaneous action
potentials (sAP-none, red) in 33Qn1-derived neuronal precursors differentiated into neurons for 21 dpp using SCM1/2 (top, n ⫽ 69 from N ⫽ 5), SCM-Base
(middle, n ⫽ 29 from N ⫽ 3), or SFDM4 (bottom, n ⫽ 21 from N ⫽ 2) protocols. B: current-clamp recordings (injected current ⫽ 0 pA), exemplifying the model
activity for 33Qn1-derived neuroprecursors differentiated into neurons for 21 dpp using SCM1/2 (top), SCM-Base (middle), or SFDM4 (bottom) protocol;
expanded time-base sections of traces are shown below each main trace. C: plots of resting membrane potential (Vm) values of means ⫾ SE (top) and individual
neurons derived from 33Qn1 neuronal precursors and differentiated for 21 dpp grouped according to the type of spontaneous activity which they each exhibited;
horizontal lines show means ⫾ SD. One-way ANOVA, Tukey’s post hoc test: **P ⬍ 0.01, ***P ⬍ 0.001, ****P ⬍ 0.0001, number of observations and
platedowns are as defined in A.

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C528 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

SCM1/2 Protocol Promotes Robust and Rapid
Electrophysiological Maturation of iPSC-Derived Neurons
To determine the functional benefits of differentiating hu-
man neural progenitors in SCM1/2 media, the electrophysio-
logical characteristics were determined of neurons differenti-
ated from 34D6 NPCs and a line that was derived using a
nonintegrating reprogramming protocol (33Qn1). The idea was
that comparison of functional readouts of these two lines could
be compared, and if they were similar, the more useful nonin-
tegrated lines, 33Qn1, would be then be used for the remaining
functional studies. Both iPSC-derived NPCs were each differ-
entiated using the three different protocols, SCM1/2, SCM-
Base, and SFDM4, and functional readouts were determined
and compared at 21 dpp.
In comparison to the two control media (SFDM4 and SCM-
Base), differentiation in SCM1/2 of 34D6 iPSC yielded a much
higher proportion of neurons that demonstrated spontaneous
electrical activity (Fig. 3, A and B). Thus 34% (25/73) of
SCM1/2 differentiated neurons were coded as sAP-full, with a
further 36% (26/73) coded as sAP-attempted. In complete
contrast, only 5% (1/19; SCM-Base) and 0% (0/19; SFDM4) of
control differentiated neurons were coded as sAP-full, with just
5% (1/19; SCM-Base) and 21% (4/19; SFDM4) coded as
sAP-attempted. Strikingly, at 28 dpp, 100% of SCM1/2 neu-

A B C SFDM4

Spikes/s
6
SCM base
SCM1/2 iAP coding at 21 dpp SCM1/2 iAP 5 SCM1/2
Voltage (mV)

40 4
100 ms
20 3
0 2
-20
1
sAP-Train -40
sAP-Attempted Train 0
-60 0 20 40 60 80 100 120 140 160 180
sAP-Single
-80 Injected current (pA)
sAP-Attempted Single D

Afterhyperpolarization (mV)
sAP-None -100

Overshoot (mV)

Amplitude (mV)
** ****
20 ns ns 0 80 ns
*
SCM-Base iAP
SCM-Base iAP coding at 21 dpp 15
Voltage (mV)

60
40
-20
20 100 ms
10 40
0
-20 -40
5 20
-40
sAP-Train -60 ns
0 -60 ** 0
sAP-Attempted Train -80 ****
SF a s e

SF ase

SF ase
sAP-Single -100
SC 1/2

SC 1/2

SC 1/2
4

4
-B

-B

-B
M

M
M
M

sAP-Attempted Single M
M

M
M
D

D
SC

SC

SC
sAP-None
E
80 0 8
Depolarization rate (V/s)

Half width (ms)

Repolarization rate (V/s)

SFDM4 iAP * *
SFDM4 iAP coding at 21 dpp ns ** ns
Voltage (mV)

ns
40
100 ms 60 6
20 -10
0
40 4
-20
-40 -20
20 2
sAP-Train -60
sAP-Single ns ns
-80 0 -30 ns 0
sAP-Attempted Single
-100
SF ase

SF ase

SF ase

sAP-None
SC 1/2

SC 1/2

SC 1/2
4

4
-B

-B

-B
M

M
M
M

M
M

M
M
D

D
SC

SC

SC

Fig. 5. SCM1/2 protocol enhances the generation of 34D6-derived neurons exhibiting induced action potential trains and augments their spike characteristics.
A: pie charts showing the proportion of neurons that exhibit trains of induced action potentials (iAP-train, dark green), attempted trains of action potentials
(iAP-attempted train, light green), single induced action potential (iAP-single, orange), attempted single induced action potential (iAP-attempted single, pink)
or no induced action potentials (iAP-none, red) in 34D6-derived neuronal precursors differentiated into neurons for 21 dpp using SCM1/2, SCM-Base, or SFDM4
protocols. Numbers of individual observations and platedowns are as defined in Table 2 and Fig. 3, respectively. B: current-clamp recordings when neurons were
held at between ⫺70 and ⫺80 mV and to 1-s injected currents of ⫺10 pA (light grey) and ⫹20 pA (medium grey) and the current producing the maximum iAP
activity (black) exemplifying the model activity for 34D6-derived neuroprecursors differentiated into neurons for 21 days dpp using SCM1/2 (top), SCM-Base
(middle), or SFDM4 (bottom) protocol; expanded time-base sections of traces are shown below each main trace. D: spike frequency plots for neurons exhibiting
trains of iAPs following differentiation of 34D6 neural precursors using SCM1/2 (closed black squares), SCM-Base (closed grey squares), or SFDM4 (open black
squares). Induced action potential spike size parameter plots for 34D6 neural precursors differentiated for 21 dpp using SCM1/2 (black bars), SCM-Base (light
grey bars), or SFDM4 (dark grey bars) showing overshoot (left), afterhyperpolarization (middle), and full amplitude (right). One-way ANOVA, Tukey’s post hoc
test: *P ⬍ 0.05, **P ⬍ 0.01, ****P ⬍ 0.0001; numbers of individual observations and platedowns are as defined in Fig. 3, respectively. E: induced action
potential spike rate parameter plots for 34D6 neural precursors differentiated for 21 dpp using SCM1/2 (black bars), SCM-Base (light grey bars), or SFDM4 (dark
grey bars) showing depolarization rate (left), repolarization rate (middle), and half width (right). One-way ANOVA, Tukey’s post hoc test: *P ⬍ 0.05, **P ⬍
0.01; numbers of individual observations and platedowns are as defined in Fig. 3, respectively.

