BioResearch Open Access
Volume 3, Number 4, August 2014
ª Mary Ann Liebert, Inc.
DOI: 10.1089/biores.2014.0019
ORIGINAL RESEARCH ARTICLE
Directing Human Induced Pluripotent Stem Cells
into a Neurosensory Lineage
for Auditory Neuron Replacement
Niliksha Gunewardene,1 Nicole Van Bergen,2 Duncan Crombie,2 Karina Needham,1
Mirella Dottori,3 and Bryony A. Nayagam 2,4,5
Abstract
Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem
cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection.
As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction
protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9)
toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression
of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESCderived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker
(PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin,
Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The
hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural
markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically
active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels
of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the
differentiation of hiPSCs into the required lineages.
Key words: stem
cell; hiPSC; differentiation; cochlear implant; auditory neuron; deafness
pursued over the last decade with promising results using
human embryonic stem cells (hESCs).1–3 However, the intrinsic ethical and practical limitations associated with the
use of hESCs has prompted investigation into the use of
autologous stem cell types, such as nasal-4 or bone marrowderived5–9 mesenchymal stem cells and induced pluripotent
stem cells (iPSCs)10,11 for AN replacement. The use of autologous cells would avoid the need for immunosuppression
treatment and eliminate the complications associated with
tissue rejection by transplanting sensory neurons generated
from patient cells.12
To explore the potential of human iPSCs (hiPSCs), it is
vital that their capacity to differentiate into an appropriate
Introduction
P
rolonged exposure to loud noise, administration of
ototoxic drugs and aging can all contribute to the degeneration of sensory hair cells and auditory neurons (ANs) in
mammals. As the mammalian inner ear cells do not regenerate, the loss of these cells leads to permanent hearing loss.
Presently, the cochlear implant is the only routinely available
clinical treatment for deafness, but its function relies upon
the presence of a healthy population of ANs. A treatment
strategy to regenerate or replace damaged ANs would therefore be useful in cases of severe AN degeneration. Stem cell–
based therapies for AN replacement have been extensively
1
Department of Otolaryngology, 2Centre for Eye Research Australia, and 5Bionics Institute, University of Melbourne, East Melbourne,
Victoria, Australia.
3
Centre for Neural Engineering, and 4Department of Audiology and Speech Pathology, University of Melbourne, Parkville, Victoria,
Australia.
1
2
lineage is thoroughly characterized. A reasonable approach
to determine the efficiency with which they differentiate
into an AN lineage is to probe for the expression of key factors involved in AN development. The inner ear originates
from a non-neural ectodermal region called the otic placode.13 As development progresses, the otic placode invaginates to form an otic vesicle that gives rise to all the cell
types of the inner ear, including the ANs.13 The transcription
factors Pax2/8,14–16 Eya1/Six1,17–19 and Sox220–22 have been
shown to upregulate expression of Neurogenin 1 (Neurog1)23
in otic progenitor cells, directing them toward a proneurosensory lineage. As development progresses, these Neurog1positive cells delaminate from the otic vesicle to form the
ANs medially and the vestibular ganglion neurons laterally.24 The delaminated neurons begin to show limited expression of Neurog1 and start to sequentially express the
neurogenic differentiation factor 1 (NeuroD1),25–27 brainspecific homeobox/POU domain protein 3a (Brn3a),28–30
and transcription factor Gata331–33 during their differentiation into sensory neurons.34,35 Subsequently, the mature
ANs express the characteristic pan neuronal cytoskeletal
markers Neurofilament kDa 160 (NFM), ßIII-tubulin, the vesicular glutamate transporter (VGLUT1), and the tyrosine receptor kinase B (TrkB) and C (TrkC) for brain-derived
neurotrophic factor (BDNF) and neurotrophin 3 (NT3).36
There have recently been several advances in specifying
stem cells toward an otic lineage.37–40 For instance, Koehler
and colleagues demonstrated the potential to guide murine
embryonic stem cells sequentially toward a non-neural,
pre-placodal and otic placode-like lineage using a complex
three-dimensional culture system.40 While promising, the
molecular signals and combinations of transcription factors
required to prompt the differentiation of stem cell–derived
otic placodal cells into ANs remains to be elucidated.
There have been a series of attempts to differentiate stem
cells toward an AN-like lineage using combinations of signaling molecules [basic fibroblast growth factors (bFGFs),
bone morphogenetic proteins, sonic hedgehog, and Y27632]
and the neurotrophins (BDNF and NT3) involved in normal
AN development.2,39,41–44 Of the studies conducted using
human stem cells, it has been demonstrated that human stem
cell–derived neurons express combinations of several AN developmental markers, including NEUROG 1, BRN3A, ßIIItubulin, and NFM42; PAX2/8, FOXG1, SOX1, NESTIN,
and BRN3A3; BRN3A, GATA3, and PERIPHERIN2; and
PAX2, BRN3A, PERIPHERIN, and NFM.44 Importantly,
we have recently reported the use of an induction protocol
for generating a mixed population of neurosensory progenitors from hESCs.44 Sensory neurons derived from this protocol exhibited electrophysiological properties akin to embryonic
mammalian ANs45,46 and made synapses on appropriate peripheral targets in vitro.44
The use of this protocol is supported by the recent literature
indicating that placodal and neural crest–derived sensory neurons share similar molecular and phenotypic properties during
development.44,46,47 Given their close association during development, it is possible that the neurons derived from this
heterogeneous sphere-forming assay contain auditory-like
precursors.12 This study therefore aimed to use this established
protocol to determine if hiPSCs could differentiate into a neurosensory-like lineage in a similar manner to that previously
reported using hESCs.44,46 We chose to characterize the
GUNEWARDENE ET AL.
potential of two hiPSC lines to differentiate into a neurosensory lineage, using hESCs as a control. To our knowledge,
this is the first study reporting the potential for hiPSCs to differentiate toward an auditory neuron-like lineage.
