Modeling human early otic sensory cell development with induced pluripotent stem cells
Modeling human early otic sensory cell development with induced pluripotent stem cells
Hanae Lahlou 0 1 2
Alejandra Lopez-Juarez 0 1 2
Arnaud Fontbonne 0 1 2
Emmanuel Nivet 0 2
Azel Zine 0 1 2
0 Current address: Harvard Medical School, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary , Boston , United States of America
1 Aix Marseille Universit eÂ , CNRS, LNIA UMR 7260, Marseille, France, 2 Aix Marseille Universit eÂ, CNRS, NICN UMR 7259, Marseille , France , 3 UniversiteÂ de Montpellier, Facult eÂ de Pharmacie , Montpellier , France
2 Editor: Bruce B Riley, Texas A&M University , UNITED STATES
The inner ear represents a promising system to develop cell-based therapies from human induced pluripotent stem cells (hiPSCs). In the developing ear, Notch signaling plays multiple roles in otic region specification and for cell fate determination. Optimizing hiPSC induction for the generation of appropriate numbers of otic progenitors and derivatives, such as hair cells, may provide an unlimited supply of cells for research and cell-based therapy. In this study, we used monolayer cultures, otic-inducing agents, Notch modulation, and marker expression to track early and otic sensory lineages during hiPSC differentiation. Otic/placodal progenitors were derived from hiPSC cultures in medium supplemented with FGF3/ FGF10 for 13 days. These progenitor cells were then treated for 7 days with retinoic acid (RA) and epidermal growth factor (EGF) or a Notch inhibitor. The differentiated cultures were analyzed in parallel by qPCR and immunocytochemistry. After the 13 day induction, hiPSC-derived cells displayed an upregulated expression of a panel of otic/placodal markers. Strikingly, a subset of these induced progenitor cells displayed key-otic sensory markers, the percentage of which was increased in cultures under Notch inhibition as compared to RA/EGF-treated cultures. Our results show that modulating Notch pathway during in vitro differentiation of hiPSC-derived otic/placodal progenitors is a valuable strategy to promote the expression of human otic sensory lineage genes.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The research leading to these results has
received funding from the European Community's
Seventh Framework Programme under grant
agreement No. 603029 (Project OTOSTEM). ªLa
Fondation Pour l'Auditionº provided support for the
final year Ph.D. fellowship rewarded to HL." The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
Hearing loss and vestibular dysfunction are the most common sensory deficits in humans [
The inner ear is a highly specialized sensory organ containing auditory and vestibular hair
cells (HCs) that transduce mechanical energy into electrical energy for transmission to the
central nervous system [
]. During otic development, HCs in the inner ear are derived from the
differentiation of early otic progenitor cells through a precise temporally and
spatially-coordinated pattern of gene expression orchestrated by complex signaling cascades [3_4]. A normal
human cochlea contains approximately 16,000 sensory HCs forming one row of inner HCs
and three rows of outer HCs. They are limited in number and are susceptible to damage from
a variety of insults, ranging from ototoxic drugs to loud noise exposure, genetic mutations, or
the effects of aging. In contrast to the avian cochlea able to regenerate lost HCs [5±6], the
mature mammalian cochlea is unable to spontaneously regenerate HCs leading to permanent
Over the past few years, stem cell-based therapy approaches aiming to emulate otic
development in the production of HCs from stem cells have received substantial interest [7±8]. The
generation of replacement HCs from a renewable source of otic progenitors remains one of
the principal requirements for the successful development of a cell-based therapy within the
inner ear. Murine embryonic stem cells (mESCs) have already demonstrated their capability of
differentiating into otic epithelial lineage in vitro [9±15]. Furthermore, previous studies with
human embryonic stem cells (hESCs) have revealed their ability to differentiate along an otic
neurogenic lineage, giving rise to neurons with a partial functional restoration of HC
innervation in an animal model of auditory neuropathy [16±17].
There is also evidence that hESCs are able to differentiate into cells of otic epithelial lineage
when grown in aggregate/embryoid body (EB)- or adherent cell cultures [18±19]. Recently, the
concept of differentiating hESC-derived HC-like cells has been elegantly demonstrated by the
ability of these hESCs to differentiate self-guided when cultured in hydrogels as extracellular
matrix mimics for three-dimensional (3D) cell culture [
]. These EB/aggregate and
3D-organoid in vitro guidance methods did allow the generation of HC-like cells displaying stereocilia
bundles from pluripotent stem cells. However, they were found to be complex and
time-consuming with variable efficiency and were not appropriate for the isolation of dissociated otic
progenitors required for the development of cell-based therapies.
Human ESCs challenged with retinoic acid (RA), epidermal growth factor (EGF), and other
growth factors have previously been shown to differentiate into HC-like cells [
this study was mainly focused on otic neural progenitors and thus did not explain or
characterize the presumptive otic/placodal progenitors. The available differentiation protocols remain
unsatisfactory and require further investigation in order to obtain higher yields of otic sensory
progenitors. Despite enormous progress made towards unraveling the signaling cascades
governing otic sensory differentiation, and their sequential orchestration during development,
much of otic cell fate determination remains not fully understood yet. The key to the in vitro
production of otic/placodal progenitors and their further differentiation into human otic
sensory cells is the identification of critical developmental pathways, as well as how and when they
have to be modulated.
In this line, human pluripotent stem cells, either from embryonic origin [
] or obtained by
cell reprogramming [
], namely human induced pluripotent stem cells (hiPSCs), have
received considerable attention for use as a cellular platform for the in vitro production of cell
types of interest, including otic cells, by recapitulating their developmental steps. Indeed,
thanks to their self-renewal and pluripotency features, they represent a suitable source for in
vitro generation of a large number of otic progenitors and their derivatives, including initial
HCs. Moreover, in the future, hiPSCs may serve as an autologous source of replacement for
HCs and/or neurons in the injured inner ear, if their differentiation to early otic/placodal
progenitors and otic sensory lineage is first successful in vitro.