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS
rons (17/17) were coded as sAP-full and this high level of
activity was maintained through to 35 dpp (6/6); in SCM-Base,
the proportion of cells with spontaneous activity remained at 0
from 28 to 35 dpp (Fig. 3A). These data are consistent with the
observation that differentiation by each of the protocols gen-
erated populations of cells that had mean resting Vm values that
were significantly different (one-way ANOVA, P ⬍ 0.05) and
that SCM1/2 generated neurons with a relatively more hyper-
polarized Vm than did SCM-Base (P ⬍ 0.05; Fig. 3C). How-
ever, rather modest values of resting Vm, and the differences
observed between the populations as a whole, mask the real
differences which could be observed when the populations
were analyzed according to their sAP codes. Independently of
protocol, neurons coded as sAP-full were strikingly and signif-
icantly more hyperpolarized than those coded as sAP-at-
tempted (P ⬍ 0.0001) or sAP-none (P ⬍ 0.0001; Fig. 3C).
Thus the mean resting Vm for SCM1/2 neurons coded as
sAP-full was ⫺46.9 ⫾ 1.6 mV (n ⫽ 25), those coded sAP-
attempted was ⫺36.8 ⫾ 1.41 (n ⫽ 31), and those coded
sAP-none was ⫺32.6 ⫾ 1.71 mV (n ⫽ 53).
Similarly, 33Qn1 iPSC-derived neurons differentiated using
SCM1/2 demonstrated a substantially augmented proportion of
neurons showing spontaneous electrical activity than did those
differentiated in either SCM-Base or SFDM4 (Fig. 4, A and B).
Thus SCM1/2 generated 57% (39/69) of neurons, which were
coded as sAP-full, with 16% (11/69) sAP-attempted and 28%
(19/69) of sAP-none. Only 7% (2/29; SCM-Base) and 0%
(0/21; SFDM4) of neurons were coded as sAP-full, with 38%
(11/29; SCM-Base) and 10% (2/21; SFDM4) coded as sAP-
attempted. Likewise, differentiation of 33Qn1 NPCs using
each of the protocols generated populations of neurons that had
mean resting Vm values that were significantly different (one-
way ANOVA, P ⬍ 0.0001), with SCM1/2 generating neurons
with a relatively more hyperpolarized Vm than those differen-
tiated in SCM-Base (P ⬍ 0.0001; Fig. 4C). Analyses of 33Qn1
iPSC-derived neurons based on sAP codes demonstrated that
sAP-full were significantly more hyperpolarized than those
coded as sAP-attempted (P ⬍ 0.01) or sAP-none (P ⬍ 0.0001;
Fig. 4C). Thus, mean resting Vm for SCM1/2 neurons coded as
sAP-full was ⫺47.5 ⫾ 1.3 mV (n ⫽ 39), those coded sAP-
attempted was ⫺37.5 ⫾ 3.4 (n ⫽ 11), and those coded
C529
SCM-Base (63% 12/19) but was iAP-train for SCM1/2 (59%,
43/73). Where iAP-trains could be recorded, spike frequency
was much higher in 34D6 neurons differentiated in SCM1/2
than in either SCM-Base or SFDM4 (Fig. 5C), although there
was only a modest decrease in mean input resistance (Rin) in
the neurons differentiated in SCM1/2 media compared with
SCM-Base or SFDM4 (data not illustrated). Interestingly, in-
dependently of which protocol was employed to differentiate
the neurons, there was clear correlation between sAP code and
iAP code for each individual neuron but, most importantly,
SCM1/2 produced dramatically more sAP-full/iAP-train
(100%) than did either SFDM4 or SCM-Base (Table 2).
Examining all neurons within each protocol that fired at least
one iAP, deeper analysis of the first spike revealed a significant
effect of differentiation protocol on overshoot, afterhyperpo-
larization, amplitude, depolarization rate, and half-width (P ⬍
0.01 or lower by one-way ANOVA). Specifically, SCM1/2
generated neurons with significantly (P ⬍ 0.01) shortened
half-width compared with SCM-Base neurons (Table 3). As
was seen in the earlier Vm analysis (Fig. 3), some of these
differences appeared modest when considering the entire pop-
ulation of neurons differentiated with each protocol. However,
once neurons were analyzed according to their sAP code, there
was, by one-way ANOVA, a significant effect of sAP code on all
of the spike parameters, showing that sAP-full neurons had larger
and faster action potentials than those coded sAP-none (Table 3).
Since in 34D6-derived neurons SCM1/2 generated the highest
proportion of sAP-full neurons, it is clear that the SCM1/2 proto-
col forced more cells to express a mature neuronal phenotype,
synchronizing the maturation across a higher proportion of iPSC-
neurons, than did the SCM-Base or SFDM4 protocols.
Again, in agreement with the data from 34D6-derived neu-
rons, differentiating 33Qn1-derived NPCs using the SCM1/2
protocol increased the proportion of functionally active neu-
Table 2. Proportions and percentages of 34D6-derived
neurons that demonstrated each of the different induced
activity codes
sAP-None sAP-Attempted sAP-Full

sAP-none was ⫺34.5 ⫾ 1.6 mV (n ⫽ 19). The data obtained Proportion % Proportion % Proportion %
with Q33n1-derived neurons (Fig. 4) consistently mirrored SCM1/2
those described above for 34D6 iPSC-derived neurons (Fig. 3), iAP-none 2/22 9% 0/26 0% 0/25 0%
with SCM1/2 supporting an accelerated and more complete iAP- attempted single 2/22 9% 0/26 0% 0/25 0%
transition to functional maturity; a notion supported by the data iAP-single 5/22 23% 0/26 0% 0/25 0%
iAP-attempted train 8/22 36% 13/26 50% 0/25 0%
shown in Figs. 3C and 4C, which plot resting Vm against sAP iAP-train 5/22 23% 13/26 50% 25/25 100%
code for each different differentiation protocol at 21 dpp and SCM-Base
demonstrate clearly how the SCM1/2 protocol generates many iAP-none 2/17 12% 0/1 0% 0/1 0%
more sAP-full and sAP-attempted coded neurons than do the iAP-attempted single 1/17 6% 0/1 0% 0/1 0%
iAP-single 12/17 71% 0/1 0% 0/1 0%
other two protocols. iAP-attempted train 1/17 6% 1/1 100% 1/1 100%
The ability of the SCM1/2 protocol to generate a larger iAP-train 1/17 6% 0/1 0% 0/1 0%
proportion of functionally active neurons than the control SFDM4
protocols was also dramatically reflected in the quantity and iAP-none 4/15 27% 0/4 0% 0/0 0%
quality of iAPs. Once again, the SCM1/2 protocol was seen to iAP-attempted single 0/15 0% 1/4 25% 0/0 0%
iAP-single 10/15 67% 2/4 50% 0/0 0%
promote a more mature neuronal phenotype, as demonstrated iAP-attempted train 0/15 0% 0/4 0% 0/0 0%
by the larger proportion of 34D6-derived neurons that exhib- iAP-train 1/15 7% 1/4 25% 0/0 0%
ited iAP-train activity in the SCM1/2 protocol (59%, 43/73)
Proportions and percentages of 34D6-derived neurons that demonstrated
than in either the SCM-Base (5%, 1/19) or the SFDM4 (11%, each of the different induced activity codes (iAP), split according to their
2/19) protocols (Fig. 5A and exemplified in Fig. 5B); indeed, spontaneous activity codes (sAP) following differentiation in SCM1/2, SCM-
the modal pattern was iAP-single for SFDM4 (63%, 12/19) and Base, or SFDM4 at 21 days postplate-down (dpp).

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C530 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

Table 3. Analysis of passive and active parameters of induced action potentials of 34d6 IPSCs differentiated using SCM1/2,
SCM-Base, and SFDM4 protocols at 21 dpp
SCM 1/2 (n‡ ⫽ 73) SCM-Base (n‡ ⫽ 19) SFDM4 (n‡ ⫽ 19)

Action Potential Parameters Mean SE n Mean SE n Mean SE n

sAP-full
Passive
Vm, mV ⫺46.8 1.7 25 ⫺49.7 — 1 — — —
Rin, G⍀ 0.8 0.1 19 0.3 — 1 — — —
Cp, pF 18.9 1.7 25 4.1 — 1 — — —
Spike analysis
Threshold, mV ⫺39.6 1.0 24 ⫺41.1 — 1 — — —
Overshoot, mV 22.3 2.4 24 1.9 — 1 — — —
Afterhyperpolarization, mV ⫺56.3 1.2 24 ⫺78.9 — 1 — — —
Amplitude, mV 78.6 3.0 24 80.8 — 1 — — —
Depolarization rate, V/s 79.1 7.4 24 50.4 — 1 — — —
Repolarization rate, V/s ⫺24.9 11.4 24 ⫺33.6 — 1 — — —
Half width, ms 3.2 0.2 24 2.7 — 1 — — —
sAP-attempted
Passive
Vm, mV ⫺35.5 1.5 26 ⫺43.4 — 1 ⫺43.2 2.6 4
Rin, G⍀ 0.8 0.1 20 1.7 — 1 1.6 0.4 4
Cp, pF 14.6 1.6 24 7.8 — 1 8.4 1.6 4
Spike analysis
Threshold, mV ⫺40.0 1.3 26 ⫺49.3 — 1 ⫺35.4 3.6 3
Overshoot, mV 12.6 2.0 26 13.0 — 1 6.0 5.4 3
Afterhyperpolarization, mV ⫺52.4 1.6 26 ⫺55.0 — 1 ⫺38.5 8.1 3
Amplitude, mV 65.0 3.2 26 67.9 — 1 44.5 13.2 3
Depolarization rate, V/s 58.1 9.1 26 50.1 — 1 37.1 8.9 3
Repolarization rate, V/s ⫺23.2 4.0 26 ⫺13.2 — 1 ⫺23.9 2.8 3
Half width, ms 4.2 0.3 26 4.6 — 1 5.9 0.5 3
sAP-none
Passive
Vm, mV ⫺33.6 1.7 22 ⫺30.8 1.6 17 ⫺31.5 2.5 15
Rin, G⍀ 0.9 0.1 20 0.9 0.1 17 0.9 0.1 14
Cp, pF 17.5 2.3 20 6.5 0.8 17 10.9 1.3 12
Spike analysis
Threshold, mV ⫺35.1 1.9 19 ⫺39.7 2.0 15 ⫺39.8 2.4 10
Overshoot, mV 12.4 2.4 19 9.2 3.1 15 4.1 1.3 11
Afterhyperpolarization, mV ⫺52.6 1.6 19 ⫺45.5 4.1 15 ⫺34.8 5.2 11
Amplitude, mV 65.0 3.3 19 54.7 6.6 15 39.0 6.2 11
Depolarization rate, V/s 36.3 6.4 19 34.5 5.8 15 27.2 6.8 11
Repolarization rate, V/s ⫺18.1 2.7 19 ⫺13.7 3.4 15 ⫺18.4 6.0 11
Half width, ms 4.8 0.5 19 6.0 0.8 15 5.6 0.9 11
The parameters obtained for each set of neurons has been subdivided according to their sAP coding: sAP-full, sAP-attempted, or sAP-none. Vm, resting
membrane potential; Rin, input resistance; Cp, whole cell capacitance. n‡ ⫽ Total number of cells recorded; n ⫽ number of cells falling in each category.