Methods
Cell lines
The iPS (foreskin) clone 1 and clone 2, abbreviated iPS1
and iPS248 (WiCell) and the hESC line H9 (WA-09, WiCell)
were used in this study. For the iPS1 cell line, cells [iPS1(A)
and iPS1(B)] were obtained from two different laboratories
[The iPS1(A): Centre for Neural Engineering, University
of Melbourne and iPS1(B): Neuroregeneration Research
Unit, Centre for Eye Research Australia] but were maintained and cultured using identical experimental procedures.
The passage numbers for the stem cell lines used ranged from
71 to 94 [iPS1(A)]; 55 to 92 [iPS1(B)]; 33 to 48 (iPS2); and
85 to 140 (H9). All tissue culture procedures were performed
using aseptic techniques in class 2 biological safety cabinets.
Stem cells were maintained at 37C, 5% CO2 and differentiated at 37C, 10% CO2 in humidified incubators.
Neural differentiation
The protocol used to differentiate the human stem cells toward a neurosensory lineage is depicted in Fig. 1. The hESC
and hiPSC cell lines were cultured as colonies on a mitotically inactivated human fibroblast feeder layer in gelatin
coated organ culture dishes (BD Bioscience). The cells
were cultured in Knockout Serum Replacement media (1:1
DMEM:F12 with GlutaMax, Knockout Serum Replacement,
10 mM nonessential amino acids, and 55 mM ß-mercaptoethanol; all purchased from Life Technologies) supplemented
with 10 lg/mL of bFGF (Peprotech). The human stem cell
colonies were routinely mechanically passaged each week.
To differentiate hESCs and hiPSCs toward a neural crest lineage, the stem cell colonies were transferred into Noggin media
containing Neurobasal media (NBM; Neurobasal A with 1% N2,
2% B27, 2 mM l-glutamine and 0.5% penicillin/streptomycin;
all purchased from Life technologies), 500 ng/mL of Recombinant Noggin (R&D Systems) and 4 ng/mL of bFGF. The cells
were cultured for 14 days without passage and Noggin media
was replaced every other day. After 14 days, the Noggin
media was replaced with fresh phosphate buffered saline
(PBS). The colonies were mechanically cut into grids using
two needles (23 gauge). The dissected pieces were washed in
an organ culture dish containing NBM, epidermal growth factor
(EGF; Peprotech), and bFGF (20 ng/mL each) and then transferred into a low attachment 96 well plate (1–2 pieces per
well) containing NBM supplemented with EGF and bFGF
(20 ng/mL each) to promote neurosphere formation. The neurospheres were maintained for a period of 4 days at 37C with 5%
CO2 in a humidified incubator.
After 18 days in vitro (DIV), the neurospheres were plated
onto gelatinized organ culture dishes that contained a layer of
mitotically inactivated human fibroblast feeders and NBM
supplemented with EGF and bFGF (20 ng/mL each), followed
by treatment with the Rho-kinase inhibitor Y27632 (25 lM;
Sigma-Aldrich) at 19 and 21 DIV. From this time point onward, media was changed every other day with NBM only
to promote sensory neural differentiation (24–35 DIV).
HUMAN IPSC INTO AUDITORY-LIKE NEURONS
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FIG. 1. Timeline of neural differentiation and the auditory neuron developmental markers examined in vitro. (A) The
hiPSCs and hESCs (control) were treated with Noggin media supplemented with bFGF for 2 weeks (neural induction). Subsequently, dissected colony pieces were treated with EGF and bFGF (neurosphere induction). The neurospheres were plated
on a layer of HFF and treated with EGF and bFGF at 18 DIV, followed by treatment with Y27632 at 19 and 21 DIV (neural
crest induction). From 24 DIV onward, the neurospheres were cultured in NBM only (sensory neural induction). Note: The
hiPSCs and hESCs were analyzed for AN marker expression using immunochemical and qRT-PCR techniques at the time
points highlighted in red. (B) The transcription factors analyzed at different stages of AN development. AN, auditory neuron;
bFGF, basic fibroblast growth factor; DIV, days in vitro; EGF, epidermal growth factor; hESCs, human embryonic stem cells;
HFF, human foreskin fibroblasts; hiPSCs, human induced pluripotent stem cells; NBM, neurobasal media; qRT-PCR, quantitative real time polymerase chain reaction.