Among the molecular pathways that regulate otic specification and competence during
development, the Notch pathway is a critical player [23±28]. Numerous studies have reported
that Notch inhibition, in damaged cochleae, increases the conversion of adult stem cells to
HCs by a mechanism involving the upregulation of Atoh1 [29±30]. The Notch pathway is a
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highly conserved signaling pathway that regulates cell fate and development in a variety of
metazoan tissues [
] and plays a crucial role in the regulation of stem cell behavior [32±33].
In this study, we investigated whether a disruption of the Notch pathway could affect the
differentiation of hiPSC-derived otic/placodal cells into otic sensory cells as compared to RA/
EGF treatment in monolayer cultures [
]. To monitor the generation of human otic sensory
cell-lineage, we measured the expression of a comprehensive panel of early otic/placodal and
sensory otic lineage markers in parallel using quantitative real time-PCR and
Our results show that timely inhibition of the Notch pathway during the differentiation of
hiPSC-derived otic progenitor cells is a valuable strategy to efficiently generate human inner
ear otic sensory cells in vitro. Our work improves our understanding of the mechanisms
controlling otic sensory lineage differentiation and may contribute towards developing a
cellbased therapy approach for inner ear disorders.
Materials and methods
Human induced pluripotent culture and maintenance method
The hiPSCs ChiPSC-4 line was provided by Cellartis (GoÈteborg, Sweden). This cell line was
derived from fibroblasts of healthy human donors and reprogrammed by using polycistronic
retrovirus technology, based on the transduction of Oct3/4, Sox2, Klf4, and c-Myc
transcription factors [
]. The hiPSCs were maintained using a proprietary feeder-free culture
system, they were plated at a density of 40,000±50,000 cells/cm2 onto coated dishes with a
DEF-CS™ COAT-1 matrix (1:20, Cellartis) diluted in D-PBS (+/+) (Gibco by Life
Technologies). Cells were expanded in DEF-CS™ 500 basal medium, daily supplemented with
DEF-CSTM GF-1 (1:333), GF-2 (1:1000) and GF-3 (1:1000) additives (Cellartis). When the
cells were confluent at 80±90% (about 5±7 days), they were passaged using TrypLE Select1
(Life Technologies). The cells grew quickly, and in less than a week, we could proceed to the
sof cells. Then, they were frozen or differentiated into inner ear otic cell lineage.
Early otic placode induction
We performed initial otic placodal induction from hiPSCs using a monolayer culture system
previously described for the differentiation of human embryonic stem cells (hESCs) into
neuronal and epithelial cells [
]. In our study, undifferentiated hiPSCs, from passages 18±21,
were seeded at 30,000 cells/cm2 onto coated laminin flasks (1.5 μg/cm2, R&D Systems). Cells
were cultured in DFNB basal medium (DMEM/F12 with N2 and B27, Gibco by Life
Technologies), supplemented with fibroblast growth factors, FGF3 (50 ng/ml) and FGF10 (50 ng/ml)
(R&D Systems) from the first day until the end of the induction period (i.e. day 13). The
medium was replaced every 2 days.
Sensory otic cell differentiation
To induce differentiation into otic sensory cells, dissociated otic/placodal progenitors obtained
from the early otic induction were transferred onto gelatin coated flasks at 80,000 cells/cm2
and cultured in DFNB medium, supplemented with either RA (1 mM, Sigma) and EGF (20
ng/ml; R&D Systems) or with a gamma-secretase inhibitor at 5 μM, i.e.
difluorobenzeneacetamid (DBZ, Tocris Bioscience). Exposure to either RA/EGF or DBZ was initiated from day 14
to day 20 and the medium was replaced every 2 days. At different culture periods, qPCR and
immunostaining were performed on differentiated cells and analyzed for the expression of
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early otic/placodal markers (i.e. PAX2, PAX8, DLX5, GATA3) and otic sensory markers (i.e.
ATOH1, POU4F3, MYO7A).
Total RNA was extracted from undifferentiated hiPSCs at day 0 and differentiated cells at day
13 and day 20 in vitro using the PureLink1 RNA Mini Kit (Life Technologies) according to
manufacturer's instructions. cDNA was synthesized from 1 μg of RNA per sample, using
High-Capacity RNA-to-cDNA™ Kit (Life Technologies), and 5 μl of cDNA were submitted to
qPCR reaction. Quantitative RT-PCR was performed with TaqMan1 Fast Real Time PCR
System (Applied Biosystems). Primer pairs used are listed in S1 Table (in the supporting
information). Samples were run in duplicates and analyzed with the 7500 Software v2.0 (Applied
Biosystems). Relative expression levels were determined according to the ΔΔCt method, the
GAPDH gene serving as endogenous control for normalization. RNA extractions were
performed from three independent cultures and the reported values are the mean of these three
independent experiments, each performed in duplicate.
All values are presented as the mean ± sem. Statistical differences between groups were
analyzed by one-way ANOVA and the Student's t-test using GraphPad Prism 6. Statistical
difference was reported for p-values below 0.05, an asterisk indicates significant differences between
means ( p < 0.05; p < 0.01; p < 0.001).
RNA was isolated from hiPSCs and differentiated cells as for standard qRT-PCR using the
PureLink1 RNA Mini Kit (Life Technologies) according to manufacturer's instructions [
We used 1 μg of total RNA from undifferentiated and differentiated cultures for cDNA
synthesis using High-Capacity RNA-to-cDNA™ Kit (Life Technologies). For each sample, 6.5 ng
cDNA was used to perform 14 cycles of pre-amplification using of TaqMan PreAmp Master
Mix (Applied Biosystems) and a 96 pooled PCR primer mix (final 0.2μM of each).