rons compared with those differentiated using the control
protocols. SCM1/2-treated 33Qn1 cells exhibited 90% (37/41)
iAP-train activity, which was much higher than observed in
SCM-Base (10%, 16/230) or in SFDM4 (37%, 7/19) (Fig. 6A
and exemplified in Fig. 6B). Likewise, where iAP-trains could be
recorded in 33Qn1-derived neurons, spike frequency was much
higher in SCM1/2 than in either SCM-Base or SFDM4 (Fig. 6C).
Again, independently of which protocol was employed to differ-
entiate the 33Qn1 NPCs, there was clear correlation between sAP
code and iAP code for each individual neuron and as previously
seen in the 34D6 neurons, SCM1/2 produced dramatically more
sAP-full/iAP-train than SFDM4 and SCM-Base (Table 4). Spike
analyses of the neurons (Fig. 6, D and E) according to their sAP
code by one-way ANOVA displayed a significant effect of sAP
code mostly on amplitude parameters and depolarization rate of
iAP, showing that sAP-full neurons had larger and faster action
potentials than those coded sAP (Table 5).
In 34D6 neurons, there was a significant effect of differen-
tiation protocol (P ⬍ 0.0001) on mean cell capacitance (Cp),
with the SCM1/2 protocol generating neurons with signifi-
cantly higher Cp, at 17.0 ⫾ 1.1 pF, than those differentiated in
either SCM-Base (6.4 ⫾ 0.7 pF; P ⬍ 0.0001) or SFDM4
(10.3 ⫾ 1.1 pF; P ⬍ 0.001). Surprisingly, neither Na⫹ nor K⫹
current densities were significantly affected by the differenti-
ation protocol (Fig. 7, A and B). However, the SCM1/2 proto-
col generated neurons that displayed significant differences in
the activation/inactivation profiles of the voltage-activated
Na⫹ currents, resulting in SCM1/2 neurons exhibiting larger
availability windows with high G/Gmax maxima and a higher
proportion of neurons with Vm values falling within those
windows than did either SCM-Base or SFDM4 neurons (Fig.
7C). Strikingly, neurons differentiated by the SCM1/2 protocol
exhibited a very small mean difference between half-activation
and half-inhibition voltages (Va50 ⫺ Vi50 ⫽ 7.1 ⫾ 1.2 mV; an
inverse measure of Na⫹ current availability), which was sig-
nificantly lower than values for either SCM-Base (23.6 ⫾ 2.4
mV; P ⬍ 0.0001) or SFDM4 neurons (17.9 ⫾ 2.9 mV);
SCM-Base and SFDM4 were not different.
Similarly, 33Qn1-derived neurons differentiated using any
of the three protocols also resulted in no significant differences

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS C531

A B SCM1/2 iAP
C
8

Spikes/s
SCM1/2 iAP coding at 21 dpp 80 SFDM4

Voltage (mV)
60 SCM-Base
100 ms SCM1/2
40 6
20
0 4
-20
-40
sAP-Train 2
-60
sAP-Single
-80
-100 0
0 20 40 60 80 100 120 140 160 180
Injected current (pA)
SCM-Base iAP D
80
Voltage (mV)

Afterhyperpolarization (mV)
SCM-Base iAP coding at 21 dpp

Overshoot (mV)
50 * 0 120 *

Amplitude (mV)
60 ns ns
100 ms ** 100
*
40 40
-20
20 80
30
0 -40 60
-20 20
40
-40 -60
sAP-Train 10 20
ns ns
-60
sAP-Attempted Train 0 -80
ns
0
-80

SF ase
SF ase

SF ase
sAP-Single

SC 1/2

4
SC 1/2

SC 1/2
-B
4

4
-B

-B
sAP-Attempted Single -100

M
M

M
M
M

D
M

M
M

M
D

D
SC
SC

SC
sAP-None
SFDM4 iAP E
80
Voltage (mV)

* *

Depolarization rate (V/s)

120

Half width (ms)

3

Repolarization rate (V/s)

SFDM4 iAP coding at 21 dpp 0 ns ns
60 ns ns
40 100
100 ms -20
20 80 2
0 60 -40
-20
40 1
-40 -60
ns ns
sAP-Train -60 20
sAP-Attempted Train *
-80 0 -80 0
SF ase

SF ase

SF ase
sAP-Single -100
SC 1/2

SC 1/2

SC 1/2
4

4
-B

-B

-B
M

M
sAP-Attempted Single
M
M

M
M

M
D

D
SC

SC

SC
sAP-None
Fig. 6. SCM1/2 protocol enhances the generation of 33Qn1-derived neurons exhibiting induced action potential trains and augments their spike characteristics.
A: pie charts showing the proportion of neurons that exhibit trains of induced action potentials (iAP-train, dark green), attempted trains of action potentials
(iAP-attempted train, light green), single induced action potential (iAP-single, orange), attempted single induced action potential (iAP-attempted single, pink),
or no induced action potentials (iAP-none, red) in 34D6-derived neuronal precursors differentiated into neurons for 21 dpp using SCM1/2, SCM-Base, or SFDM4
protocols. Numbers of individual observations and platedowns are as defined in Fig. 4. B: current-clamp recordings when neurons were held at between ⫺70
and ⫺80 mV and to 1-s injected currents of ⫺10 pA (light grey) and ⫹20 pA (medium grey) and the current producing the maximum iAP activity (black)
exemplifying the model activity for 33Qn1-derived neuroprecursors differentiated into neurons for 21 dpp using SCM1/2 (top), SCM-Base (middle), or SFDM4
(bottom) protocol; expanded time-base sections of traces are shown below each main trace. C: spike frequency plots for neurons exhibiting trains of iAPs
following differentiation of 33Qn1 neural precursors using SCM1/2 (closed black squares), SCM-Base (closed grey squares), or SFDM4 (open black squares).
D: induced action potential spike size parameter plots for 33Qn1 neural precursors differentiated for 21 dpp using SCM1/2 (black bars), SCM-Base (light grey
bars), or SFDM4 (dark grey bars) showing overshoot (left), afterhyperpolarization (middle), and full amplitude (right). One-way ANOVA, Tukey’s post hoc test:
*P ⬍ 0.05, **P ⬍ 0.01; numbers of individual observations and platedowns are as defined in Fig. 4. E: induced action potential spike rate parameter plots for
33Qn1 neural precursors differentiated for 21 dpp using SCM1/2 (black bars), SCM-Base (light grey bars), or SFDM4 (dark grey bars) showing depolarization
rate (left), repolarization rate (middle), and half width (right). One-way ANOVA, Tukey’s post hoc test: *P ⬍ 0.05; numbers of individual observations and
platedowns are as defined in Fig. 4.

in the maximal voltage-activated Na⫹ current densities of the
neurons (Fig. 8, A and B). Likewise, the maximal K⫹ current
densities of 33Qn1 neurons differentiated using SCM1/2 and
SCM-Base were not significantly different. However, the max-
imal K⫹ current densities of 33Qn1 neurons differentiated
using SFDM4 was significantly smaller, which may reflect the
lower variance observed in these experiments. Differences in
voltage-activated current magnitudes of the 33Qn1 neurons
notwithstanding, higher excitability in SCM1/2 was, like in the
34D6 cohort, accompanied by an increased percentage of
neurons with Vm values fitting within the Na⫹ current avail-
ability window than in SCM-Base and SFDM4. (Fig. 8C).
Taken together, these data suggest that the SCM1/2 protocol
enhances functional maturation by two major biophysical en-
hancements: hyperpolarizing the resting Vm to enable higher
spontaneous activity and increasing the Na⫹ current availabil-
ity to facilitate regenerative iAP-train activity.
Spontaneous Neural Network Activity Develops by 21 Days
of Differentiation in SCM1/2
In addition to the hyperpolarizing influence of SCM1/2
media, sAP activity may also reflect the rate and extent of
synaptogenesis and functional neural network activity. To
examine whether each protocol was able to support functional

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C532 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

Table 4. Proportions and percentages of 33Qn1-derived neurons that demonstrated each of the different induced activity
codes
sAP-None sAP-Attempted sAP-Full

