MYC Gene Delivery to Adult Mouse Utricles Stimulates
Proliferation of Postmitotic Supporting Cells In Vitro
Joseph C. Burns*, James J. Yoo, Anthony Atala, John D. Jackson
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America
The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can
lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells
within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become
depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy
for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/
4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a
first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ
cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and
the degradation-resistant T58A mutant of c-Myc (c-MycT58A) triggered significant levels of supporting cell S-phase entry as
assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the
proliferative response. The number of BrdU-labeled cells plateaued between 5–7 days after infection, and then decreased
,60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture
medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and
accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in
these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell
fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and
differentiation may restore regenerative potential to supporting cells within the adult mammalian inner ear.
Citation: Burns JC, Yoo JJ, Atala A, Jackson JD (2012) MYC Gene Delivery to Adult Mouse Utricles Stimulates Proliferation of Postmitotic Supporting Cells In
Vitro. PLoS ONE 7(10): e48704. doi:10.1371/journal.pone.0048704
Editor: Domingos Henrique, Instituto de Medicina Molecular, Portugal
Received August 6, 2012; Accepted October 1, 2012; Published October 30, 2012
Copyright: ? 2012 Burns et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Rena Shulsky David Foundation. The funder had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The Wake Forest Institute for Regenerative Medicine has filed a U. S. Provisional Patent Application on the technology described herein
(submitted March 7, 2012). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: email@example.com
The sensory epithelia within the inner ears of adult mammals
and humans are highly differentiated, postmitotic, and regenera-
tion deficient. Thus, the loss of sound- and acceleration-detecting
hair cells from auditory or vestibular sensory epithelia leads to
permanent hearing or balance impairments, respectively. In
contrast, the less differentiated sensory epithelia within the inner
ears of developing mice and non-mammals of all ages are capable
of more significant hair cell regeneration after damage, and non-
mammals can recover sensory function [1–5].
During sensory epithelial development and regeneration, cells
that morphologically resemble supporting cells act as otic
progenitors that can self-renew and give rise to new hair cells. In
vitro and in vivo evidence suggests that the progressive, postnatal
depletion of these progenitors, likely via terminal differentiation,
limits regeneration in mammals [4,6–19].
Ectopic, long-term expression of the four transcription factors,
Oct3/4, Sox2, Klf4, and c-Myc reprograms isolated somatic cells
into induced pluripotent stem cells (iPSCs) [20–23]. The initial
stages of the reprogramming process result in a partially
dedifferentiated, ‘‘pre-iPSC’’ state, and transient expression of
the iPSC factors has recently been utilized to directly reprogram
somatic cells into lineage-restricted, multipotent progenitor/stem
cells [24–29]. Therefore, applying the iPSC reprogramming
technology – typically used with isolated somatic cells – to intact
inner ear organs may be a novel approach for dedifferentiating
adult mammalian supporting cells while they remain in situ.
Here, we show that adenoviral-mediated expression of a
degradation-resistant mutant form of the iPSC factor, c-Myc,
induces robust S-phase entry of supporting cells in cultured utricles
from adult mice. In contrast, supporting cells remained postmitotic
after ectopic expression of the three other iPSC factors, Oct3/4,
Klf4, and Sox2. We present evidence that at least a portion of the
cells were able to progress into M-phase, and a small number of
cells replicating their DNA were found 21 days post-virus (DPV);
however, many of the cycling cells appeared to enter apoptosis
between 7 and 14 DPV. Switching from growth medium to serum-
free differentiation medium could prevent the loss of cycling cells,
but the protective effect of serum deprivation was temporary and
subsided by 14 DPV. Within the protective time window, a modest
but significant increase in the area of the sensory epithelium was
detected at 10 DPV, and a very small number of cells that had
replicated their DNA labeled with antibodies to the hair cell
marker myosin VIIA. These results provide evidence that ectopic
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expression of c-MycT58A in inner ear organs may restore
proliferative plasticity to postmitotic supporting cells.
Materials and Methods
Animals and dissection of utricles
All animal work was approved by the Animal Care and Use
Committee of Wake Forest University (protocol number: A11–
222). Swiss Webster mice, adults of either sex (.6 weeks old) and
timed-pregnant females, were obtained from Charles River
(Wilmington, MA). Labyrinths were dissected from temporal
bones in ice-cold DMEM/F-12 (Invitrogen, Carlsbad, CA), the
utricles were isolated, and the roof, otoconia, and nerve were
mechanically removed under aseptic conditions. The dissected
organs contained the entire sensory epithelium, a small portion of
the surrounding non-sensory epithelium, and the underlying
Organ culture and infection with adenoviral vectors
Adenoviruses containing vectors encoding Oct3/4, Klf4, Sox2,
c-MycT58A, or GFP under the control of a cytomegalovirus
(CMV) promoter were obtained from Stemgent (Cambridge, MA).