Pre-amplified PCR products were then treated with 4U of Exo I (NEB) in order to remove free primers
resulting from pre-amplification. Finally, cleaned pre-amplification was diluted 1:5 with 1X
TE Buffer. The qPCR experiments were performed using microfluidic 96.96 Dynamic Array
chip and the BioMark™ HD System following manufacturer procedure. Final reaction volume
of qPCR was 6.75 nL, with 500 nM primer final concentration and 1:18 sample final
concentration. Primer pairs used are listed in S1 Table (in the supporting information). The data was
acquired using Real-Time PCR Analysis Software in the BioMark instrument. Ct values were
processed by automatic threshold for all assays, with derivative baseline correction using
BioMark Real-Time PCR Analysis Software 4.1.3 (Fluidigm).
All data analyses were performed with R software v3.0 and ΔΔCt package v1.12. Data have
been analyzed using the ΔΔCt package algorithm which was built on the 2−(ΔΔCt) Method [
All values are presented as the mean ± sem. Statistical differences between groups were
analyzed by one-way ANOVA and the Student's t-test. Statistical difference was reported for
pvalues below 0.05, an asterisk indicates significant differences between means ( p < 0.05;
p < 0.01; p < 0.001).
Cochlea whole-mount surface preparation
The use of animals in this study was in accordance with the guidelines of CNRS (Centre
National de la Recherche Scientifique). Animal housing and experiments were conducted in
accordance with French national legislation (JO 87±848) and approved by our local ethics
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committee named ªDirection DeÂpartementale de la Protection des Populations, PreÂfecture des
Bouches du RhoÃneº, France (Permit No. B13-055-25).
The cochlear epithelium preparations were collected from postnatal day 1 Swiss wild-type
mice. After decapitation, the temporal bones containing the inner ears were removed from the
skull, the cochleae were opened and the sensory epithelium was separated from the modiolus
Immunocytochemistry and microscopy
In vitro differentiated cells and cochlear epithelium whole mount-preparations were fixed with
4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature.
Unspecific binding was blocked in 0.3% Triton X-100, 10% normal donkey serum and 1%
bovine serum albumin in PBS for 30 min. Then, samples were incubated overnight at 4ÊC with
specific primary antibodies (S2 Table, in the supporting information) diluted in the same
buffer without Triton X-100. They were then washed and incubated with AlexaFluor
(Molecular Probes) secondary antibodies and the nuclei were counterstained with Hoechst. Control
experiments including negative control (i.e. without primary antibodies) were processed in
parallel. Samples were mounted using Prolong Gold Antifading reagent on Superfrost glass
slides (Life Technologies). The images were acquired with a Zeiss confocal microscope LSM
710 NLO Zeiss and Zen software (Zeiss, Jena, Germany).
Cell counting and statistical analysis
The cells were counted manually using Graphic tools of Zeiss computer software (Zen 2012,
blue edition). The fraction of immuno+ cells among the total number of cells identified by
Hoechst was used to label all nuclei in at least five fields per coverslip in each in vitro condition.
Three independent experiments were conducted for each determination and data were
expressed as mean ± sem. Data were analyzed using the Student's t-test or one-way ANOVA.
Statistical difference was reported for p-values below 0.05, an asterisk indicates significant
differences between means ( p < 0.05; p < 0.01; p < 0.001).
Efficient differentiation of hiPSC-derived otic/placodal progenitors
Placodal development and otic induction is a complex process involving multiple signaling
steps during which, the specification and competence of individual cells become restricted
until the progenitors are able to continue their differentiation towards lineage of defined cell
]. During otic development, many markers including non-neural ectoderm,
preplacodal ectoderm, and early otic lineage genes are expressed in a fluctuating manner. Indeed some
early gene markers are transiently down-regulated and later re-expressed, whereas, other
genes are only transiently expressed or are continuously up-regulated during early otic
]. This pattern of gene expression may interfere with a clear chronological
identification of cell populations during otic/placodal development in an in vitro environment. We
therefore used the term ªotic/placodal progenitorsº to refer to all generated early otic lineage
cell phenotypes in our in vitro differentiation.
With the aim to study the conversion of hiPSCs to otic/placodal progenitors and their
subsequent differentiation into otic sensory lineage, we used a fully characterized hiPSC line
expressing the hallmark markers of pluripotency as confirmed by immunolabeling for
NANOG, OCT3/4, SSEA4 and SOX2 (S1 Fig). We used a guided strategy with hiPSCs growing
as adherent monolayers (Fig 1A±1D). First, in an attempt to derive early otic/placodal
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Fig 1. Schematic summary outlining the otic differentiation from hiPSCs in monolayer culture. (A) As a first step (step1), hiPSCs at day 0 were
exposed to FGF3 and FGF10 growth factors until day 13 for early otic/placodal induction and, in a second step (step2) were then differentiated into otic
sensory cells by exposure to either RA/EGF or DBZ until day 20. (B-D) Morphological characteristics of hiPSC-derived otic progenitor cells after FGF3/
10, RA/EGF and DBZ treatments respectively. Scale bars, 200 μm. Abbreviations: DFNB, DMEM/F12 supplemented with N2 and B27; RA, retinoic
acid; DBZ, difluoro-benzeneacetamide; EGF, epithelial growth factor; FGF, fibroblast growth factor.
progenitors from hiPSCs, we tested whether enriching culture medium with FGF3/FGF10 was
sufficient to trigger their induction into early otic lineage, as was previously reported with
]. The hiPSCs underwent rapid and profound morphological changes upon FGF3/
FGF10 treatment suggesting their differentiation (Figs 1B and 2A). To determine whether the
cells had started differentiating toward early otic lineage, we assessed the dynamic expression
of a panel of lineage markers known to be expressed during otic/placodal development. We
focused on the changes in the expression of transcription factors belonging to the PAX, DLX
and GATA gene families, among which some are key markers known to define otic placode
]. More specifically, we used a combined expression of PAX2, PAX8, GATA3
and DLX5 gene markers to track the differentiation of hiPSCs towards early otic/placodal
lineage. Analysis of transcript relative expression of these genes showed that differentiated cells
from hiPSCs started expressing these markers by day 6 in vitro (Fig 2B). Confirming these first
observations, immunocytochemistry analyses revealed that PAX2, GATA3, DLX5 and PAX8
were also expressed at the protein level in a subset of the differentiated cells (Fig 2C±2F).