Proportion % Proportion % Proportion %

SCM1/2
iAP-none 0/8 0% 0/7 0% 0/26 0%
iAP- attempted single 0/8 0% 0/7 0% 0/26 0%
iAP-single 4/8 50% 0/7 0% 0/26 0%
iAP-attempted train 0/8 0% 0/7 0% 0/26 0%
iAP-train 4/8 50% 7/7 100% 26/26 100%
SCM-Base
iAP-none 1/13 8% 0/9 0% 0/1 0%
iAP- attempted single 1/13 8% 0/9 0% 0/1 0%
iAP-single 1/13 8% 1/9 11% 0/1 0%
iAP-attempted train 3/13 23% 0/9 0% 0/1 0%
iAP-train 7/13 53% 8/9 89% 1/1 100%
SFDM4
iAP-none 2/17 12% 0/2 0% 0/0 0%
iAP- attempted single 2/17 12% 0/2 0% 0/0 0%
iAP-single 5/17 29% 0/2 0% 0/0 0%
iAP-attempted train 3/17 18% 0/2 0% 0/0 0%
iAP-train 5/17 29% 2/2 100% 0/0 0%
Proportions and percentages of 33Qn1-derived neurons that demonstrated each of the different iAP codes, split according to their sAP codes following
differentiation in SCM1/2, SCM-Base, or SFDM4 at 21 dpp.

synaptogenesis, the generation of spontaneous synaptic cur-
rents and the exhibition of evoked postsynaptic currents and/or
neurotransmitter-evoked changes in intracellular calcium con-
centration ([Ca2⫹]i) were determined in neurons differentiated
from the two cell lines 34D6 and 33Qn1.
Neurons differentiated from either line using either the
SFDM4 protocol for 21 dpp never exhibited miniature synaptic
currents. The SCM-Base protocol only rarely supported the
generation of GABAergic minis and then only in Q33n1
neurons (2 of 14 cells). In contrast, between 30 and 75% of
neurons differentiated from either cell line by the SCM1/2
protocol exhibited robust GABAergic and modest glutamater-
gic minis (Fig. 9). Thus the mean amplitude of spontaneous
GABAergic synaptic events recorded at ⫺40 mV was 6.3 ⫾
0.8 pA (n ⫽ 3/11) and 13.9 ⫾ 1.4 pA (n ⫽ 15/17), with
interevent interval values of 3.7 ⫾ 0.6 s (n ⫽ 3/11) and 0.5 ⫾
0.1 s (n ⫽ 15/17), for 34D6- and 33Qn1-derived neurons,
respectively; these currents were effectively and reversibly
blocked by 10 ␮M bicuculline (Fig. 9A). These observations
are consistent with the almost uniform GABA immunostain-
ing, as shown in Fig. 2G. The very low number of glutama-
tergic events observed in both lines differentiated using any
protocol made robust analysis unreliable; this is not surprising
since the prepatterning protocol using SLI/SL media was
actually designed to generate a preponderance of GABAergic
ventral forebrain NPCs.
GABA (100 ␮M) evoked inhibitory postsynaptic currents in
100% of 33Qn1-derived neurons differentiated in SCM1/
SCM2, SCM-Base, or SFDM4 with mean current densities
at ⫺40 mV of 50.5 ⫾ 6.7 pA/pF (n ⫽ 16/16), 50.6 ⫾ 5.8
pA/pF (n ⫽ 13/13), and 29.0 ⫾ 4.8 pA/pF (n ⫽ 9/9), respec-
tively (Fig. 9B). Similarly, 100% of 34D6-derived neurons
differentiated using SCM1/2 showed inhibitory postsynaptic
currents with mean current densities at ⫺40 mV of 41.1 ⫾ 5.3
pA/pF (n ⫽ 18/18) (data not shown).
In 33Qn1-derived neurons differentiated using the SCM1/2,
SCM-Base, or SFDM4 protocol, 5-s applications of 100 mM
NMDA/10 mM glycine evoked postsynaptic currents with
mean current densities at ⫺40 mV of ⫺2.4 ⫾ 0.6 pA/pF (n ⫽
15/16), ⫺1.5 ⫾ 0.3 pA/pF (10/13), and ⫺1.1 ⫾ 0.3 pA/pF
(4/12), respectively (Fig. 10A). In comparison, after differen-
tiation in SCM1/2, the mean current density of the NMDA-
evoked postsynaptic currents in 34D6 neurons was ⫺1.7 ⫾ 0.5
pA/pF (n ⫽ 3/4) (data not shown). Further evidence for
NMDA receptor expression is shown in Fig. 10B, which shows
punctate, presumably synaptic, staining of NR1, NR2A, and
NR2B NMDA receptor subunits. The idea that the SCM1/2
protocol is prosynaptogenic was supported by the SEM data
that showed highly complex arborizing neuronal networks,
demonstrating distinct dendritic spines at high power (Fig.
10C), and by extensive staining of the pre- and postsynaptic
markers synaptophysin and PSD95, with evidence of their
coregistration indicating apposition of pre- and postsynaptic
terminals (Fig. 10D).
SCM1/2 Protocol Promotes Maturation of Calcium Signaling
in iPSC-Derived Neurons
Concentrating on the nonintegrating iPSCs, Ca2⫹ imaging of
33Qn1 neurons that had been differentiated using the three
different protocols revealed markedly faster maturation rates in
cells differentiated using the SCM1/2 protocol (Fig. 11, A and
B). The neurons were imaged at different time points after
plating, and the responses to high potassium (60 mM KCl, a
depolarizing stimulus), 300 ␮M glutamate/30 ␮M glycine, and
300 ␮M GABA in normal and low (7.5 mM) extracellular
chloride were recorded (sample traces for SCM1/2 neurons are
presented in Fig. 11C). At the day of the plating, 0 dpp, only
10.9 ⫾ 3.8% of cells responded to high potassium, 5.7 ⫾ 5.7%
of cells responded to glutamate, and 1.0 ⫾ 1.3% of cells
responded to GABA in low chloride (Fig. 11D, D0, n ⫽ 4).
Even by the end of week 1 postplating (7 dpp), the vast
majority of the cells from all three protocols responded to the
high potassium challenge, demonstrating the early functional

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS C533
Table 5. Analysis of passive and active parameters of induced action potentials of 33Qn1-derived neurons differentiated
using SCM 1/2, SCM-Base, and SFDM4 protocols at 21 dpp
SCM1/2 (n‡ ⫽ 69) SCM-Base (n‡ ⫽ 29) SFDM4 (n‡ ⫽ 21)

Action Potential Parameters Mean SE n Mean SE n Mean SE n

sAP-full
Passive
Vm, mV ⫺47.5 1.3 39 ⫺53.7 — 2 — — —
Rin, G⍀ 0.6 0.1 32 0.6 — 1 — — —
Cp, pF 19.7 1.6 34 9.0 — 1 — — —
Spike analysis
Threshold, mV ⫺37.9 1.6 26 ⫺36.7 — 1 — — —
Overshoot, mV 40.7 2.9 26 39.0 — 1 — — —
Afterhyperpolarization, mV ⫺56.6 2.1 26 ⫺59.8 — 1 — — —
Amplitude, mV 97.3 3.5 26 98.8 — 1 — — —
Depolarization rate, V/s 101.2 6.3 26 117.5 — 1 — — —
Repolarization rate, V/s ⫺59.9 5.5 26 ⫺56.5 — 1 — — —
Half width, ms 2.0 0.1 26 1.9 — 1 — — —
sAP-attempted
Passive
Vm, mV ⫺37.5 3.4 11 ⫺36.9 2.1 11 ⫺32.5 — 2
Rin, G⍀ 0.9 0.2 11 0.6 0.1 9 0.9 — 2
Cp, pF 15.6 2.7 10 17.1 1.5 9 29.9 — 2
Spike analysis
Threshold, mV ⫺38.3 2.4 7 ⫺38.3 2.0 9 ⫺26.9 — 2
Overshoot, mV 39.4 5.2 7 24.7 4.4 9 38.2 — 2
Afterhyperpolarization, mV ⫺54.6 2.3 7 ⫺58.6 1.7 9 ⫺54.9 — 2
Amplitude, mV 93.9 6.5 7 83.3 4.6 9 93.2 — 2
Depolarization rate, V/s 99.0 16.0 7 85.9 12.1 9 98.1 — 2
Repolarization rate, V/s ⫺61.8 10.8 7 ⫺45.6 5.1 9 ⫺49.7 — 2
Half width, ms 1.8 0.2 7 2.2 0.2 9 2.0 — 2
sAP-none
Passive
Vm, mV ⫺34.5 1.6 19 ⫺27.7 1.5 16 ⫺23.8 1.4 19
Rin, G⍀ 1.0 0.1 15 0.5 0.1 13 0.6 0.1 17
Cp, pF 16.4 3.0 12 13.3 1.6 9 13.4 2.2 12
Spike analysis
Threshold, mV ⫺36.9 4.0 8 ⫺37.1 2.5 11 ⫺33.0 1.8 13
Overshoot, mV 21.4 6.3 8 24.7 3.3 11 23.1 4.1 13
Afterhyperpolarization, mV ⫺53.9 5.1 8 ⫺55.1 2.9 11 ⫺53.6 2.5 13
Amplitude, mV 75.2 7.5 8 79.8 5.9 11 76.6 6.1 13
Depolarization rate, V/s 64.5 12.2 8 79.5 8.4 11 65.2 10.3 13
Repolarization rate, V/s ⫺37.6 5.1 8 ⫺41.8 4.8 11 ⫺38.0 5.6 13
Half width, ms 2.4 0.2 8 2.3 0.2 11 2.6 0.2 13
The parameters obtained for each set of neurons has been subdivided according to their sAP coding: sAP-full, sAP-attempted, or sAP-none. n‡ ⫽ Total number
of cells recorded; n ⫽ number of cells falling in each category.