Immunocytochemistry
Neurospheres were fixed at 19–35 DIV with ice-cold 4%
paraformaldehyde for 10 minutes and washed thrice in PBS
(5 minutes each). The neurospheres were immunostained
with the following primary antibodies: mouse a PAX7 (Developmental Hybridoma Bank; 1:20), rabbit a PAX2 (Covance; PRB-276P; 1:200), rabbit a SOX2 (Abcam; AB97959;
1:100), chicken a neurofilament-M (Millipore; AB5735;
1:1000), chicken a b-III tubulin (Millipore; AB9354; 1:1000),
mouse a BRN3A (Millipore; MAB1585; 1:1000), goat a
ISLET1 (Millipore; AF1837; 1:200) and rabbit a VGLUT1
(Invitrogen; 48-2400; 1:3000). The primary antibodies were
prepared in primary blocking solution [0.1% Triton-X
(Sigma-Aldrich) in PBS and 2% normal goat (AbacusALS) or donkey serum (Millipore)]. The diluted primary
antibodies were added to fixed cells and stored overnight at
4C in a humidified container. The next day, the cells were
rinsed thrice in blocking solution (5 minutes each). Appropriate Alexa-Fluor labeled secondary antibodies (Life Technologies) were diluted in the secondary blocking solution
[0.1% Tween (Sigma-Aldrich) in PBS and 2% goat or donkey serum] and added to the cells (200 lL per coverslip).
The cells were left for 2 hours at room temperature with
gentle rotation. After 2 hours, the cells were washed thrice
in PBS (5 minutes each) and mounted with ProLongGold antifade reagent containing the nuclear stain 4¢,
6-diamidino-2-phenylindole (Invitrogen). Confocal images
were taken using an LSM 510 META confocal scanning
laser system with a Zeiss AxioImagerZ1 microscope. Zen
digital imaging software (Carl Zeiss) was used to process
and analyze the images.
The immunocytochemistry results were analyzed by calculating the percentage of the total number of neurospheres
that had positive immunolabeling for the markers analyzed.
A minimum of eight neurospheres were analyzed for each
marker and time point (19, 21, 24, 28, and 35 DIV). Statistically significant differences were evaluated using a t-test and
the data presented as the mean – standard deviation from at
least three independent experiments. Statistical significance
was determined as follows: p < 0.05 (*), p < 0.01 (**), and
p < 0.001 (***).
Quantitative real time polymerase chain reaction
Undifferentiated and differentiating hiPSCs and hESCs
were collected for quantitative real time polymerase chain
reaction (qRT-PCR) at days 0 (undifferentiated), 19, 21, 24,
28, and 35. The cells were harvested by manual scraping,
washed in PBS, and resuspended in RNAlater (Sigma Aldrich)
for storage at 20C. The SV Total RNA isolation kit
(Promega Corporations) was used to extract total RNA from
the cell samples following the manufacturer’s instructions.
The RNA was quantified using a NanoDrop spectrophotometer
4
(Thermoscientific). For each sample, equal amounts of total
RNA (60 ng) were reverse transcribed to single stranded
cDNA using the High Capacity RNA-to-cDNA kit (Life
Technologies) following the manufacturers protocol.
The qRT-PCR reaction was carried out using the TaqMan
gene expression master mix (Life Technologies). The commercially available probes included PAX2 (Hs01057416_
m1: FAM dye), NEUROD1 (Hs01922995_s1: FAM dye),
BRN3A (POU4F1; Hs00366711_m1: FAM dye), GATA3
(Hs00231122_m1_FAM dye), and SLC17A7 (gene encoding
VGLUT1; Hs00220404_m1: FAM dye), and ß-ACTIN as the
housekeeper reference gene (4326315E: VIC dye, primer limited; all from Life Technologies). For each probe, a standard
curve was generated from a 1:10 serial dilution of control
cDNA sample. The reaction efficiencies were > 97% for all
the probes except for PAX2, which had a reaction efficiency
of 90% (reaction efficiencies between 0.90 and 1.0 were considered acceptable, as recommended by Applied Biosystems).
The experimental samples had a final cDNA concentration
of 60 ng/lL and were diluted to 6 ng/lL (1:10) for the qRTPCR experiment.
The qRT-PCR reactions were run using a RotorGene Q instrument (Qiagen) at the following thermocycler conditions:
1 cycle of 50C for 2 minutes then 95C for 10 minutes, followed by 40 cycles of 95C for 15 seconds then 60C for 1
minute. The reactions were run in duplex (gene of interest
and housekeeping probe present in each vial). For each reaction, no template controls (water only) and reverse transcriptase negative samples (without cDNA) were included as
negative controls.
The Rotor-gene 6000 software (Version 1.7, Corbitt
Research) was used to analyze the data. The relative quantification of each sample was determined using the 2DDCt
delta-delta analysis algorithm.49 The cycle threshold (Ct)
values of the experimental samples were obtained using
the thresholds from each probe’s standard curves. To calculate the DCt value, the expression levels of the experimental
samples were measured in triplicate and normalized against
the endogenous control, ß-ACTIN. Next, the DCt values were
standardized against the calibrator’s DCt to determine the
DDCt value. The relative quantification was then calculated
as 2DDCt. Note that for all the genes except NEUROD1,
the undifferentiated cells were used as the calibrator sample.
As NEUROD1 could not be detected in the undifferentiated
cells, 19 DIV cells were chosen as the calibrator sample.
Statistical analyses of the qRT-PCR data were performed
using GraphPad Prism. The relative quantification results
were analyzed using the one-way analysis of variance analysis of each gene at defined time points. A Kruskal–Wallis
test was performed on the nonmatched, nonparametric
data. Results are presented as the mean – standard deviation.
Statistical significance was determined as follows: p < 0.05
(*), p < 0.01 (**), and p < 0.001 (***).