To construct its adenoviruses, Stemgent uses the AdEasy
adenoviral vector system, which allows for insertion of a promoter
and gene of interest into the E1-, E3-deleted backbone of
adenovirus serotype 5. The cDNA plasmids cloned into the viral
genome are described in Stadfeldt et al.  and can be obtained
from Addgene. The Addgene plasmid ID numbers and final
concentration of adenoviruses (transduction units per mL, TU/
mL) used for the co-infection experiments were: mouse Oct3/4
(ID: 19768; titer: 56107TU/mL), mouse Klf4 (ID: 19770; titer:
26108TU/mL), mouse Sox2 (ID: 19767, titer: 56107TU/mL),
and human c-Myc T58A mutant with a hemagglutinin (HA) tag
(ID: 19769, titer: 26108TU/mL). The stock adenovirus solution
comes stored in a suspension buffer consisting of 25 mM Tris
(pH 7.5), 2.5 mM MgCl2, and 1 M NaCl. Stemgent titers its
adenovirus using the immunoassay titration method.
For organ culture and adenovirus infection, dissected utricles
were adhered to glass-bottom dishes (Mat-Tek, Ashland, MA; two
utricles per dish) coated with Cell-Tak (BD Biosciences, San Jose,
CA) as described  and maintained at 37uC and 5% CO2in
100 mL of growth medium consisting of DMEM/F12, 5% FBS
(Invitrogen), 0.25 mg/mL Fungizone (Invitrogen), and 10 mg/mL
ciprofloxacin (Bayer, Berlin, Germany). Utricles were allowed to
stabilize in culture for 24 h, after which the growth medium was
replaced with infection medium (DMEM/F12, 0.25 mg/mL
Fungizone, and 10 mg/mL ciprofloxacin). Adenoviruses were then
added at the indicated concentrations for 8 h. Following
adenovirus washout, utricles were cultured in 2 mL of growth
medium until fixation. To label cells in S-phase, BrdU (3 mg/mL,
Sigma) was added for the periods indicated. In some instances, the
growth medium was replaced with 2 mL of differentiation
medium to promote hair cell differentiation. Differentiation
medium was based on a described formula  and consisted of
DMEM/F12, N2 supplement (Invitrogen), 0.25 mg/mL Fungi-
zone, and 10 mg/mL ciprofloxacin.
The following antibodies were used: rabbit anti-myosin VIIA
(1:200; Proteus Biosciences, Ramona, CA; # 25-6790) and mouse
anti-myosin VIIA (1:100; Developmental Studies Hybridoma
Bank, Iowa City, Iowa; # MYO7A 138-1) to label hair cell soma;
mouse anti-BrdU (1:50; BD Biosciences; # 347580) to label cells
that had incorporated BrdU during S-phase; rabbit anti-Ki67
(1:200; Thermofisher Scientific, Kalamazoo, MI; # RM-9106-S0)
to label cells in the active G1, S, G2, and M phases of the cell
cycle; mouse anti-phospho-histone H3 (Ser10) (PH3-Ser10; 1:200,
Cell Signaling Technology, Danvers, MA; # 9706) to label cells in
M phase; rabbit anti-Oct3/4 (1:200; Santa Cruz Biotechnology,
Santa Cruz, California; # SC-5279); mouse anti-Klf4 (1:200;
Abcam, Cambridge MA; # AB75486); rabbit anti-Sox2 (1:200;
Millipore, Billerica, MA; # AB5603); mouse anti-c-Myc (1:200;
Santa Cruz Biotechnology; # SC-40); mouse anti-HA (1:200;
Abcam; # AB18181); and rabbit anti-activated-caspase 3 to label
cells undergoing apoptosis (1:200; Abcam; # AB3623).
For immunocytochemistry, utricles were fixed in fresh 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at
room temperature (RT). After fixation, specimens were washed in
PBS then permeabilized and blocked for 1 h at RT in PBS with
0.2% Triton X-100 (PBS-T) and 10% normal goat serum (NGS;
Invitrogen). Samples to be labeled with anti-BrdU were digested
with DNAse I (0.5 kunitz/mL; Sigma) for 1 h at 37uC before
adding the blocking solution. Samples were then incubated in the
appropriate primary antibodies in PBS-T with 2% NGS
overnight, followed by 3 rinses in PBS-T and labeling with
AlexaFluor-conjugated secondary antibodies (1:200, Invitrogen) in
PBS-T for 3 h at RT. Where indicated, AlexaFluor-conjugated
phalloidin (5 U/mL, Invitrogen) and/or DRAQ5 (1:1000, Cell
Signaling) were included with the secondary antibodies to detect
F-actin and nuclei. Utricles were rinsed in PBS 3 times and
mounted in SlowFade (Invitrogen). Specimens were imaged using
a Zeiss LSM 510 confocal microscope.