expression of voltage-gated Ca2⫹ channels at this time point
(n ⫽ 6 –11; Fig. 11A, top). However, by week 2 the intensity of
the responses to high potassium was significantly lower for
neurons differentiated using both SFDM4 and SCM-Base neu-
rons than it was for neurons differentiated using SCM1/2 (n ⫽
8 –13), and the difference remained significant through to 3
wk (n ⫽ 7–14; Fig. 11B, top). The percentage of cells
responding to 300 ␮M glutamate/30 ␮M glycine was sig-
nificantly higher for SCM1/2 than for SFDM4 and SCM-
Base neurons at weeks 1 and 3 postplating (Fig. 11A,
middle), and the intensity of the responses was also signif-
icantly higher for SCM1/2 cohort than SFDM4 and SCM-
Base at weeks 2 and 3 postplating (Fig. 11B, middle). The
percentage of the cells responding to GABA in low extra-
cellular chloride remained significantly lower for SFDM4
and SCM-Base treatments compared with SCM1/2 neurons
during the whole differentiation period, and reached only
56.5 ⫾ 12.0% for SFDM4, 72.0 ⫾ 10.6% for SCM-Base
neurons, but 98.8 ⫾ 0.7% for SCM1/2 neurons at week 3
(Fig. 11A, bottom). The changes in the intensity of the
responses to GABA in low chloride over the maturation
period were similar to those seen in response to high
potassium, being lower for both SFDM4 and SCM-Base
neurons than for SCM1/2 neurons at weeks 2 and 3 post-
plating (Fig. 11B, bottom).
Since SCM1/2 supported much faster neuronal maturation, we
performed day-to-day imaging of the responses during the first 10
days and then at 14 and 21 dpp for this cohort. Ca2⫹ imaging of
33Qn1 cells differentiated using the SCM1/2 protocol revealed
that cells responded remarkably consistently to neuronal stimuli,
producing an almost homogenous pattern of responses across the
entire population by 21 dpp (n ⫽ 4 –14; Fig. 11C). Specifically,
the response to high potassium evoked Ca2⫹ influx in 36.2 ⫾
12.2% of cells at 1 dpp; this proportion rose to 97.6 ⫾ 1.6% by 6
dpp and was at 99.8 ⫾ 0.2% by 14 dpp, where it remained
thereafter (Fig. 11D). Moreover, the magnitude of the Ca2⫹ influx
also rose during differentiation and reached a plateau at 14 dpp
(Fig. 11E). The proportion of cells responding to glutamate/
glycine application also increased in a time-dependent manner
from 3.50 ⫾ 2.2% at 1 dpp to 96.9 ⫾ 1.5% at 9 dpp (Fig. 11D).

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C534 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

A SCM1/2 B SCM1/2
C SCM1/2

G/Gmax
Current Density
1.0
250
-47.2 mV

(pA/pF)
200 0.8
150
100 0.6
1 nA
50
0.4
0.
50 ms
-120
-100 -80 -60 -40 -20 20 40 60
-50 0.2
Voltage (mV)
-100
-150
0.0
-120 -100 -80 -60 -40 -20 0
-200
Voltage (mV) Vm = -39.0 ± 1.1 mV (n = 75)

G/Gmax
SCM-Base SCM-Base 1.0

Current Density
250 SCM-Base

(pA/pF)
200 0.8
-57.3 mV
150
100 0.6
1 nA 50
0.4
50 ms -120 -100 -80 -60 -40 -20 20 40 60
-50 0.2
0.
-100
Voltage (mV)
-150
0.0
-120 -100 -80 -60 -40 -20 0
-200 Voltage (mV) Vm = -33.7 ± 1.7 mV (n = 28)
SFDM4 SFDM4
Current Density

SFDM4 1.0

G/Gmax
250
-44.9 mV
(pA/pF)

200 0.8
150
1 nA
100 0.6
50 ms
Voltage (mV)

50
0.4
0
-50 -120 -100 -80 -60 -40 -20 20 40 60
-50 0.2
0.
-100 Voltage (mV)
-100
-150
0.0
0 100 200 300 400 -120 -100 -80 -60 -40 -20 0
-200 Vm = -34.0 ± 2.3 mV (n = 19)
Time (ms) Voltage (mV)
Fig. 7. SCM1/2 protocol augments voltage-activated Na⫹ current availability in 34D6-derived neurons. A: exemplar families of whole cell currents evoked by
the voltage activation/inactivation protocol (as shown below the lower, SFDM4, trace) in 34D6-derived neuronal precursors differentiated into neurons for 21
dpp using SCM1/2 (top), SCM-Base (middle), or SFDM4 (bottom) protocols. B: mean current density vs. voltage plots derived for maximum voltage-activated
Na⫹ (circles) and K⫹ currents (squares) derived from traces exemplified in A. C: mean fractional conductance (G/Gmax) plots for voltage activation (closed
squares) and inactivation (open circles) of Na⫹ currents derived from traces exemplified in A. Peak G/Gmax is indicated by the arrow. Individual Vm values are
plotted on each graph by the open triangles.

The magnitude of glutamate-evoked Ca2⫹ influx also increased
with time to 21 dpp (Fig. 11E). Although the time-dependent
changes in responses to GABA application were broadly similar
to those evoked by glutamate or high potassium, the analysis of
the responses was more complex. In physiological extracellular
solution, a GABA-evoked rise in Ca2⫹ can be due to 1) GABAA-
evoked depolarization-dependent opening of voltage-activated
Ca2⫹ channels, and 2) GABAB-evoked Ca2⫹ release (38). In
physiological solution, GABA evoked Ca2⫹ influx in 28.1 ⫾
12.6% of cells at 1 dpp, which rose to 95.1 ⫾ 4.1% by 9 dpp.
Although the magnitude of the GABA responses increased in a
time-dependent manner, it was more than 10-fold lower than
when GABA was applied in the presence of low chloride con-
centration, suggesting a major and increasing role for GABAA. In
addition, lastly, despite the percentage of cells responding to
GABA in low extracellular chloride remained ⬃99% at 14
and 21 dpp, the percentage of cells responding to GABA in
normal extracellular chloride dropped from 62 ⫾ 9.4% at 14
dpp to 31.6 ⫾ 9.8% at 21 dpp (P ⬍ 0.05), perhaps indicating
increased amount of inhibitory GABA receptors signaling
towards the end of the SCM1/2 maturation protocol (Fig.
11D).
Evidence that the neurons formed excitatory synaptic net-
works whose activity was limited by an ongoing inhibitory
GABAergic tone, indicative of a mature GABAA phenotype,
was provided by experiments using multiwell MEA plates
(Fig. 12). Glutamate (3–15 ␮M) and NMDA (5 ␮M) both
produced transient increases in neuronal firing when applied to
neurons differentiated with the SCM1/2 protocol (Fig. 12, B
and C, and Table 6). The transient nature was most likely due
to depolarizing block. Application of 5 ␮M gabazine, a phar-
macological blocker of GABAA receptors, resulted in a sub-
stantial and maintained increase in the rate of neuronal firing
(Fig. 12D). Application of TTX (500 nM) eliminated all
spiking activity (Fig. 12B and Table 6). In total, increased
spiking activity was detected from 23 electrodes (4 wells) in
the presence of glutamate, 29 electrodes (12 wells) in the
presence of NMDA, and 20 electrodes (5 wells) in the presence
of gabazine. Decreased activity was detected from 13 elec-
trodes (5 wells) in the presence of TTX.