In vitro electrophysiology
The stem cell–derived neurospheres and cultures of primary ANs (prepared from P4-P7 Wistar rat pups, as previously described46,50) were plated onto glass coverslips
[10 mm diameter, Menzel-Glaser; precoated with polyornithine (500 lg/mL; Sigma-Aldrich) and mouse laminin
(0.01 mg/mL; Invitrogen)] and grown in vitro at 37C, 10%
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CO2. As previously described,44,46 coverslips were transferred to a recording chamber fitted to an AxioExaminer
D1 microscope (Carl Zeiss) for electrophysiological recordings. The cells were superfused at 1–2 mL/min with a solution containing 137 mM NaCl, 5 mM KCl, 10 mM HEPES,
1 mM MgCl2, 2 mM CaCl2, 10 mM glucose (pH 7.35; 300–
305 mOsmol/kg). Recordings in stem cell cultures were
made from neurons with a bipolar morphology (soma diameter of *10 lm), between 28 and 35 DIV. Recordings
were made at room temperature using borosilicate microelectrodes (2–6 MO; 1.0 mm O.D.; 0.58 mm I.D., Sutter) filled
with an internal solution containing 115 mM K-gluconate,
10 mM HEPES, 7 mM KCl, 0.05 mM EGTA, 2 mM Na2ATP,
2 mM MgATP, and 0.5 mM Na2GTP (pH7.3; 290–295 mOsmol/kg). All chemicals were purchased from Sigma-Aldrich.
Signals were recorded with a MultiClamp 700B amplifier
( Molecular Devices), data acquisition system (Digidata
1440A, Molecular Devices), and AxoGraph X analysis software (AxoGraph Scientific).
Results
Both hiPSC- and hESC-derived neurosensory
progenitors express auditory neural markers
The stepwise differentiation protocol and the subsequent
examination of transcription factor/protein expression are
shown in Fig. 1. Given that the inner ear cells arise from
the dorsal hindbrain region during development, we initially
probed for expression of the dorsal hindbrain marker, PAX7,
for gross lineage confirmation. A significant increase in the
percentage of neurospheres with PAX7 immunopositive
cells was observed at 24 DIV [compared with control cells
at 19 DIV; iPS1(A) and iPS1(B): p < 0.01, iPS2 and H9:
p < 0.001; Supplementary Fig. S1]. At 24 and 35 DIV,
there was an increase in the expression of PAX7 relative to
19 DIV and this was consistent for all the cell lines. However, a decrease in the percentage of hESC-derived neurospheres with PAX7 positive cells was observed at 35 DIV
(compared with 24 DIV), but the decrease was not statistically significant.
To investigate whether the hiPSC and hESC neural progenitors derived from this assay were differentiating toward
an otic placodal lineage, the expression of PAX2 was also
examined (Fig. 2A). Immunocytochemical results indicated
that there was a significant increase in the percentages of
neurospheres that had PAX2 positive cells at 35 DIV [compared with 19 DIV; iPS1(A) and H9: p < 0.001, iPS1(B):
p < 0.01, and iPS2: p < 0.05; Fig. 2B, C-I]. Notably, at 35
DIV, all neurospheres examined contained PAX2-positive
cells. However, variability in the levels of PAX2 expression was noticed between the hiPSC lines (iPS1 and iPS2),
between samples of the same hiPSC line [iPS1(A) and
iPS1(B)], and between the hiPSCs and hESCs. To quantify
the expression levels of PAX2, qRT-PCR was employed.
Consistent with the immunocytochemical data, the qRTPCR results revealed an upregulation of PAX2 in the hiPSC
and hESC-derived neurosensory progenitors at 19 DIV
(iPS2: p < 0.05; H9: p < 0.001) and 35 DIV [when compared
with the undifferentiated controls; iPS1(B) and H9: p < 0.05;
Fig. 2C-II]. Although variabilities in the mean fold changes
of PAX2 were also observed between the hiPSC lines, samples of the same hiPSC line, and between the hiPSC and
HUMAN IPSC INTO AUDITORY-LIKE NEURONS
5
FIG. 2. Expression of PAX2 in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) Pax2 is expressed in otic placodal
cells during the early stages of AN development. (B) Using immunostaining, an increase in the expression of PAX2 was observed at 35 DIV. Scale bar = 100 lm (relative to all images). (C-I) There was a significant increase in the percentages of
hiPSC and hESC neurospheres that had PAX2 immunopositive cells. Data obtained from at least three independent experiments
and expressed as means – SEM of triplicates of each sample. (C-II) The qRT-PCR data show the fold changes of PAX2 in the
cell lines at 19 and 35 DIV. The results were presented as fold changes relative to the endogenous control, ß-ACTIN, and the
undifferentiated stem cells. Each point on the graph represents mean – SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001. DIV, days
in vitro; DAPI, 4¢,6-diamidino-2-phenylindole; SD, standard deviation; SEM, standard error of the mean.
hESC lines, collectively, these findings indicated that PAX2
mRNA and protein could be detected in both the hiPSC- and
hESC-derived neural progenitors across different time points
during differentiation.
In addition to Pax2, the transcription factor Sox2 has
been found to play a critical role during early AN development in promoting otic epithelial cells into a proneurosensory
lineage (Fig. 3A).21,22 The coexpression of SOX2 with the
sensory neural marker BRN3A was analyzed in all neurospheres at 19 and 35 DIV (Fig. 3B). We found that SOX2
was expressed in all the hiPSC and hESC neurospheres
analyzed at 19 and 35 DIV. Coimmunostaining with both
SOX2 and BRN3A showed subpopulations of cells that
were BRN3A + /SOX2 + , BRN3A/SOX + , or BRN3A + /
SOX2 (Fig. 3C). This may represent different progenitor
subpopulations and/or progenitors that are at different temporal stages of differentiation.