To quantify the percentage of GFP-expressing cells in Ad.GFP-
infected utricles, the number of GFP-positive/myosin VIIA-
negative supporting cells and GFP-positive/myosin VIIA-positive
hair cells in 50 mm 650 mm regions were separately counted at
nine different locations spaced along the anterior-posterior axis of
the medial edge, striola, and lateral edge of each utricle. The
average for the nine regions was computed and then divided by
previous estimates of the mean density of supporting cells and hair
cells in adult mouse utricles in vivo [4,32]. For total BrdU counts
and cell cycle phase analysis, all BrdU-, Ki67-, and PH3-Ser10-
labeled nuclei in the sensory epithelium were manually counted for
each utricle using the Cell Counter plugin in ImageJ (U.S.
National Institutes of Health, Bethesda, MD). Macular area was
measured by using ImageJ to trace the outline of the sensory
epithelium in confocal images of utricles labeled with anti-myosin
OriginPro 7.5 was used to conduct sigmoidal equation fits,
Student’s t-tests, and one-way or two-way ANOVAs followed by
Tukey’s Test of Multiple Comparisons (alpha level =0.05 in all
cases). All descriptive statistics are presented as mean 6 s.e.m. To
calculate nonlinear least squares fits, OriginPro uses Levenberg-
Marquardt chi-squared minimization with automatic parameter
initialization. To fit equations to the data, 200 iterations of this
minimization routine were performed. The following sigmoidal
equation was used for the fits:
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Type 5 adenovirus transduces supporting cells and a
small fraction of hair cells in adult mouse utricles in vitro
The inner ear sensory epithelium is difficult to transfect using
techniques such as electroporation and lipofection [33–35]. Some
viruses efficiently infect supporting cells and/or hair cells [36,37],
and ectopic gene expression in supporting cells has been observed
after infecting adult mouse utricles with type 5 adenovirus in vitro
[38–41]. To characterize the efficiency of ectopic expression
versus adenovirus concentration, we infected utricles cultured
from mice .6 weeks old with 106–109transduction units per mL
(TU/mL) of adenovirus that contained a vector encoding green
fluorescent protein (Ad.GFP) under the control of a CMV
promoter. The cultures were fixed at 3 days post virus (DPV),
and the percentage of GFP-expressing supporting cells that did not
label with antibodies for the hair cell marker myosin VIIA were
quantified by sampling multiple regions within the sensory
106TU/mL resulted in little to no GFP expression, but the
percentage of supporting cells expressing GFP increased for
concentrations ranging from 107–109TU/mL (Fig. 1A–D, H;
n=2 utricles per concentration). Adenoviral-mediated GFP
expression in utricular hair cells from neonatal mice has previously
been modeled with a sigmoidal function . Nonlinear least
squares fitting to our data showed that the percentage of GFP-
expressing supporting cells in adult mouse utricles could similarly
be modeled with a sigmoidal function (r2=1), which had a half-
maximal transduction efficiency at 1.36108TU/mL and a
maximum efficiency of 77.2% (Fig. 1H, Table 1). Within the
range of Ad.GFP concentrations tested, the utricular macula
maintained epithelial and cytoskeletal integrity as revealed with
fluorescent phalloidin labeling (Fig. 1A’–D’). The GFP-expressing
supporting cells displayed typical, cylindrical shapes that were
more expanded at their apical and basal ends and compacted at
the level of the hair cell nuclei (Fig. 1E–F).
Labeling of hair cells with antibodies to myosin VIIA allowed us
to distinguish between hair cells and supporting cells, and we
found that GFP expression was mostly restricted to supporting
cells (Fig. 1E–F). However, we did find occasional GFP-positive/
myosin VIIA-positive hair cells with characteristic flask-like shapes
(Fig. 1G). The intensity of GFP-expression in these cells appeared
weaker than in neighboring supporting cells, which may contrib-
ute to difficulty in identifying them since previous studies have
reported that adenovirus type 5 exclusively expresses in supporting
cells within adult mouse utricles [38–40]. The percentage of hair
cells expressing GFP also fit with a sigmoidal function (r2=1) at
virus concentrations ranging from 106–109TU/mL, but the
maximum hair cell transduction efficiency was only 3.4%, which
was 23-times lower than for supporting cells (Fig. 1H, Table 1).