Interestingly, some of these differentiated cells displayed double immunolabeling for DLX5 and
GATA3 markers (Fig 2F). As early as day 6 in vitro, quantitative analysis revealed that PAX2,
GATA3, DLX5 and PAX8 were respectively expressed by 13.68% ± 2.44%, 10.32% ± 1.57%,
23.03% ± 4.28% and 6.89% ± 4.28% of differentiated cells (Fig 2C). These results suggest that a
subset of differentiating cells was rapidly engaged toward otic/placodal cell fate. We then
checked whether extending the exposure time to FGF3/FGF10 could contribute towards
improving the outcome of otic differentiation (Fig 3A±3C). RT-qPCR analyses of a panel of
otic placode markers indicated that PAX2, GATA3 and DLX5 transcripts significantly
increased after 13 days of differentiation, with greater PAX2 and GATA3 expression levels
compared to those at day 6 (Fig 3A). On the contrary, we observed that PAX8 expression was
lost, confirming the early and transient expression of PAX8 during otic development. To
validate our results, we next quantified PAX2+ cells at day 13 of in vitro differentiation (Fig 3B).
Interestingly, the temporal analysis of PAX2+ cells showed a significant and progressive
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Fig 2. Development of otic/placodal competence and expression of early lineage markers in cultures at day 6 in
vitro. (A) A bright-field view of hiPSC-derived otic/placodal progenitors after FGF3/10 exposure. An example of a
colony displaying cells with spindle-like shape, indicating conversion efficiency from undifferentiated hiPSCs into
early otic/placodal progenitors (insets). Scale bar, 200 μm. (B) A histogram depicting values that were expressed as fold
change relative to hiPSCs using RT-qPCR. Data were normalized to control for the amount of RNA isolated from
undifferentiated hiPSCs (day 0) and this level of mRNA expression was referred to as 1. At this stage of FGF treatment,
a highly efficient induction of otic/placodal (i.e. PAX2, PAX8 and DLX5) gene markers was observed as compared to
undifferentiated hiPSCs. Statistical differences were determined with unpaired Student's t-test when comparing
mRNA expression levels between undifferentiated (day 0) and differentiated cells (day 6) cells. P values are indicated
by p < 0.05, p < 0.01, and p < 0.001 (n = 3). (C) Quantification of the expression of early otic lineage markers by
immunocytochemistry revealed their significant upregulation (PAX2, GATA3 and DLX5) in FGF-treated cultures as
compared to in undifferentiated hiPSCs. (D-F) Representative immunostainings for PAX2, GATA3, PAX8 and DLX5
in day 6 cultures. A population of PAX2+, PAX8+ and GATA3+, DLX5+ cells are observed in these differentiated
cultures. Interestingly, in some areas, cells co-expressed DLX5 and GATA3 (dotted circle, F). Hoechst staining is
shown in blue. Scale bars, 50 μm.
increase of PAX2+ cell levels from (12.68% ± 2.12%,
p = 0.04) at day 6 up to 29.34% (±
p = 0.009) at day 13 (Fig 3C). Of importance, PAX2, a paired box transcription
factor, has previously been deemed a crucial otic/placodal lineage marker [39±40].
HMG domain transcription factor, SOX2, is another critical gene for the in vivo
development of embryonic inner ear. SOX2 expression was found at the earliest stages of development,
when the otic placode first appears, and was required for establishment of the progenitor cells
within the prosensory domain [
]. We used immunostaining to examine the co-expression of
SOX2 and PAX2, to confirm the generation of otic/ placodal progenitors at a cellular level at
day 13 (Fig 3D). Immunolabelling showed a subset of SOX2 and PAX2 double positive cells
(10% ± 2.18) at day 6 and this cell percentage increased to (15% ± 2.50) at day 13 during the
time course of FGF-treated cultures (S2 and S3 Figs).
In parallel to the upregulation of otic/placodal markers, there was a decrease in the
expression level of known gene markers of mesendoderm and other cell lineages at day 13 in vitro
(Fig 4). The expression of mesoderm (Brachyury, T) and endoderm (SOX17) genes shown
previously to be expressed in hiPSCs [
] had also significantly decreased from d13 and remained
low or absent at day 20. We also observed that the pro-neural marker SOX3 [
], was only
weakly detectable at day 13, suggesting a suppressed differentiation of pro-neural ectodermal
cells in our guidance strategy. This was associated with a decrease of known neural crest genes
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Fig 3. Molecular characterization of otic progenitor cells. (A) RT-qPCR of the expression of several otic/placodal
gene markers in FGF3/10 differentiated cells after 13 days in vitro. A highly efficient expression of key gene markers
(i.e. PAX2, GATA3 and DLX5) of otic placodal/lineage was found, while no PAX8 expression was found at this stage of
differentiation. Data were normalized to control for the amount of RNA isolated from undifferentiated hiPSCs and
this level of mRNA expression was referred to as 1. Statistical differences were determined with unpaired Student's
ttest. P values are indicated with p< 0.05, p < 0.01, and p < 0.001 (n = 3 experiments). (B-Bº) PAX2
immunoreactivity in differentiated cells further confirmed the otic placodal-like identity at day 13 in vitro. (C) Cell
quantification revealed a significant increase of PAX2 immuno (+) otic progenitor cells at day 13 when compared to
their percentage in cultures at day 6 in vitro. Statistical analyses used ANOVA multiple comparisons; different letters
(a, b, c) indicate significant differences between groups (at least p < 0.05). (D-Dº) Immunocytochemical analyses of
the expression of otic/placodal markers (PAX2 and SOX2) in cultures at day 13. A subset of differentiated cells
coexpressed SOX2 and PAX2 (arrows), suggesting placode/otic-like identity. Hoechst staining is shown in blue. Scale
bars, 50 μm in all panels.