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS C535

A B C
SCM1/2

Current Density
(pA/pF)
SCM1/2 SCM1/2

G/Gmax
1.0
600 -45.0 mV
0.8
400
0.6
200
2 nA 0.4
-120-100 -80 -60 -40 -20 20 40 60
100 ms -200 0.2
Voltage (mV)
-400 0.0
-600 -120 -100 -80 -60 -40 -20 0
Voltage (mV) Vm = -42.2 ± 1.2 (n = 71)

Current Density
(pA/pF)
SCM-Base SCM-Base SCM-Base

G/Gmax
1.0
600 -50 mV
0.8
400
2 nA 0.6
200

100 ms 0.4
-120-100 -80 -60 -40 -20 20 40 60
-200 0.2
Voltage (mV)
-400
0.0
-600 -120 -100 -80
-60 -40 -20 0
Voltage (mV) Vm = -33.0 + 1.8 (n = 29)
SFDM4 SFDM4 SFDM4
Current Density
(pA/pF)

1.0

G/Gmax
-45.0 mV
2 nA 600 0.8
400
0.6
100 ms
200
Voltage (mV)

0.4
0
-50 -120-100 -80 -60 -40 -20 20 40 60 0.2
-200 Voltage (mV)
-100
-400 0.0
0 100 200 300 400 -120 -100 -80 -60 -40 -20 0
Time (ms) -600 Voltage (mV) Vm = -24.6 + 1.4 (n = 21)
⫹
Fig. 8. SCM1/2 protocol augments voltage-activated Na current availability 33Qn1-derived neurons. A: exemplar families of whole cell currents evoked by the
voltage activation/inactivation protocol (as shown below the lower, SFDM4, trace) in 34D6-derived neuronal precursors differentiated into neurons for 21 dpp
using SCM1/2 (top), SCM-Base (middle), or SFDM4 (bottom) protocols. B: mean current density vs. voltage plots derived for maximum voltage-activated Na⫹
(circles) and K⫹ currents (squares) derived from traces exemplified in A. C: mean fractional conductance (G/Gmax) plots for voltage activation (closed squares)
and inactivation (open circles) of Na⫹ currents derived from traces exemplified in A. Peak G/Gmax is indicated by the arrow. Individual Vm values are plotted
on each graph by the open triangles.

DISCUSSION large GABA and glutamate postsynaptic currents; and 10)

To address functional deficits commonly seen in hPSC-
derived neurons differentiated under established neural culture
conditions, we have established a new protocol that both
synchronizes the neurogenesis of iPSC-derived neural progen-
itors and accelerates their functional maturation. This rapid
prosynaptogenic, in vitro differentiation protocol generates
authentic, mature synaptic networks of connected forebrain
neurons from a variety of iPSCs. Taken together, the data
presented herein show that, compared with standard neuronal
differentiation methods, the SCM1/2 protocol enhances the
rate and extent of synchronized maturation of iPSC-derived
neurons (34D6 and 33Qn1), generating neurons with 1) accel-
erated neurite extension; 2) increased cell capacitance; 3)
augmented CREB and ERK pathways activation; 4) relatively
hyperpolarized resting Vm; 5) higher spontaneous action po-
tential activity; 6) increased regenerative induced action po-
tential train activity; 7) larger Na⫹ current availability; 8)
augmented spontaneous GABA miniature synaptic currents; 9)
rapid and uniform development of a mature, inhibitory
GABAA receptor phenotype, which, when blocked, results in
dramatically augmented neuronal network activity. However,
although the SCM1/2 protocol provides optimal conditions for
the differentiation and maturation of neurons, it does not
address their long-term maintenance requirements. Recently, a
novel medium named “BrainPhys” has been optimized for the
long-term maintenance of neurons in culture (2) and this may
usefully provide a third medium for use in conjunction with
SCM1 and SCM2.
Since neurogenesis is a multistep process, characterized by
sequential phases of differentiation and maturation, we aimed
to improve NPC differentiation by developing two media for
sequential use. Culture in SCM1 medium for 1 wk was de-
signed to support better the early neurogenic phase of differ-
entiation, after which SCM2 medium supported further neuro-
nal maturation and synaptogenesis. SCM1 used as its base a
medium similar to the classical DMEM:F-12 medium with N2

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C536 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

A Bicuculline (10 µM) B GABA (100 µM)

20 pA

0.5 s 0.5 nA

5s

GABA minis GABA minis GABA IPSP

*
ns
*
20 * 5 **** 60

Current density (pA/pF)

Inter-event interval (s)
Amplitude (pA)

4
15
40
3
10
2
20
5
1

0 0 0

e
2

2
2

4
as
1/

1/
1/

1/

1/

M
M

M
M

-B

D
SC

SC
SC

SC

SC

SF
M
SC
6

n1
6

n1

n1
n1
D
D

Q
Q

n1

Q
Q
34
34

33
33

33
33

Q
33
Fig. 9. SCM1/2 protocol generates neurons characterized by spontaneous and induced postsynaptic neurotransmitter currents and maturation of inhibitory
GABAA responses in 34D6- and 33Qn1-derived neurons. A: exemplar spontaneous miniature GABAA current trace recorded from 34D6-derived neuronal
precursors differentiated into neurons for 21 dpp using SCM1/2, before and during application of 10 ␮M bicuculline, as indicated by the horizontal bar (top).
Mean amplitude (bottom left) and interepisode intervals (bottom right) of miniature synaptic GABAA currents recorded in neurons differentiated for 21 dpp from
2 separate iPSC lines, 34D6 and 33Qn1. B: exemplar (top) and mean (bottom) GABA-evoked postsynaptic inhibitory postsynaptic currents recorded from
33Qn1-derived neuronal precursors differentiated into neurons for 21 dpp using the SCM1/2 protocol, SCM-Base, or SFDM4 protocol.

and B27 supplements (12). SCM2 used the same base-medium Plating D16 NPCs in medium with 10 ␮M DAPT caused a
mixed 1:1 with Neurobasal A. Both SCM1 and SCM2 media significant reduction in cell proliferation; however, this was
included BDNF and a small molecule TrkB pathway agonist, insufficient to synchronize cell cycle exit, as seen by the
LM22A4 (32), to provide neurotrophic support to differentiat- progressive rather than abrupt loss of MKI67 immunostain-
ing neurons. Preliminary studies indicated poor neuronal sur- ing over 7 days (Fig. 1A). To complement the action of
vival at 21 dpp without neurotrophic support and that BDNF DAPT we also used PD0332991, a small molecule inhibitor
and LM22A4 promote neurite outgrowth and neuronal sur- of the cyclin-dependent kinase 4/6 that blocks progression
vival. Although LM22A4 could substitute for BDNF (data not through the G1/S checkpoint and would therefore force cell
shown), in our experiments we maintained both compounds in cycle exit (10, 50).
both media throughout the protocol. Besides the suppression of Notch effectors that maintain a
To address the stochastic nature of NPC differentiation, and proliferative progenitor state, developmental mechanisms pro-
problems in defining neuronal age associated with continued mote structural and functional programs of neuronal develop-
NPC proliferation, we first synchronized NPC neurogenesis. A ment and synaptogenesis. We previously analyzed the mecha-
critical step in the transition from neural progenitor to neuron nistic basis for astrocyte-enhanced differentiation and matura-
is the lengthening of the cell cycle, leading to cell cycle exit tion of iPSC-derived NPCs and identified critical roles for
(28, 41); therefore, we used two small molecules, DAPT and GABAA-dependent augmentation of voltage-gated calcium
PD0332991, to promote cell cycle exit and block cell cycle channel function (45). Since the neuronal maturation-promot-
progression. Previous studies have used ␥-secretase inhibitors ing properties of astrocyte conditioned medium could be mim-
(exemplified by DAPT) to block Notch pathway signaling icked by raising extracellular Ca2⫹ in ADF from 1.2 to 1.8
(7). Notch pathway inhibition is known to activate proneural mM, and by providing 300 ␮M GABA to drive GABAA-
gene expression and indirectly reduce cell proliferation. dependent (bicuculine-sensitive) signaling, these additions

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS C537

A B C

SCM1/2 SCM-Base
NMDA NMDA
NR1 MAP2ab

20 pA

10 s
NR2A MAP2ab

NMDA EPSP
e
as
2

4
-B
1/

M
M

D
SC

SC

SF
n1

n1
n1
Q

NR2B MAP2ab
Q
Q
33

33
33
Current density (pA/pF)