This efficiency was also substantially lower than the ,60%
reported for hair cells in utricles from neonatal mice . Also, the
viral concentration yielding half-maximal hair cell transduction in
adults was an order of magnitude lower than for supporting cells,
but on the same order of magnitude for neonatal hair cells
(Fig. 1H, Table 1; concentration at half-maximal infection
=1.36107TU/mL). Combined, the results suggest that adenovi-
rus transduces hair cells at similar concentrations independent of
age, the number of transduced hair cells decreases substantially
with age, and adenovirus transduces adult hair cells at lower
concentrations than adult supporting cells. It remains unclear
whether the age-related decrease in GFP-expressing hair cells
reflects a change in infectivity or CMV promoter activity.
Although the number of GFP-expressing hair cells was relatively
low, ectopic gene expression within hair cells may need to be taken
into consideration when infecting adult mouse utricles with
adenovirus in vitro.
Adenoviral gene delivery of iPSC transcription factors to
the adult mouse utricle initiates S-phase entry in
postmitotic supporting cells
After characterizing the adenoviral transduction efficiency, we
next sought to determine whether ectopic expression of any of the
four iPSC transcription factors could initiate cell cycle reentry of
postmitotic supporting cells in vitro. For this, we co-infected adult
mouse utricles with separate adenoviruses encoding mouse Oct3/4
(Ad.O, 56107TU/mL), mouse Klf4 (Ad.K, 26108TU/mL),
mouse Sox2 (Ad.S, 56107TU/mL), and the T58A mutant of
human c-Myc (Ad.MT58A, 26108TU/mL) under the control of
CMV promoters and then added BrdU to the culture medium for
the remainder of the culture period to label any cells that entered
S-phase (n=3–7 utricles per culture period). The T58A mutation
confers resistance to degradation by preventing threonine phos-
phorylation [42–45]. For controls, we infected other utricles with
Ad.GFP alone (56108TU/mL; n=2–5 utricles per culture
period). Twenty-four h after washing out the virus, antibodies to
BrdU did not label any nuclei in the sensory epithelium of utricles
co-infected with Ad.O, Ad.K, Ad.S, and Ad.MT58A (Fig. 2A, I).
However, by 3 DPV, the co-infected utricles contained many
BrdU-positive nuclei in their sensory epithelia (Fig. 2B, J). We also
found BrdU-positive nuclei in the maculae at six later timepoints
ranging from 5 to 28 DPV (Fig. 2C–H). Induction of S-phase entry
appeared to be specific to utricles co-infected with the four iPSC
transcription factors since comparatively minimal BrdU labeling
was detected in the sensory epithelium of Ad.GFP-infected utricles
Even at 28 DPV, the sensory epithelium maintained its integrity
and mitten-like shape, and supporting cells and hair cells
maintained compaction with tall aspect ratios (Fig. 2A’–H’).
Adenoviral vectors typically do not integrate into the host DNA,
and they exhibit transient expression profiles in some cells, which
may reduce the potential for tumorigenic effects from insertional
mutagenesis or transformation . Nevertheless, strong GFP
expression persisted at qualitatively similar cellular densities for all
28 DPV (Fig. 2I’–P’). GFP expression also persisted in the
surrounding non-sensory epithelium and underlying stromal
tissue, suggesting that the replication-deficient adenovectors were
not cleared or may have integrated into the host DNA. LacZ
expression from adenoviral vectors has also been observed out to
one month after delivery of adenovirus to mouse utricles in vivo
None of the BrdU-positive nuclei in the sensory epithelium
labeled with antibodies to myosin VIIA, indicating that only
supporting cells were entering S-phase and none of the labeled
cells had differentiated into new hair cells when cultured with
growth medium (Fig. 3A–B). Quantification of the mean number
of BrdU-positive supporting cells revealed that S-phase entry
peaked between 5 and 7 DPV (mean BrdU-positive nuclei at 7
DPV =10568; Fig. 3C). The mean number of BrdU-labeled
nuclei per sensory epithelium declined sharply by 45% from 7 to
10 DPV, and then decreased more gradually and eventually
stabilized between 10 and 28 DPV (mean BrdU-positive nuclei at
28 DPV =45617; Fig. 3C). Since BrdU was included in the
culture medium throughout, a decline in the number of BrdU-
positive nuclei indicates that the population of BrdU-labeled cells
somehow became depleted, likely via cell death or exclusion from
the sensory epithelium. Antibodies to activated caspase 3 co-
labeled 2–3 BrdU-positive cells per sensory epithelium at 8 DPV
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(n=2 utricles), suggesting that the depletion of BrdU-positive cells
was due to apoptosis (Fig. 3E). The small number of activated-
caspase-3-labeled cells may have been due to rapid apoptosis .