including DLX4 and the homeobox gene PHOX2B, considered as essential in establishing
neural crest identity [
Together, our results demonstrate that hiPSC differentiation in monolayer cultures with
FGF3/FGF10 treatment is able to produce presumptive early otic cells that co-express multiple
otic/placodal markers as early as day 6 in vitro. Extended exposure to the same inducing agents
contributed to a significant increase in the number of human otic progenitor-like cells by day
13 in vitro.
Notch inhibition enhances differentiation of otic sensory markerexpressing cells
We then wondered whether the newly generated hiPSC-derived otic/placodal progenitors
could be further differentiated along the otic sensory lineage to generate cells with initial
HClike phenotype. To this end, progenitor cells at day 13 of differentiation were switched to a
culture medium containing RA/EGF, modulators of two pathways that are active during inner
ear development. The RA has pleiotropic functions during embryogenesis and has been found
to expand otic competence within posterior placode during inner ear development [44±45],
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Fig 4. Reduction of meso-endodermal and other lineages during the time course of otic sensory differentiation from hiPSCs. RT-qPCR for
changes of in vitro-derived cell type markers after 13 and 20 days in comparison to hiPSCs at day 0, normalized to GAPDH gene. Culture conditions are
FGF3/10 from d0-d13 followed by DBZ from d13-d20. Expression analyses show downregulation of transcripts for mesoderm (BRACHYURY, T),
endoderm (SOX17), neural crest (DLX4, PHOX2B), pro-neural ectoderm (SOX3), and anterior placode (CXCR4) lineage markers. Statistical differences
were determined with one-way ANOVA. P values are indicated with for p < 0.05, p < 0.001, p < 0.0001 in comparison to hiPSCs (n = 3
while EGF participates towards regulating the in vitro production of HCs in embryonic and
early postnatal inner ears [46±47]. Of interest, a synergistic interaction between RA and EGF
has been found to induce the differentiation of HC-like cells from hESCs [
]. After 7 days
differentiation under RA/EGF treatment (Fig 5A), we performed qPCR assays to examine the
possible progression of hiPSC-derived otic/placodal progenitors into otic sensory cells.
Analysis of transcript expression data revealed a significant downregulation in the expression of a
panel of early otic/placodal markers (Fig 5B), suggesting a cell fate transition with decrease of
otic/placodal identity and further commitment of the hiPSC-derived otic progenitors. The
relative gene expression of PAX2 returned to basal levels (from 47.67 ± 7.41 at day 13 to
3.79 ± 1.87) at day 20 and was accompanied with a significant upregulation of some initial HC
markers, such as ATOH1 (Fig 5C). The downregulation of pluripotency gene markers
confirmed that during in vitro differentiation, most cells had lost their pluripotency features by
day 20 (S4 Fig). The overall results of these gene expression analyses reveal that in parallel to
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Fig 5. Expression of late otic cell markers. (A) Otic/placodal progenitors from day 13 were maintained for one more week in culture medium
supplemented with RA/EGF. (B) The differentiated progenitor cells at day 20 exhibited a significant decrease of a subset of otic/placodal markers as
determined by RT-qPCR. (C) In contrast, in these RA/EGF cultures, we observed a notable increase in some gene markers (ATOH1, POU4F3, MYO7A)
that define the otic sensory fate with a significant upregulation of ATOH1 expression. Statistical differences were determined with unpaired Student's
ttest. P values are indicated with p< 0.05 and p < 0.01 (n = 3 experiments).
the loss of early otic placode markers upon RA/EGF treatment, hiPSC-derived otic
progenitorlike cells start expressing a subset of otic sensory markers.
In order to assess whether other treatments could promote the differentiation of otic
progenitors into cells displaying an otic sensory phenotype, we decided to evaluate the impact of
Notch inhibition as compared to RA/EGF treatment in hiPSC cultures. Indeed, previous data
from our laboratory and others have demonstrated a crucial role of Notch signaling in otic
development and cell-fate decisions [23±28].
To evaluate the impact of Notch inhibition on differentiation along the otic sensory lineage
in vitro, hiPSC-derived otic progenitors from day 13 were subjected to a treatment with
gamma-secretase inhibitor (DBZ compound) over a one-week period (Fig 6A). At day 20,
differentiated cells were harvested to analyze and compare the expression levels of otic sensory
markers between DBZ and RA/EGF-treated age-matched cultures (Fig 6B±6E). Similarly to
what we observed after RA/EGF treatment, qPCR assays on DBZ-treated cells showed a
decrease in expression levels of a subset of otic/placodal markers. Remarkably, PAX2
downregulation was significantly greater in DBZ compared to RA/EGF-treated cultures.
In the culture condition in which hiPSCs were differentiated in DFNB medium, the relative
expression of early otic/placodal was very low for PAX2 at day 13 and increased for GATA3,
DLX3/5 at day 20. Of interest, the relative expression levels of the late otic markers ATHO1
and MYO7A remained undetectable during the time course of in vitro differentiation in DFNB
culture medium (S4 Fig).