To further evaluate the potential of hiPSCs and hESCs to
differentiate into an AN-like lineage, the expression of
ISLET1 and BRN3A was analyzed using immunocytochemistry (Fig. 4A, B). It was found that there was an increase in
the percentages of neurospheres with ISLET1 and BRN3A
immunopositive cells at 35 DIV within both the hiPSC and
hESC neurospheres [iPS1(A) and iPS2: p < 0.05, Fig. 4C-I;
H9: p < 0.001, Fig. 4C-II]. The colocalization of Brn3a and
Islet1 is known to be critical for sensory neural differentiation.51 Here, the presence of BRN3A + /ISLET1 + immunopositive cells was noticed within the neurospheres at 35
DIV [Fig. 4B-II, B-III]. Given the differences in the relative
expression levels of the sensory neural proteins between the
cell lines, qRT-PCR was used to quantify the expression of
NEUROD1, BRN3A, and GATA3. The qRT-PCR results
revealed that NEUROD1 was upregulated in the hiPSC and
hESC lines at 35 DIV [compared with the control cells at
6
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FIG. 3. Expression of SOX2 and BRN3A in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) SOX2 is expressed
during the proneurosensory stage of AN development. (B) SOX2 immunopositive cells were observed in the hiPSC and hESC
neurospheres at all the time points analyzed. (C) The box inserts of the merged images of (B) show that at 19 DIV and 35
DIV, there were some cells that were BRN3A + /SOX2 + (arrows); however, the majority were either BRN3A + /SOX2
and/or BRN3A/SOX2 + . Scale bar = 100lm (all images).
19 DIV; iPS1(B) and H9: p < 0.05; Fig. 4D-I]. Similarly,
BRN3A was upregulated in the hiPSC- and hESC-derived
neural progenitors at 35 DIV [compared with the undifferentiated cells; iPS1(A), iPS1(B), and iPS2: p < 0.05 and H9:
p < 0.001; Fig. 4D-II]. Conversely, GATA3 was downregulated at 19 DIV in the hiPSC- and hESC-derived neural
progenitors [compared with the undifferentiated controls;
iPS1(A): p < 0.001, iPS1(B) and iPS2: p < 0.05; Fig. 4DIII]. However, at 35 DIV, GATA3 was significantly upregu-
lated in the iPS2-derived neural progenitors (compared
with the expression at 19 DIV; p < 0.05). Consistent with
the immunocytochemistry, there were differences in the
fold changes of NEUROD1, BRN3A, and GATA3 between
the hiPSC and hESC lines over the time course examined.
Overall, the trend of AN marker expression remained similar
between the hiPSC and hESC lines, but there were variabilities in the mRNA and protein expression levels of these
markers between the cell lines.
‰
FIG. 4. Expression of NEUROD1, ISLET1, BRN3A, and GATA3 in the hiPSC and hESC neurospheres at 19 and 35 DIV.
(A) NeuroD1, Islet1 and Brn3a are expressed during the ganglion neuron stage of AN development and Gata3 is expressed in
ANs. (B-I) Using immunostaining, an increase in the expression of ISLET1 and BRN3A was observed at 35 DIV. Scale bar =
100 lm (relative to all images). (B-II, B-III) Notably, there were several ISLET1 and BRN3A immunopositive cells at 35
DIV. (C-I, C-II) There was a significant increase in the percentages of hiPSC and hESC neurospheres that had ISLET1 and
BRN3A immunopositive cells. Data obtained from at least three independent experiments and expressed as means – SEM of
triplicates of each sample. (D-I) The qRT-PCR data show the fold changes of NEUROD1 in the cell lines at 24 and 35 DIV
relative to the control cells at 19 DIV. (D-II) The qRT-PCR results indicate the fold changes of BRN3A in the cell lines at 19
and 35 DIV relative to the undifferentiated controls. (D-III) The fold changes of GATA3 in the cell lines at 19 and 35 DIV.
The qRT-PCR results were presented as fold changes relative to the endogenous control, ß-ACTIN. Each point on the graph
represents mean – SD (n = 3). *p < 0.05; ***p < 0.001.
HUMAN IPSC INTO AUDITORY-LIKE NEURONS
7
8
As expected, the neural specific markers ßIII-tubulin and
NFM were expressed in both the hiPSC and hESC neurospheres throughout the differentiation timeline. Interestingly,
a decrease in the expression levels of ßIII-tubulin was noticed
at the later time points of differentiation within all cell lines (35
DIV; Fig. 5B). The decrease in ßIII-tubulin expression was
most pronounced in the iPS2- and hESC-derived neurospheres.
Furthermore, the presence of bipolar neurons (as determined
by positive staining for BRN3A and NFM) emanating from
the periphery of both the hiPSC and hESC neurospheres
were observed as early as 24 DIV (Fig. 5C), supporting the differentiation to a relevant phenotype for AN replacement.
Given the glutamatergic phenotype of ANs, the temporal
expression of the vesicular glutamate transporter, VGLUT1,
was also analyzed in the hiPSC and hESC lines (Fig. 6A).