Many of the BrdU-labeled nuclei were present in pairs, triplets,
or quadruplicates, suggesting that some cells were completing
mitosis and dividing (Fig. 3A–B, D). Quantification showed that
the percentage of BrdU-positive nuclei per sensory epithelium
appearing in doublets also peaked between 5 and 7 DPV (Fig. 3D;
percentage of doublets at 7 DPV =6868%). The percentage of
doublets declined thereafter, following a similar trend as the mean
number of BrdU-labeled nuclei (Fig. 3D; percentage of doublets at
28 DPV =3163%). Together, the results suggest that many cells
Figure 1. Adenovirus primarily infects supporting cells but also some hair cells in adult mouse utricles in vitro. (A–D) Low
magnification (206/0.75 NA) confocal images of utricles from adult mice that were incubated with increasing concentrations of adenovirus
engineered to express green fluorescent protein (GFP, green) under the control of a CMV promoter. Utricles were fixed and labeled 3 d after
adenovirus was washed out. Hair cells are labeled with antibodies to myosin VIIA (magenta). (A9–D9) Fluorescent phalloidin labeling in the utricles
from A–D. Scale bar for A–D’, 100 mm. (E) High-resolution (636/1.4 NA) confocal section taken at the apical surface of the sensory epithelium shows a
cluster of GFP-positive supporting cells (green). The supporting cell apical surfaces have characteristic polygonal shapes compared to the circular
profile of hair cells labeled with anti-myosin-VIIA (magenta). (F) View of a confocal image stack parallel to the apical-basal axis of supporting cells and
hair cells shows a supporting cell expressing GFP (green), but its neighboring myosin-VIIA-labeled hair cell (magenta) does not. Scale bar, 5 mm. (G)
Same view as in F shows a GFP-expressing hair cell. Scale bar, 5 mm. (H) A graph showing quantification of the percentage of GFP-expressing
supporting cells (black circles) and hair cells (magenta circles) with increasing concentration of adenovirus. Myosin VIIA labeling was used to
distinguish between supporting cells and hair cells, and sigmoidal equations (black and magenta lines) were fit to the data points (see Table1 for
equation and coefficients).
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were passing through S-phase, completing M phase and cytoki-
nesis, and then dying. Since the levels of BrdU labeling eventually
stabilized and some doublets were still detected at 28 DPV, a
fraction of cells may be capable of surviving after induction of cell
cycle reentry with iPSC transcription factors.
Of the four iPSC transcription factors, c-MycT58A is both
necessary and sufficient for inducing reentry of
supporting cells into the cell cycle
Antibody labeling of 3 DPV utricles that were separately
infected with Ad.O, Ad.K, Ad.S, or Ad.MT58A (each at 56108
TU/mL) showed increased protein levels of Oct3/4, Klf4, and c-
Myc in some supporting cell nuclei (Fig. 4A, C, G). Oct3/4, Klf4,
and c-Myc antibody labeling was not detectable in utricles infected
with Ad.GFP, which indicated that the increased protein levels
were a result of ectopic expression from Ad.O, Ad.K, and
Ad.MT58A, respectively (Fig. 4B, D, H). Sox2 is expressed in
supporting cells and a subset of hair cells in the adult mouse utricle
in vivo , and the intensity of Sox2 antibody labeling did not
appear to differ between Ad.S- and Ad.GFP-infected utricles
(Fig. 4E, F).
Antibodies to Klf4 labeled the greatest number of nuclei,
whereas c-Myc antibodies labeled ,10 nuclei per sensory
epithelium (Fig. 4A, G). Ad.MT58A-infected utricles fixed and
labeled with c-Myc antibodies at 1, 2, 5, and 10 DPV also
contained similarly low numbers of c-Myc-positive supporting
cells, as did antibody labeling for the hemagglutinin (HA) tag that
was engineered into the c-MycT58A transgene (data not shown).
Since the ectopic protein levels appeared to differ for each
factor, we next sought to determine whether cell cycle reentry was
dependent on one individual factor by separately infecting adult
mouse utricles with Ad.O (56107TU/mL), Ad.K (26108TU/
mL), Ad.S (56107TU/mL), or Ad.MT58A (26108TU/mL) at
viral concentrations identical to those used for the co-infection
experiments. BrdU was included in the culture medium for the
remainder after virus washout. Fixing the cultures at 5 DPV and
labeling for BrdU revealed significant numbers of supporting cells
that had reentered the cell cycle in utricles infected with
Ad.MT58A compared to those infected with Ad.O, Ad.K, or
Ad.S (Fig. 5A–D; mean BrdU-positive supporting cells in
Ad.MT58A-infected utricles =79610; p,0.05, One-way AN-
OVA with Tukey’s Test of Multiple Comparisons; n=9 utricles).
Increasing the concentration of Ad.MT58A 5- or 10-times
significantly enhanced the numbers of BrdU-labeled nuclei at 5
Table 1. Coefficient values for sigmoidal curve fits.