To examine the role of Notch signaling in differentiation along the otic sensory lineage, we
then evaluated the gene expression levels of major downstream effectors of the Notch pathway
with known involvement in cell fate within the developing inner ear [48±50]. Our qPCR
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Fig 6. Enhancing the number of human otic sensory cells by Notch inhibition. (A) The otic/placodal progenitors from day 13 were exposed for 7
days (i.e. up to day 20) either to RA/EGF or DBZ compound. (B) Otic/placodal markers were significantly downregulated in DBZ-treated when
compared to RA/EGF-treated cells. (C) Downregulation of a subset of Notch signaling genes (HES5, HES7, LFNG) in DBZ-treated when compared to
RA/EGF-treated cells. (D) The decrease of Notch pathway components after DBZ treatment was accompanied with a significant upregulation of two
otic sensory markers (ATOH1, MYO7A) transcripts. (E) Quantification of MYO7A+ cells in differentiated cells from DBZ-treated and RA/EGF
treatedcultures revealed a much larger fraction of differentiated cells expressing MYO7A (50%) in DBZ-treated when compared to RA/EGF-treated cells (4%).
The individual bars show the % of immuno(+) cells to the total number of Hoechst-labeled cells. Shown are mean values and standard deviations
determined in three independent experiments for each data set. Statistical differences were determined with unpaired Student's t-test. P values are
indicated with for P < 0.05. (F-G) Representative immunostaining patterns for MYO7A in RA/EGF and DBZ-treated cultures at day 20 in vitro. Scale
bar, 20 μm.
analyses revealed that DBZ treatment leads to a significant downregulation in expression of
HES5 and LFNG, two crucial components of the Notch pathway (Fig 6C). This decrease was
accompanied by a significantly greater increase in the expression of the initial HC marker
ATOH1 in DBZ as compared to RA/EGF-treated cultures, suggesting a greater effect of Notch
inhibition on otic sensory commitment. In addition, we observed a significant upregulation of
MYO7A in DBZ-treated cultures (Fig 6D). MYO7A being one of the specific initial HC
proteins, we next assessed MYO7A protein expression in the DBZ-treated cells.
Immunocytochemistry analysis confirmed the expression of MYO7A in DBZ-treated cultures. Strikingly,
around half (50.15% ± 9.75%, p = 0.04) of the differentiated cells were immunopositive for
MYO7A following DBZ treatment (Fig 6E±6G) in contrast to only 4.83% (± 0.96%) of those
following RA/EGF treatment (Fig 6E and 6F).
Furthermore, co-immunostaining analysis revealed that MYO7A+ cells co-expressed
another initial HC marker i.e., POU4F3. Of importance, the specificity of the MYO7A and
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Fig 7. Expression pattern of otic sensory markers in vitro mimics that in early developing cochlear epithelium. (A-Aº) After one week of treatment
with DBZ, a subset of differentiated cells displayed co-expression MYO7A in the cytoplasm (shown in red) and POU4F3 in the nucleus (shown in
green). (B-Bº) The expression pattern of MYO7A and POU4F3 in in vivo mouse cochlear whole-mount preparations. Scale bars, 20 μm.
POU4F3 immunophenotype in DBZ-differentiated cells (Fig 7A±7Aº) was corroborated by
their expression in whole-mount preparations of postnatal day 1 mouse cochlea (Fig 7B±7Bº).
In addition, we found the same expression pattern to that in vivo, with POU4F3 confined to
the nucleus and MYO7A located in the cytoplasm. Noticeably, the expression of MYO7A in
the differentiated cells was mainly confined in a perinuclear cap-like structure, perhaps
indicating a polarization of the generated cells (Fig 7Aº) in line with a HC-like phenotype, though
it may also reflect their immaturity when compared to their in vivo counterparts showing
broader distribution within the entire cytoplasm.
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We also tested for the expression of genes characteristic of other epithelial cells in the inner
ear such as supporting cells. There was evidence that at least some supporting cell
differentiation was taking place under our culture conditions. In the differentiated cells obtained at day
13 and day 20 in vitro, genes characteristic of supporting cells (S100A1, JAG1, SOX2, GJB1, and
LGR5) were also observed (S5 Fig).
Moreover, GJB1, encoding CONNEXIN 32 and forming part of the gap junction between
supporting cells in vivo, was highly detected at day 20 after exposition of progenitor cells to
DBZ compound as compared to other conditions. The SOX2 is detected at day 20, however, its
expression is prominent in FGF3/10 cultures at day 13, consistent its dual role in early otic
placode specification and supporting cell determination during inner ear development. In
addition, we detected a population of SOX2 immuno+ cells intermixed with differentiated cells
that became Myosin7A immuno+ cells at day 20 in vitro (S6 Fig).
Altogether our data suggest that Notch inhibition promotes the differentiation of
hiPSCderived otic/placodal to an enriched set of cells that had expression profiles characteristic of
otic sensory cells in vitro.