Immunocytochemistry illustrated numerous VGLUT1-positive
puncta on NFM positive neurons, in both the hiPSC and
hESC neurospheres (Fig. 6B). Furthermore, by 21 DIV, all
of the hESC and hiPSC neurospheres showed expression of
VGLUT1 (data not shown). Accordingly, the qRT-PCR results revealed an upregulation of the gene encoding VGLUT1
(SLC17A7) at 35 DIV [when compared with the undifferentiated controls; iPS1(B) and H9: p < 0.05; Fig. 6C]. However,
variabilities in the fold changes of VGLUT1 were also observed between the hiPSC lines, samples of the same
hiPSC line, and between the hiPSC and hESC lines. For instance, at 35 DIV, the range in fold changes of VGLUT1 expression was between 7.47 and 44 [compared with the
undifferentiated controls; iPS1(A): 7.47, iPS1(B): 33.7, iPS2:
13.6, and H9: 44]. In summary, these data indicate that
VGLUT1 could be detected in both the hiPSC and hESCderived neural progenitors. However, variabilities in the
absolute levels of VGLUT1 expression were found between
the cell lines.
The hiPSC and hESC-derived neurons
were electrophysiologically active
The physiological function of stem cell–derived neurons
was assessed using whole cell patch clamp electrophysiological recordings (Fig. 7A). Similar to the electrical profile of
early postnatal ANs, the typical pattern seen in response to
membrane depolarization was a single action potential for
both the hiPSC- and hESC-derived neurons (Fig. 7B, E).
Voltage clamp recordings in all populations revealed a transient inward current at stimulus onset followed by a sustained
outward current during membrane depolarization (Fig. 7F).
The inward current was abolished in the presence of tetrodotoxin (a sodium channel blocker; Fig. 7F), while the outward
component of the current could be attenuated with the addition of the potassium channel blockers tetraethylammonium
and 4-aminopyridine (Fig. 7G). These findings illustrate the
presence of both sodium and potassium currents (INa and IK
respectively) in these populations of stem cell–derived sensory neurons.
Discussion
The development of a stem cell–based therapy for deafness requires overcoming several major challenges, one
being to successfully differentiate stem cells to an appropriate neurosensory lineage. It has previously been reported that
GUNEWARDENE ET AL.
exposing hESCs to human feeders and a combination of
EGF, bFGF, and Y27632 prompts their differentiation into
electrically active neurosensory progenitors in vitro.46 Here
we aimed to determine whether hiPSCs would behave in a
similar manner. It was found that the neural induction protocol
used was efficient at directing hiPSCs toward a neurosensorylike lineage in vitro, confirmed by the expression of a cohort
of relevant sensory neural mRNA and proteins throughout
the differentiation timeline. Notably, the highest expression
levels of the sensory neural markers were observed at 35
DIV, but there were differences in the absolute expression
levels between the cell lines.
Human iPSCs have the potential to differentiate
into a neurosensory lineage in vitro
We analyzed the expression of a cohort of AN developmental markers at different stages of differentiation using a
combination of immunocytochemical and qRT-PCR techniques. To assess if the neural assay was producing dorsal,
otic progenitors, expression of the dorsal hindbrain marker
PAX7 and the otic placodal marker PAX2 were initially examined. The peak expression of PAX7 was observed at 35
DIV in the hiPSC neurospheres and 24 DIV in the hESC neurospheres (relative to 19 DIV), hence confirming gross lineage specification. Given that PAX7 is an early neural marker,
its expression at the later time points of differentiation was
surprising. However, it must be noted that this neural assay
was generating a heterogeneous population of neural phenotypes at different temporal stages of differentiation; therefore, it is probable that it was producing other neural
lineages (in addition to dorsal hindbrain lineages) that have
later onset and persistent PAX7 expression. Furthermore,
the decrease in the percentage of hESC-derived neurospheres
that had PAX7 expression observed at 35 DIV is likely to reflect the specification and differentiation of the cells into
more mature lineages. It has previously been reported that
Pax2 has a critical role in the differentiation of ANs and
their peripheral innervation in situ.16 Furthermore, Pax2 animal knockout models have reported a complete loss of
ANs,14–16 clearly demonstrating the importance of this transcription factor for normal AN development. Our findings
indicated that at 35 DIV, all of the neurospheres analyzed
had PAX2 immunopositive cells. Consistently, qRT-PCR
revealed an upregulation of PAX2 mRNA in the hiPSC and
hESC lines at 35 DIV, relative to the undifferentiated cells.
Overall, these results suggest that the neural populations generated from this neural assay contained correctly specified
dorsal, otic progenitors.
All inner ear cells, including the cochlear hair cells, supporting cells, and sensory neurons, arise from a common
neurosensory domain within the otic vesicle. The neurosensory domain of the otic vesicle can be recognized very early
in development by the expression of Sox2.20 It has been
reported that Sox2 is expressed in both neuronal and sensory
progenitors but downregulated in differentiated hair cells
and neurons.20–22 Our observations that SOX2 was highly
expressed in the hiPSC and hESC-derived neurospheres
throughout the differentiation timeline indicated that the
neural progenitors were being specified toward a proneurosensory lineage. Furthermore, the mutual exclusivity of
BRN3A and SOX2 in some cells analyzed at 19 and 35
FIG. 5. Expression of ßIII-tubulin and NFM in the hiPSC- and hESC-derived neurospheres. (A) The neural specific markers ßIII-tubulin and NFM are expressed in the ganglion neuron and at AN stages of development. (B) Both the hiPSC and
hESC neurospheres showed some level of ßIII-tubulin expression over the time course examined. Note that at 35 DIV,
the levels of ßIII-tubulin had declined in the hiPSC line iPS2 and hESC line H9 cell lines (arrows). Scale bars = 100lm (relative to all images). (C) Bipolar BRN3A + /NFM + neurons were observed from as early as 24 DIV in the hiPSC and hESCderived neural progenitors (arrows). Scale bar = 50 lm (relative to all images). NFM, Neurofilament kDa 160.