Supporting cells 0.577.2 1.36108
Hair cells0 3.41.26107
Figure 2. Co-infection of Ad.O, Ad.K, Ad.S, and Ad.MT58A induces cell cycle reentry of postmitotic supporting cells in adult mouse
utricles in vitro. (A–F) Low magnification (206/0.75 NA) confocal images of co-infected utricles from adult mice at various days post-virus (DPV).
BrdU was included in the culture medium for the entire period after virus washout, and utricles were fixed and labeled at the times indicated. Hair
cells and nuclei that entered S-phase are labeled with antibodies to myosin VIIA (magenta) and BrdU (green), respectively. (A’–F’) Fluorescent
phalloidin labeling in the utricles from A-F. Scale bar for A–F’, 100 mm. (I–P) Low magnification (206/0.75 NA) confocal images of control utricles
infected with Ad.GFP. Few to no BrdU labeled nuclei are present in the sensory epithelium. (I’–P’) GFP expression in the utricles from I–P. Scale bar for
I–P’, 100 mm.
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DPV (Fig. 5D–G; mean BrdU-positive supporting cells at 16109
TU/mL =357665, mean BrdU-positive supporting cells at
26109TU/mL =564619; p,0.05, One-way ANOVA with
Tukey’s Test of Multiple Comparisons; n=2–4 utricles), and
increasing the concentration of Ad.O, Ad.K, or Ad.S to 16109
TU/mL did not result in significant S-phase entry (data not
shown). Thus, of the four transcription factors, c-MycT58A
appears to be both necessary and sufficient for the induction of cell
The levels of S-phase entry in Ad.MT58A-infected (26108TU/
mL) utricles were similar to those in utricles co-infected with Ad.O
(56107TU/mL), Ad.K (26108TU/mL), Ad.S (56107TU/mL),
and Ad.MT58A (26108TU/mL; Figs. 3C, 5G). When we double-
labeled Ad.MT58A-infected utricles with antibodies to c-Myc and
Ki-67, a protein that is upregulated during the active phases of the
cell cycle , we detected one nucleus in five specimens that
labeled with both antibodies, indicating that few actively cycling
cells had detectable levels of c-MycT58A protein. While there
were relatively few c-Myc-positive cells compared to the large
number of proliferating cells, it remains possible that autonomous
expression of c-MycT58A in supporting cells induces cell cycle
reentry, but c-MycT58A protein gets degraded to undetectable
levels as the cells transition out of quiescence. Therefore, we were
not able to determine whether cell cycle reentry is driven by cell
autonomous expression of c-MycT58A or whether ectopic
expression in neighboring cells stimulates the proliferative
response via paracrine signaling.
Supporting cells in Ad.MT58A-infected utricles can
proceed to mitosis
Many of the BrdU-positive nuclei in utricles infected with just
Ad.MT58A appeared in doublets (Fig. 5F, inset), suggesting these
cells completed all phases of the cell cycle and divided. To further
characterize the effects of Ad.MT58A infection on cell cycle
progression in supporting cells, we co-labeled Ad.MT58A-infected
utricles (16109TU/mL) fixed at 5, 7, and 10 DPV with antibodies
to BrdU and Ki-67. Since BrdU is permanently incorporated into
DNA during replication in S-phase, cells that label with antibodies
to both Ki67 and BrdU have replicated their DNA and are still
actively cycling. Cells that label for just BrdU have replicated their
DNA and exited the cell cycle. Cells that label for just Ki67 have
not yet replicated their DNA and are presumed to be in G1.
Similar to co-infected utricles, the mean number of BrdU-
labeled nuclei per sensory epithelium in Ad.MT58A-infected
utricles increased moderately from 5 to 7 DPV, and then declined
by 47% between 7 and 10 DPV (Figs. 3C, 6A–B; mean BrdU-
positive supporting cells at 7 DPV =414654, mean BrdU-positive
supporting cells at 10 DPV =228644; n=4 utricles per culture
period). The mean number of Ki-67-labeled nuclei per sensory
epithelium exhibited a similar temporal pattern, but the percent
decrease (34%) was smaller between 7 and 10 DPV (Fig. 6A–B).