Regenerative medicine offers reasonable expectations for the potential treatment of inner ear
disorders through the replacement of lost or damaged sensory cells. Initial advances in the
differentiation of murine ESCs/iPSCs into HC-like cells [9±15] have paved the way for similar
progress with pluripotent stem cells of human origin. Compelling evidence accumulated over
the last decade supports hiPSC technology as offering a promising future for stem cell research,
disease modeling and cell-based therapies in different types of tissues. In the case of the inner
ear, one of the challenges is to better define the developmental pathways and their sequential
activation/inactivation to allow the efficient in vitro production of human inner ear
otic/placodal progenitors and their further differentiation into otic sensory cell-like cells. In the present
study, our aim was to promote human otic/placodal induction processes and the generation of
otic sensory lineage cells by exploring the effects of timely modulations of major pathways
during hiPSC differentiation. Our results show that the expression of otic/placodal markers can be
induced through the activation of the FGF signaling pathway by FGF3 and FGF10 ligands,
suggesting that human placodal development and otic induction from hiPSCs is also an
FGFdependent process, as previously demonstrated with lineage guidance of both mESCs [9±14]
and hESCs [17±20]. FGF activation has emerged as a prominent player in promoting
otic-epibranchial progenitor identity during early development [51±52]. Indeed, ectopic expression of
FGF3 or FGF10 during mouse embryogenesis induces the formation of ectopic otic vesicles
expressing some otic markers i.e., PAX2 [
]. Complementary loss-of-function approaches in
zebrafish revealed that high levels of PAX2A and PAX8 favor otic differentiation, whereas low
levels increase cell numbers in epibranchial ganglia [
]. In addition, previous data reported
that FGF signaling is important at early stages to induce expression of PAX2 and PAX8
required for otic induction through differential regulation of competence factors FOXI1, SOX3
and FGF24 [
]. Interestingly, in our gene expression analysis of differentiated cells at day 13,
we found a significant upregulation of PAX2 in parallel to a downregulation of SOX3, known
as a pro-neural ectoderm lineage marker. In addition to PAX2/PAX8 expression, our results
showed a population of hiPSC-derived otic/placodal progenitors that co-expressed other
markers, such as DLX5 and GATA3 which are generally found in the native otic placode.
While not specific individually, the combined expression of multiple otic/placodal gene
markers is thought to be a good indicator of otic lineage identity [10±13]. Our results suggest that
FGF treatment of hiPSCs in monolayer cultures promotes human otic/placodal progenitors
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while reducing or suppressing mesendoderm and pro-neural ectoderm lineages. The
generation of different otic/placodal lineages implies the homogeneous nature of differentiation
allowed by adherent monolayer system [
] compared to EB-aggregates or 3D-based
culture strategies [
]. In the second step of the procedure, we tested which in vitro conditions
would enhance the ability of hiPSC-derived otic progenitors to differentiate into human otic
sensory cells. To this end, we explored the differentiation potential of otic progenitor cells
under Notch inhibition (i.e. DBZ-treated) as compared to RA/EGF treatment previously used
to induce HC-like cells in vitro from hESCs [
]. We used multiple initial HC gene markers
(ATOH1, POU4F3 and MYO7A) to examine a possible sensory cell identity after challenging
human otic progenitors with either DBZ compound or RA/EGF supplements for an additional
7 days in culture. The otic sensory lineage specification depends on the proneural gene
ATOH1 and its interactions with other transcription factors [
]. ATOH1 is necessary and in
some contexts sufficient for early inner ear HC development [
]. The POU4F3 and MYO7A
have been found expressed in differentiating HCs during early inner ear development [57±58]
shortly after ATOH1 and are considered to be among the crucial initial HC markers in otic
sensory lineage studies [
]. Of interest, our qPCR analysis revealed a significant
upregulation of ATOH1 expression when early otic progenitor cells were exposed to RA/EGF. This
observation fits with the roles of EGF and retinoid pathways in inner ear development and
with a previous study on hESCs . RA has also been shown to regulate otic vesicle formation
in vivo [44±45], whereas EGF ligands i.e., EGF/TGF-alpha promote proliferation and/or
maintenance of inner ear progenitor cells in vitro [46±47]. In contrast, for otic progenitors
maintained 7 days in vitro under Notch inhibition, we observed a significant increase in the relative
gene expression of both ATOTH1 and MYO7A as compared to their levels in age-matched RA/
EGF-treated cultures. In addition, challenging otic progenitors with DBZ resulted in the
generation of half (~50% of total) being MYO7-immunopositive compared to around 5% within
the RA/EGF-treated cultures. Our immunostaining results provide additional insight and
support the efficient promotion of otic sensory lineage achieved under Notch modulation, which
led to the differentiation of sensory cell populations that were double immuno+ for MYO7A
and POU4F3. Interestingly, using an embryoid body model, Costa et al. [
HClike cells (i.e., MYO7A immuno+) using mESCs by genetic programming through combined
overexpression of ATOH1, GFI1 and POU4F3 transcription factors. Interestingly, compared to
RA/EGF cultures, DBZ cultures showed a concomitant decrease in the expression of the
bHLH gene HES5 and the Fringe gene LNFG both of which are components of the Notch
pathway. This result is in accordance with our previous observation of a significant downregulation
of HES5 following pharmacological inhibition of Notch in mouse inner ear tissue
specificstem cells differentiated in a sphere model [
]. Furthermore, a recent study demonstrated
that initial pro-sensory cell fate is regulated by Fringe activity, requires low levels of Notch
signaling and is sensitive to changes in Notch signaling in the developing organ of Corti [
Another subsequent cell fate decision operates via a lateral inhibition mechanism to sort out
HCs and supporting cells during inner ear development [23_25]. This second cell fate process
was reported as independent of Fringe activity and much less sensitive to small changes in
Notch activity [
]. Taking into account the increase in the expression of otic sensory markers
and downregulation of HES5 and LNFG known to modify Notch signal transduction
], it is reasonable to presume that in our in vitro differentiation system, a
pharmacological reduction of Notch activity would affect the Fringe-dependent otic sensory cell fate
process. Our results revealed that hiPSC-derived otic progenitors were capable of
differentiation into cells expressing markers for otic sensory lineage. This differentiation ability towards
otic sensory lineage was enhanced by a pharmacological modulation of Notch pathway in
vitro. Although, human otic progenitors differentiated either under EGF/RA or DBZ expressed
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Fig 8. Schematic representation of the generation of inner ear otic sensory cells from hiPSCs by dual stepwise activation of FGF and inhibition of
Notch signaling pathways in adherent monolayer cultures.
initial HC markers MYO7A and POU4F3, their expression was not sufficient to promote the
formation of stereocilia, as reported with inner ear organoids [
]. However, the 3D culture
protocol used to generate functional HC-like cells from hESCs in this previous study involved
multiple steps and was time-consuming (up to 75 days) as compared to the monolayer culture
system used the present study (20 days). In addition, mature HCs differentiated in organoid
aggregates may not be appropriate for further cell transplantation experiments that require
hydrogel-free culture of differentiated cell progenitors. The lack of hair bundle-like structures
in our hiPSC culture system suggested that human otic sensory cells were at a nascent state of
commitment to HC phenotype and had failed to continue final maturation.