0
D
-1

-2

-3

-4
PSD95 SYN HOECHST PSD95 SYN
Fig. 10. SCM1/2 protocol promotes postsynaptic NMDA responses and dendritic spine formation. A: exemplar (top) and mean (bottom) NMDA-evoked
postsynaptic excitatory currents recorded from 33Qn1-derived neuronal precursors differentiated into neurons for 21 days dpp using the SCM1/2, SCM-Base,
or SFDM4 protocol. B: double immunocytochemistry for NMDA receptor subunits (NR1, NR2A, and NR2B; red) and Map2ab (green) showing colocalization
of NMDA receptor subunits on mature neuron processes. C: scanning electron microscope images from 21 dpp differentiated neurons showing the presence of
elaborated processes populated with synaptic spines. Top: ⫻250; bottom: ⫻20,000. D: exemplar immunoreactivity to PSD95 and synaptophysin (SYN) in 21
dpp 33Qn1 neurons differentiated in SCM1/2 media. Confocal image at right shows neurite coregistration of presynaptic and postsynaptic markers SYN (green)
and PSD95 (red). Scale bars ⫽ 100, 50, or 5 ␮M as indicated.

were incorporated into the differentiation medium SCM1.
GABAA receptor activation leads to Ca2⫹-dependent CREB
and ERK phosphorylation, which in turn plays a central role in
regulating neurogenic gene expression (34, 52). Importantly,
the presynaptic phase of neuronal differentiation in vivo is
characterized by tonic GABA stimulation and sustained Ser-
133 phospho-CREB activity (24). Therefore, to help maintain
stable levels of CREB pathway activation during the initial
neurogenic phase of the differentiation protocol, SCM1 was
also supplemented with 10 ␮M FSK, a small molecule agonist
of the CREB pathway (1, 43). By following a developmental
rationale for protocol design and media formulation, there was
still scope for further enhancement of in vitro neuronal matu-
ration allowing for further improvement, hence the inclusion of
the prosynaptic small molecule antagonist of GSK3␤
CHIR99021.
In our experiments, neurons were essentially ventral fore-
brain, due to the use of SLI differentiation media during the
first 16 days to rosette formation. However, the SCM1/2
media were not designed as a method by which to specify
neuronal subtypes, rather they have been developed for
accelerating and enhancing the differentiation of any prepat-
terned NPCs to produce functionally active neurons in the
shortest possible time. In this regard, SCM1/2 protocol
results in rapid neuronal differentiation and morphological
maturation (Fig. 2, F–L) with expression of pre- and post-
synaptic markers and the development of spine-like struc-
tures (Fig. 10, B–D).
Although there have been numerous neuronal differentiation
protocols published to date, those that have systematically
determined the functional characteristics of the resulting cell
type have been surprisingly scarce, and none have integrated
the information with MEA-based network analysis. Neverthe-
less, several robust and carefully performed studies have pro-
vided evidence for functional maturation using a variety of cell
physiological endpoints (29, 48, 49).

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C538 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS

A B C KCl KCl
KCl response KCl response GABA
D21
2.5 Glut D14
100 SCM1/2 1.2 SCM1/2 low Cl-

F
F
% cells responding

*** D9
Base 1.0 *** * Base 2.0 GABA D8
80 GABA
D7
SFDM4 0.8 SFDM4 1.5
60 D6
0.6 21 dpp D5
40 1.0 D4
0.4 D3
20 0.5 D2
0.2
D1
0 0.0 0.0 0 dpp D0
0 200 400 600 800 1000
1

3
k

k
Time (s)
ee

ee

ee

ee

ee

ee
W

W
Glutamate response Glutamate response D
**

Total % cells responding

100 * SCM1/2 100
0.8
% cells responding

F SCM1/2 KCl
* ***
80 Base Base 80 Glutamate
* 0.6 *** ***
SFDM4 SFDM4 GABA
60 *** 60
GABA low Cl-
0.4
40 40

20 0.2 20
0 0.0 0

D4
21
1

2
3
4

8
9
1

7
D

1
D

D
D
D

D
D
D

D
k

k
k

D
ee

ee

ee
ee

ee

ee

Day post plate-down

W

W
W

GABA response in low Cl- GABA response in low Cl- E KCl

** *** Glutamate
*** *** * 1.2
100 SCM1/2 0.8 SCM1/2 GABA

F
% cells responding

***
F

Base 1.0 GABA low Cl-

80 Base
0.6
SFDM4 *** SFDM4 0.8
60 ***
0.4 ** 0.6
40
0.4
0.2
20 0.2
0 0.0 0.0

D4
21
0

2
3
4

8
9
1

7
1

1
D

D
D
D

D
D
D

D
k

k
k

D
ee

ee

ee
ee

ee

ee

Day post plate-down

W

W
W

Fig. 11. SCM1/2 protocol promotes time-dependent maturation of postsynpatic calcium responses. A: time course of Ca2⫹ influx responses of SCM1/2,
SCM-Base, and SFDM4 neurons to 60 mM KCl (top), 300 ␮M glutamate/30 ␮M glycine (middle), and 300 ␮M GABA in 7.5 mM chloride (bottom) shown
as percentages of cells responding. B: time-course of Ca2⫹ influx responses of SCM1/2, SCM-Base, and SFDM4 neurons to 60 mM KCl (top), 300 ␮M
glutamate/30 ␮M glycine (middle), and 300 ␮M GABA in 7.5 mM chloride (bottom) shown as change in intensity of the responses of the responding cells. C:
exemplar, time-slipped fura-2 recordings of Ca2⫹ influx during separate applications of 60 mM KCl, 300 ␮M glutamate/30 ␮M glycine (Glu), 300 ␮M GABA
in physiological extracellular chloride (GABA), and 300 ␮M GABA in 7.5 mM chloride (GABA low Cl⫺), as indicated. Each trace represents a recording from
a single 33Qn1-derived neuron cell during the differentiation process. The dpp range in shown to the right of the traces, while that actual dpp values are as shown
on the x-axes of the other panels. D: time course of Ca2⫹ influx responses of SCM1/2 neurons to 60 mM KCl, 300 ␮M glutamate/30 ␮M glycine, 300 ␮M GABA,
and 300 ␮M GABA in 7.5 mM chloride shown as percentages of cells responding. E: time course of Ca2⫹ influx responses of SCM1/2 neurons to 60 mM KCl,
300 ␮M glutamate/30 ␮M glycine, 300 ␮M GABA, and 300 ␮M GABA in low chloride shown as change in intensity of the responses of the responding cells.
Summary data (A, B, D, and E) are shown as mean ⫾ SE, where N is number of experiments. Statistical comparisons were performed by two-way ANOVA with
Tukey post hoc test: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

One of the most complete basic electrophysiological analy-
ses of hPSC-derived neurons was performed by Song et al.
(48), which elegantly described the time courses of iPSC and
excitatory synaptic current (ESC)-derived neuronal differenti-
ation in terms of the many important determinants of neuronal
maturation, including passive properties, iAP behavior, basic
spike characteristics, voltage-gated currents, sAP behavior, and
basic synaptic current properties. Although their data are in
broad agreement with those presented herein, there are signif-
icant differences, the most important of which centre around
the rate, extent, and uniformity of the functional maturation
process. Thus, by 21 dpp, the Song protocol produced neurons
of which only 18% exhibited repetitive iAPs, more similar to
our SFDM4 protocol at 11%, whereas the SCM1/2 protocol
generated a population of neurons with 59% (34D6) and 90%
(33Qn1) able to fire repetitive iAPs (see iAP-train analysis
above and Figs. 5 and 6). Even after a further week of
differentiation, the Song protocol was still only able to support
repetitive iAPs in 44.5% of the neurons; likely due to the lack
of synchronization of the differentiation program by cell cycle
exit as employed in the SCM protocol. Moreover, the action
potential parameters obtained at 28 dpp in neurons differenti-
ated by the Song protocol were, where available, reminiscent
of a more immature neuronal phenotype compared with those
of the SCM1/2 protocol at 21 dpp; specifically, they exhibited
smaller spike amplitudes, extremely low depolarization rates,
and long half-widths (see Tables 3 and 5 vs. Song et al. 2013
Table 1). Again, those spike parameters were more reminiscent
of neurons generated at 21 dpp by the SCM4 and SCM-Base
protocols than here with our SCM1/2 protocol (Tables 3 and
5). However, the Song neurons were particularly hyperpolar-
ized, even at 14 dpp, an observation that was attributed to high

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 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS C539

A B
Control Glutamate 3 M Glutamate 15 M TTX 0.5 M

Spikes (Hz)
60
30 V

1 ms
30 1 min

C D
Control NMDA 5 M Control Gabazine 5 M
Spikes (Hz)