Analysis of the percentage of Ki-67-positive supporting cells that
did not label with antibodies to BrdU showed that cells in G1
accumulated from 5 to 7 DPV, and then many entered S-phase,
exited the cell cycle, or died between 7 and 10 DPV (Fig. 6C;
percentage of Ki-67-positive cells that were negative for BrdU at 7
Figure 3. BrdU-labeled cells in utricles co-infected with Ad.O, Ad.K, Ad.S, and Ad.MT58A are present after four weeks in culture and
many appear in doublets. (A–B) High-resolution (636/1.4 NA) views both orthogonal (A) and parallel to the apical-basal axis of supporting cells
and hair cells in a co-infected utricle that was cultured for 7 days post-virus. BrdU was included in the culture medium for the entire period after virus
washout. Hair cells and nuclei that have entered S-phase are labeled with antibodies to myosin VIIA (magenta) and BrdU (green), respectively. The
two BrdU-positive nuclei in B appear to be a division pair, with one positioned at the level of hair cell nuclei and the other at the supporting cell
nuclear layer. Scale bar in A, 20 mm. Scale bar in B, 5 mm. (C) Graph shows quantification of the mean number of BrdU-positive nuclei per sensory
epithelium versus time in culture. Data from co-infected utricles are shown in gray, and data from control utricles infected with GFP are shown in
black. (D) Graph shows the percentage of BrdU-positive nuclei that appeared as doublets in co-infected utricles (same utricles used for the gray data
points in C). Subtracting the percentage from 100 yields the percentage of nuclei that appeared as singlets. (E) Confocal image of a co-infected utricle
fixed at 8 days post-virus and labeled with antibodies to BrdU (green) and activated caspase 3 (red). Arrow points to a pyknotic nucleus that labeled
with both antibodies. Scale bar, 10 mm.
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DPV =4767%; percentage of Ki-67-positive cells that were
negative for BrdU at 10 DPV =3067%). We also analyzed the
percentage of the BrdU-positive population that did not label with
antibodies to Ki-67 and found a steady increase from 4868% at 5
DPV to 6065% at 10 DPV, indicating cells progressively exited
the cell cycle with increased time in culture (Fig. 6C). Regression
analysis showed the increase was linear with an exit rate of 2.3%
per day (r2=0.94).
The nuclei of cells that have just completed cytokinesis are
typically smaller than nuclei in later phases of the cell cycle ,
and the majority of BrdU-positive/Ki-67-negative nuclei appeared
smaller than their BrdU-positive/Ki67-positive counterparts
(arrows in Fig. 6A). To further assess whether most cycling cells
were proceeding to mitosis, we fixed other cultures at 7 DPV and
co-labeled them with antibodies to phosphorylated serine 10 on
histone H3 (PH3-Ser10) and Ki-67. Antibodies against PH3-Ser10
recognize chromatin condensation of mitotic cells in M phase .
Quantification showed the mean number of PH3-Ser10-labeled
supporting cells and the percentage of the Ki-67-positive
population that was also PH3-Ser10-positive were both low
(Fig. 6D, F; mean PH3-Ser10-positive nuclei per sensory
epithelium =561, percentage of PH3-Ser10-positive/Ki-67-
positive nuclei =1.760.4%; n=6 utricles). Preliminary analysis
showed that the numbers of PH3-Ser10-positive nuclei were
similar at 5, 6, and 8 DPV (n=2 utricles per culture duration, data
not shown). While these data do not rule out that progression to M
phase may be inefficient in Ad.MT58A-infected utricles, they
indicate that at least some supporting cells that reenter the cell
cycle can proceed to mitosis.
The percentage of cells in M phase is similar in
Ad.MT58A-infected utricles and developing utricles from
M phase is typically the most rapid phase of the cell cycle [51–
53], so immunocytochemisty with antibodies to PH3-Ser10 may
detect few mitotic cells, particularly if a population of proliferating
cells is not cell cycle synchronized. Thus, we sought to determine
how the percentage of mitotic supporting cells in Ad.MT58A-
infected utricles compared to those in the sensory epithelium of
embryonic utricles developing in vivo. For this, we fixed utricles
from embryonic day 17.5 (E17.5) mice and labeled them with
antibodies to Ki-67 and PH3-Ser10 to quantify the number of
actively cycling cells in M phase within the sensory epithelium at
the time of fixation. Terminal mitoses and expansion of the
utricular sensory epithelium in mice do not cease until several days
after birth, and significant numbers of cycling cells can still be
detected at E17.5 [4,19].
The mean number of PH3-Ser10-positive nuclei in the E17.5
sensory epithelium was significantly higher than in utricles infected
with Ad.MT58A (Fig. 6E–F; mean PH3-Ser10-positive cells =
Figure 4. Infection with Ad.O, Ad.K, or Ad.MT58A leads to detectable increases in Oct3/4, Klf4, or c-Myc protein levels in some
supporting cells. Utricles were separately infected with Ad.O, Ad.K, Ad.S, Ad.MT58A, or Ad.GFP (56108TU/mL) and fixed at 3 days post-virus.
Shown are single confocal slices (0.75 mm z-thickness) at the level of the supporting cell nuclei. Supporting cell nuclei are labeled with the DNA dye,
DRAQ5 (blue). The same amplifier gain and offset settings used to acquire images of the treated samples were used for acquiring images of their
respective Ad.GFP controls. Note the similar intensity of Sox2 labeling in utricles infected with Ad.S and Ad.GFP. Scale bar for A–H, 10 mm.