However, once improved, the in vitro differentiation procedure used in the present study
might provide a simple and rapid approach to generating human inner ear HC-like cells
harboring functional hair bundles. In conclusion, our findings provide a useful human induced
pluripotent stem cell differentiation assay to generate large numbers of otic/placodal
progenitors with a propensity to give rise to otic sensory cells (Fig 8). This study could provide the
foundations on which to build a more solid understanding of the mechanisms controlling otic
15 / 20
sensory differentiation and represents a useful step towards future explorations of the
transplantation potential of characterized human otic progenitors.
S1 Fig. Characterization of pluripotency markers of cultured hiPSCs. Immunostaining of
undifferentiated hiPSCs with antibodies specific for pluripotency markers: NANOG, SSEA4,
OCT4, SOX2, and AP activity. (A-C) The pluripotency marker molecules were expressed in
virtually all the propagated cells. The immunostaining also revealed the lack of the early
differentiation marker SSEA1 in hiPSCs maintained in DEF-CSTM 500 culture medium. These
immunostainings are representative for three hiPSC propagation experiments. Nuclei were
stained with Hoescht (blue). Scale bars, 50 μm. Abbreviations: hiPSCs, human induced
pluripotent stem cells; AP, alkaline phosphatase.
S2 Fig. Generation of otic/placodal cells from hiPSCs cultures at day 13 of in vitro differen
tiation. Representative double immunostainings for PAX2 and SOX2 (A-Aº) and for PAX2
and DLX5 (B-Bº) in FGF3/10 cell cultures. A population of PAX2 and SOX2 double immuno
+ cells (dotted circle) are observed in these differentiated cultures. In some areas within the
colonies, cells co-expressed DLX5 and PAX2 (arrows). Hoechst staining is shown in blue.
Scale bars, 20 μm (A-Aº); 50 μm (B-Bº).
S3 Fig. Quantification of otic/placodal labeled cell expression of differentiated cells in
FGF3/10 cultures at day 6 and day 13 in vitro. The individual bars visualize the fraction of
positive immunolabelled cells to the total number of Hoechst labeled-cells examined in eleven
randomly selected distinct fields from five coverslips (n = 1).
S4 Fig. Analysis of pluripotency and otic gene markers by RT-QPCR during the time
course of hiPSC differentiation. (A) A progressive downregulation in the relative gene
expression of a subset of pluripotency factors during differentiation processes following
exposition to FGF3/10 and RA/EGF at day 13 (B) and day 20 (C) cultures respectively. (D)
Expression of early otic/placodal and late otic markers at day 13 and day 20 of in vitro differentiation
in DFNB medium alone. Note the increase in the relative expression of GATA3, DLX3/5 at day
20 and a very low expression level of PAX2 at day 13 and day 20. For late otic markers (i.e.
ATHO1 and MYO7A) their expression levels remained undetectable during the time course of
differentiation in DFNB medium. Statistical differences were determined with unpaired
Student's t-test (n = 3 experiments for B, C). Significant differences are indicated by p< 0.05. For
D, one experiment with 2 biological duplicates/ culture condition.
S5 Fig. A gene expression panel of six known supporting cell markers during the time
course of hiPSC differentiation. RT-qPCR for changes of supporting cell markers at day 13
and day 20 of differentiation in comparison to undifferentiated cells at day 0, normalized to
GAPDH gene. Expression analyses show increase in transcripts of S100A1, LGR5, JAG1,
HEY1 and SOX2 in FGF3/10 cultures. After exposition to DBZ (1±5 μM), we noticed an
increase in the expression of GJB1 and a decrease of HEY1 transcripts. Statistical differences
were determined with unpaired Student's t-test (n = 3 experiments).
16 / 20
S6 Fig. Otic sensory cell cultures previously exposed to DBZ were maintained for one additional week (day-27) in DFNB culture medium. These differentiated cultures displayed
MYO7A + cells (shown in red) intermixed with a large population of SOX2 expressing cells
(shown in green) (A-A'). Scale bar = 50 μm.
S1 Table. List of gene-specific primers used for RT-qPCR for gene expression studies.
S2 Table. List of antibodies used in immunocytochemical marker expression studies.
We thank Professors B. Fritzsch (Iowa University) and T. Van De Water (Miami University)
for their critical reading. We thank Dr. M. Khrestchatisky and Prof. F. FeÂron (NICN, AMU,
France) for use of their lab facilities. The manuscript has been revised by an independent
scientific English language editing service.
Formal analysis: Hanae Lahlou, Alejandra Lopez-Juarez, Arnaud Fontbonne, Emmanuel
Conceptualization: Azel Zine.
Nivet, Azel Zine.
Funding acquisition: Azel Zine.
Investigation: Hanae Lahlou, Azel Zine.
Project administration: Azel Zine.
Supervision: Azel Zine.
Methodology: Hanae Lahlou, Alejandra Lopez-Juarez, Arnaud Fontbonne, Azel Zine.
Visualization: Alejandra Lopez-Juarez, Arnaud Fontbonne, Emmanuel Nivet.
Writing ± original draft: Hanae Lahlou, Azel Zine.
Writing ± review & editing: Azel Zine.
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