9
10
GUNEWARDENE ET AL.
FIG. 6. Expression of VGLUT1 in the hiPSC and hESC neurospheres at 19 and 35 DIV. (A) VGLUT1 is expressed in developing ANs. (B) Using immunostaining, an increase in the expression of VGLUT1 was observed at 35 DIV. Scale bar =
50lm (relative to all images). (C) The qRT-PCR data show the fold changes of the gene encoding VGLUT1 (SLC17A7) at 19
and 35 DIV. The results are presented as fold changes relative to the endogenous control ß-ACTIN and the undifferentiated
controls. Each point on the graph represents mean – SD (n = 3). *p < 0.05; ***p < 0.001.
DIV suggests that SOX2 might have been downregulated in
the differentiated BRN3A positive neurons, consistent with
their expression pattern during AN development. Collectively, it is evident that the sphere-forming neural differentiation assay used contained a portion of correctly specified
neurosensory progenitors.
The basic helix-loop-helix transcription factors Neurog1
and NeuroD1 and the homeodomain transcription factors
Brn3a and Islet1 have been found to be critical for sensory
neural differentiation.51 The lack of any one of these factors
has been demonstrated to impact the maturity and function-
ality of the sensory dorsal root ganglion neurons.51 In the cochlea, knockout of NeuroD1 or Brn3a has been shown to
significantly reduce the number of ANs in mice.25–30 Furthermore, Islet1 has been indicated to have an important
role in specifying sensory and neuronal lineages in the developing inner ear.52 The increase in the expression and number
of cells immunopositive for ISLET1 and BRN3A protein in
both the hiPSCs and hESCs at 35 DIV, indicate that the cells
were differentiating into a neurosensory-like lineage. These
findings were supported by the qRT-PCR data, where it
was found that the sensory neural markers NEUROD1 and
HUMAN IPSC INTO AUDITORY-LIKE NEURONS
11
FIG. 7. Electrophysiological properties of hiPSC- and hESC-derived neurons. (A) Bipolar iPS2 neuron with recording microelectrode attached (Scale bar = 20lm). Typical current-clamp recordings from early postnatal auditory neurons cultured
in vitro (B), iPS1 (C), iPS2 (D), and H9-hESC (E) neurons. (F, G) Voltage-clamp recordings of stem cell-derived neurons
reveal transient tetrodotoxin-sensitive sodium current (arrow) followed by a sustained outward potassium current evoked by
membrane depolarization.
BRN3A were upregulated in the hiPSCs and hESCs at 35
DIV (when compared with the undifferentiated or 19 DIV
controls). It was evident from these findings that a higher
number of cells were differentiating into a more mature
neurosensory-like lineage by 35 DIV.
The AN marker Gata3 has been found to be a critical factor
involved in AN specification.31,33,53 Furthermore, in Gata3 conditional knockout mice, a significant reduction in the number of
ANs in the inner ear has been reported.54,55 Using qRT-PCR,
we detected GATA3 mRNA in undifferentiated hiPSCs and
hESCs. The presence of GATA3 in undifferentiated human
stem cells has previously been reported.56 Relative to the undifferentiated controls, GATA3 was downregulated at 19 DIV in
the stem cell–derived neural progenitors, which was not unexpected given that GATA3 is also a mesodermal lineage specifier. However, at 35 DIV, an overall trend of increasing
GATA3 expression was detected, particularly in the iPS2 cell
line. Given that Gata3 is expressed at a more mature stage of
AN development,31,33 it is plausible that the increase in expression observed at 35 DIV was due to the stem cell–derived neural progenitors achieving a more mature AN-like phenotype.
The type I afferent ANs have several characteristic traits,
among them, their bipolar nature and glutamatergic phenotype. Here, we found that both the hiPSCs and hESC neuro-
spheres contained bipolar BRN3A + /NFM + neurons from
as early as 24 DIV through to the latest time point examined
(35 DIV). It has previously been indicated that mouse iPSCs
transplanted into the cochlea differentiate into glutamatergic
neurons (evidenced by their expression of VGLUT1), but the
percentage of cells that acquired this lineage was considerably low (2.3%).10 Our data revealed an overall increase in
the expression of VGLUT1 over the time course examined,
with the highest expression observed at 35 DIV, hence suggesting that the cells were being specified into a glutamatergic
neuronal lineage. These findings are particularly promising
when considered in terms of this recent data, since bipolar
and glutamatergic characteristics are key attributes for a replacement AN derived from a patient-specific source. Furthermore, given the evidence that glutamate plays a key role in the
formation of peripheral synapses in the cochlea,57–59 it is possible that transplantation of a glutamatergic neuronal population would facilitate synaptogenesis in the cochlea.