Spikes (Hz)
10 30

1 min 1 min
5 15

0 0
Fig. 12. SCM1/2 protocol supports spontaneous and regulatable neural network activity of 33Qn1-derived neurons. A: exemplar unit responses from 7 of the 12
electrodes within 1 well of a 24-well multielectrode array (MEA) plate. Each panel plots 50 simultaneously detected units from each electrode, laid out in a cross
configuration, as shown. B: spike rate plotted vs. time for positive or negative-going spikes detected from single electrodes in MEA wells to that illustrated in
A. In the control no drugs were added, the electrodes detected sAP of 2–3 Hz. Application of glutamate 3 and 15 ␮M resulted in augmentation of spontaneous
activity by ⬃2-fold sustained and ⬎ 10-fold transiently, respectively. TTX (500 nM) eliminated all detectable spikes. C: spike rate plot recorded in MEA wells.
In the control no drugs were present and the electrodes detected sAP arising at 2–3 Hz. NMDA (5 ␮M) increased spiking activity transiently by ⬃5-fold. D:
spike rate plot recorded in MEA wells. In the control no drugs were added and spontaneous spiking rate was ⬃6 Hz. Gabazine (5 ␮M) augmented sustained
spontaneous activity by ⬎4-fold.

and progressive upregulation of the M current seen during that study, the GABAA response of ESC and iPSC-derived
differentiation. Interestingly, although the SCM1/2 protocol forebrain neurons shifted from excitatory to inhibitory during
was more efficient at hyperpolarizing the resting Vm than was the 7-wk differentiation, and this was due to a progressive
the SFDM4 and tended to “push” a higher proportion of cells reduction in ECl as the expression of Na⫹/K⫹/2Cl⫺cotrans-
in the sAP-full category, our neurons can only be routinely porter 1 (NKCC1) and K⫹/Cl⫺ cotransporter 2 (KCC2) was,
hyperpolarized below ⫺60 mV; this maneuver promotes sAP respectively, reduced and enhanced (44, 53). Unfortunately, no
activity as Na⫹ current inactivation is removed, and the excit- measures were reported between 1 and 5 wk of differentiation.
atory synaptic input is able to evoke action potentials. In the In our study, using voltage-activated Ca2⫹ entry as an indirect
Song protocol, the reverse is true, with the 28 dpp neurons
already hyperpolarized and what synaptic input is available is
not able to induce sAP activity. Such a difference may be more Table 6. Analysis of spontaneous unit responses recorded
a function of neuronal subtype being generated, since the Song from 33Qn1-derived neurons plated in 24-well MEA plates
NPC patterning protocol employs purmorphamine to ventral- differentiated in SCM1/2 media at 21 dpp
ize, whereas our prepatterning employs the inhibitor of WNT Spikes in the presence of
response compound IWR1 to promote intermediate progenitor Spikes in Control, Hz Drug, Hz n
domain specification. Overall, although the Song protocol Glutamate (3 ␮M) 2.2 ⫾ 0.6 10.8 ⫾ 2.4* 12
produces high-quality neurons, they take longer to differenti- Glutamate (10 ␮M) 3.1 ⫾ 1.2 9.5 ⫾ 2.6* 13
ate, are more heterogeneous in their characteristics and fire Glutamate (15 ␮M) 2.2 ⫾ 0.6 37.0 ⫾ 6.2† 12
spikes, which are still not fully mature at equivalent time TTX (500 nM) 5.7 ⫾ 1.0 0 ⫾ 0‡ 38
NMDA (5 ␮M) 0.5 ⫾ 0.2 4.3 ⫾ 2.0† 17
points. Gabazine (5 ␮M) 0.9 ⫾ 0.3 6.5 ⫾ 1.4† 22
The Livesey protocol (29) was focused particularly on mat-
uration and used several important readouts, including GABAA Spike frequency was measured before application and in the presence of
glutamate, TTX, NMDA, and gabazine and reported as mean ⫾ SE. Data were
phenotype, immature excitatory vs. inhibitory mature, which is analyzed with Student’s t-test; n ⫽ number of electrodes detecting spikes.
known in rodent models to depend developmentally on GABA- Significantly different from corresponding values in control. MEA, multielec-
regulated Cl⫺ cotransporters and subsequent [Cl⫺]i (18). In trode arrays. *P ⬍ 0.05; †P ⬍ 0.01; ‡P ⬍ 0.0001.

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C540 RAPID FUNCTIONAL MATURATION OF iPSC-DERIVED NEURONS
indicator of GABAA receptor activity, there was a progressive
increase in both the proportion of cells responding to GABAA,
and in the magnitude of the individual responses, during the
first 2 wk of differentiation (Fig. 11). By 21 dpp, blockade of
GABAergic signaling with gabazine, causing disinhibition,
resulted in a dramatic increase in neuronal network activity; all
activity was eliminated by the addition of TTX (Fig. 12). In
rodent models in vitro the functional GABA switch is depen-
dent on GABA in the medium (18), a situation that is mim-
icked in the SCM1/2 protocol.
The Telias protocol (49) generated neurons only from ESCs
but employed a similar induction and differentiation time
course to that employed in the SCM1/2 protocol. Within 3 wk,
neurons demonstrated significant maturation properties includ-
ing a higher proportion of neurons able to generate iAP train
activity. However, input resistance was consistently very high,
at over 2 G⍀ throughout, and iAPs had mean amplitudes
of ⬍40 mV, compared with ⬍1 G⍀ and ⬃70 mV, respec-
tively, using SCM1/2. Unfortunately, no details of Na⫹ current
activation/inactivation were provided, although the K⫹ current
densities appear comparable. Similarly, no indication of sAP
activity was presented, although spontaneous synaptic currents
are similar, and we have no objective measure of synchroni-
zation efficiency. Their use of DAPT would be expected to
begin to synchronize the maturation of the derived neurons.
However, as we show in Fig. 1, complete blockade of prolif-
eration, at least in iPSC-derived cells, is only possible by the
further addition of PD0332991. Nevertheless, with further
characterization, the Telias protocol may well be shown to
produce high quality, synchronized mature iPSC-derived neu-
rons, but currently there is an incomplete dataset described
which has only been performed on ESCs.
In conclusion, the use of SCM1 and SCM2 media separates
two key phases of neural differentiation; the neurogenic phase
and the synaptogenic phase, each characterized by different
signaling requirements. These requirements have been met by
formulation of two defined media that when used sequentially
on plated PSC-derived NPCs lead to rapid neuronal differen-
tiation and functional maturation. Crucially, this protocol uses
expanded and frozen prepatterned neural progenitors to deliver
mature neurons within 21 days, making it scalable and trans-
ferable to high-throughput platforms.
ACKNOWLEDGMENTS
We thank Siddharthan Chandran and Dr. Bilada Bilican for providing the
34D6 iPSC and Clive Svendsen and Dr. Virginia Mattis for providing 33Qn1
iPSC line. We also thank Dr. Josep M. Rebled and Blanca Peguera from the
Electronic Microscopy Unit at the Scientific and Technological Centers of the
University of Barcelona for support and Dr. Adrian Waite for Western blot
quantitation.
GRANTS
The CHDI Foundation generously provided funding for the majority of this
work. An NC3R’s Crack-It Challenge grant funded the MEA component of the
work. The FP7 Grant Repair-HD contributed to the electrophysiological
synaptic characterization. Grants from the Ministerio de Economia y Com-
petitividad (SAF2012-37417 to J. M. Canals) and from the ISCIII-Subdirec-
ción General de Evaluación and European Regional Development Fund
(ERDF) [RETICS to J. M. Canals (RD12/0019/0002; Red de Terapia Celular),
Spain] funded the analysis of NMDA receptors and spine formation.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AJP-Cell Physiol • doi:10.1152/ajpcell.00166.2015 • www.ajpcell.org
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AUTHOR CONTRIBUTIONS
Author contributions: V.T., P.L.Y., J.M.C., A.D.R., N.D.A., and P.J.K.
conception and design of research; V.T., C.S., P.L.Y., S.Y., P.S., E.C., A.H.,
B.A.T., C.G., J.M.H., S.J., L.B., N.C.-R., A.C., M.S., G.B., A.D.R., N.D.A.,
and P.J.K. performed experiments; V.T., C.S., P.L.Y., S.Y., P.S., E.C., J.M.H.,
J.T.B., J.M.C., A.D.R., N.D.A., and P.J.K. analyzed data; V.T., P.L.Y.,
A.D.R., N.D.A., and P.J.K. interpreted results of experiments; V.T., C.S.,
P.L.Y., P.S., A.H., C.G., J.M.H., A.D.R., N.D.A., and P.J.K. prepared figures;
V.T., N.D.A., and P.J.K. drafted manuscript; V.T., C.S., P.L.Y., S.Y., P.S.,
E.C., A.H., B.A.T., N.C.-R., A.C., M.S., G.B., J.M.C., A.D.R., N.D.A., and
P.J.K. approved final version of manuscript; A.D.R., N.D.A., and P.J.K. edited
and revised manuscript.
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