Human iPSC-derived neurosensory progenitors
are electrically active
In order for hiPSCs to replace ANs, they must be capable
of faithfully sending electrical signals from the periphery to
12
the brainstem. We have previously demonstrated that hESCderived neurons obtained from this differentiation assay are
capable of firing action potentials in response to depolarization and have a phasic firing pattern, similar to the profile of
ANs cultured in vitro.44,46 Furthermore, the electrical profiles of the hESC-derived neurons were found to resemble
those of embryonic ANs cultured in vitro.45 More specifically, the action potentials generated from hESCs were
broader and exhibited higher latencies and firing thresholds,
when compared to the early postnatal ANs.46 Here, we demonstrated that neurons derived from the iPS1 and iPS2 lines
were also capable of firing action potentials and exhibited a
phasic profile of activity. These findings were consistent with
that previously reported for stem cell–derived ANs generated
using the current neural assay,44,46 other differentiation paradigms,39,42 and also early post-natal ANs.50 Promisingly,
these findings indicate that hiPSC lines can be differentiated
into appropriate neurosensory progenitors that are similarly
functionally active.
Neurosensory marker expression differed between
hESC and hiPSC-derived neural progenitors
Immunocytochemical and qRT-PCR results demonstrated
that all hiPSC and hESC lines analyzed produced functional
neurosensory-like progenitors. However, variabilities in the
levels of marker expression were observed between hiPSC
lines and within samples of the same cell line, when compared with the hESC controls. Consistent with our findings,
there is now accumulating evidence suggesting that hiPSCs
have greater variability in their differentiation potentials
when compared with their hESC counterparts.60–63 Several
factors may account for these variabilities. In addition to
the intrinsic differences that exist between human stem cell
lines, it should be noted that hESCs and hiPSCs differ in
their source (embryonic versus somatic cell) and derivation
(direct isolation versus reprogramming).64 Furthermore, the
two hiPSC lines used in this study were generated in the
same manner, by lentiviral transfection of four transcription
factors, OCT4, SOX2, NANOG, and LIN28 into skin fibroblasts.48 It has previously been reported that hiPSCs generated using the viral transfection method contain some
genetic and epigenetic mutations due to genomic integration
of the reprogramming transcription factors.65,66 It is plausible that these abnormalities influence their differentiation efficiency and even predispose them to differentiate into
specific sublineages. Although it is difficult to conclusively
determine the exact cause of the variabilities observed between the hiPSC and hESC lines, it is possible that a combination of intrinsic cell line specific variabilities and/or the
presence of genomic and epigenetic instabilities in the
hiPSCs may be significant contributing factors.
The variabilities observed between hiPSC lines in this
study bring to light some of the challenges that may arise
when using patient-specific hiPSC lines. For instance, variances may occur in the number of ANs generated from
each individual and the functional integration capacity of
these cells in vivo. Notably, in a recent study, it was reported
that hESC-derived neural progenitors could integrate into
the deaf gerbil cochlea and restore auditory function 10
weeks post transplantation.3 However, the degree of auditory
brainstem response recovery between all the stem cell trans-
GUNEWARDENE ET AL.
planted animals ranged from < 10 dB to a complete restoration of *60 dB sound pressure level. In terms of translating
this therapy into clinic, it would be undesirable if there were
significant variances in the functional outcomes patients’
achieve with the treatment. Therefore it is necessary that future studies address these variabilities prior to the clinical application of this therapy.
The presence of genomic abnormalities that may have
contributed to the differentiation variabilities observed in
hiPSCs has been implicated to increase their disposition
to form teratomas in vivo.67 Notably, Nishimura and colleagues reported the generation of teratomas following
transplantation of mouse iPSC-derived neurons into rat cochleae.11 The authors attributed the formation of teratomas
in vivo to the use of iPSC lines generated using the viral
transfection method. These findings highlight the necessity to explore the use of other autologous stem cell types
including bone marrow- or nasal-derived mesenchymal stem
cells. Whilst these stem cell types do not harbor reprogramming-induced genomic abnormalities, they have a more restricted self-renewal and differentiation capacity compared
with iPSCs. Future studies using hiPSC lines generated
using nonviral integration methods accompanied by regular
assessment of their genomic status may overcome the risks
associated with the use of hiPSCs for cell transplantation
therapies.68
In conclusion, the results presented demonstrate that the
described neurosensory induction protocol was capable of
generating a population of appropriate bipolar hiPSC-derived
neurosensory progenitors that are functionally active. These
results provide the basis for future experimentation focused
on assessing the survival and functional integration capacity
of hiPSC-derived neurosensory progenitors in deaf animal
models and represent the first step in bringing stem cells closer
to clinical transplantation in the deaf cochlea. Furthermore,
this neural assay could also be used as a model to interrogate
the expression and interactions of other critical transcription
factors involved in AN development.
Acknowledgments
We gratefully acknowledge financial support from The
Garnett Passe and Rodney Williams Memorial Foundation
Postgraduate Research Scholarship; NHMRC Project Grant
(APP1023372); Operational Infrastructure Support from the
Victorian Government; and The University of Melbourne
Departments of Otolaryngology and Audiology and Speech
Pathology.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Bryony Nayagam, PhD
Department of Audiology and Speech Pathology
University of Melbourne
550 Swanston Street
Parkville, Victoria 3010
Australia
E-mail: b.nayagam@unimelb.edu.au
Abbreviations Used
AN ¼ auditory neuron
bFGF ¼ basic fibroblast growth factor
DIV ¼ days in vitro
EGF ¼ epidermal growth factor
hESCs ¼ human embryonic stem cells
hiPSCs ¼ human induced pluripotent stem cells
NBM ¼ neurobasal media
NFM ¼ Neurofilament kDa 160
qRT-PCR ¼ quantitative real time polymerase chain reaction
SD ¼ standard deviation