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Figure 5. Infection with Ad.MT58A is both necessary and sufficient for the observed proliferative response. (A–D) Confocal images of
utricles that were individually infected with Ad.O, Ad.K, Ad.S, or Ad.MT58A and cultured in the presence of BrdU for 5 days post-virus (adenovirus
concentrations equal to those used for co-infection experiments). Significant BrdU labeling (green) is only detected in the sensory epithelium of a
utricle infected with Ad.MT58A. Hair cells are labeled with an antibody to myosin VIIA (purple). (E–F) Confocal images of utricles infected with 16109
TU/mL (56) and 26109TU/mL (10x) of Ad.MT58A. Inset in F shows a zoomed region of the sensory epithelium. Scale bar for inset, 20 mm. (D’–F’)
Confocal images of the BrdU channel in D–F without the myosin VIIA overlay. White dashed lines demarcate the borders of the sensory epithelium.
Scale bar for A–F’, 100 mm. (G) Quantification of the number of BrdU-labeled nuclei per sensory epithelium for the different adenovirus combinations
tested. O: Ad.O, K: Ad.K, S: Ad.S, M: Ad.MT58A, OKSM=co-infection with Ad.O, Ad.K, Ad.S, and Ad.M T58A. The difference in the number of BrdU-
positive nuclei in co-infected utricles and utricles infected with Ad.MT58A did not reach statistical significance, but the increases in BrdU-positive
nuclei at higher concentrations of Ad.MT58A were significant (asterisks indicate a significant difference from all other conditions; p,0.05; One-way
ANOVA with Tukey’s Test of Multiple Comparisons; n=4 utricles).
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1061; p,0.05, Student’s t-test; n=4 utricles). However, the
percentage of the actively cycling population in M phase was
comparable to that in Ad.MT58A-infected utricles at 7 DPV
(Fig. 6E–F; percentage of PH3-Ser10-positive/Ki-67-positive
nuclei in E17.5 utricles =1.060.1%; p.0.05, Student’s t-test).
The similar percentages suggest that Ad.MT58A infection may
result in significant levels of cell division. A study examining cyclin
D1 overexpression in adult mouse utricles found a similarly low
percentage of PH3-Ser10-positive/Ki-67-positive supporting cells
and concluded that M phase progression was rare and inefficient
. Precisely quantifying M phase progression efficiency may be
difficult, and cell cycle progression may be different in vitro
compared to the utricle’s natural environment in vivo; therefore,
the ultimate determination of the ability of these methods to
stimulate significant cell replacement may have to wait for tests of
recovery in cell number using an in vivo damage model.
A portion of the supporting cells that reenter the cell
cycle remain viable
Knock-down of pocket proteins and cyclin dependent kinase
inhibitors that limit cell cycle progression have been shown to
stimulate division of hair cells and supporting cells; however, the
progeny of these divisions rapidly die and can disrupt the integrity
of the sensory epithelium [54–62]. Since many proliferating
supporting cells underwent apoptosis after Ad.MT58A infection
Figure 6. Supporting cells that reenter the cell cycle after Ad.MT58A infection can progress to mitosis. (A) Confocal images show an
Ad.MT58A-infected utricle (16109TU/mL) that was fixed at 7 days post-virus (DPV) and co-labeled with antibodies to BrdU (red) and Ki-67 (green).
Phalloidin labeling (grayscale) is shown to aid in visualizing the borders of the sensory epithelium. White dashed lines demarcate the borders of the
sensory epithelium. Scale bar, 100 mm. Insets show high-resolution views of nuclei in the sensory epithelium. Arrows indicate nuclei that labeled with
antibodies to BrdU but not Ki67. Scale bar for insets, 5 mm. (B) Graph shows the mean number of BrdU-labeled nuclei (green data points) and Ki-67-
labeled nuclei (red data points) per sensory epithelium at 5, 7, and 10 DPV. (C) Graph shows quantification of the percentage of the BrdU-positive
population that did not label with Ki67 antibodies (green data points) and the percentage of the Ki-67-positive population that did not label with
BrdU antibodies (red data points). (D) Confocal image of an adult mouse utricle infected with Ad.MT58A (16109TU/mL) that was fixed at 7 DPV and
co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). (E) Confocal images of a utricle from an embryonic day 17.5 (E17.5) mouse that was
fixed in vivo and co-labeled with antibodies to PH3-Ser10 (white) and Ki-67 (red). Phalloidin labeling (grayscale) is shown to aid in visualizing the
borders of the sensory epithelium. White dashed lines demarcate the borders of the sensory epithelium, and arrows in D and E indicate PH3-Ser10/Ki-
67 co-labeled nuclei. Scale bar for D–E, 100 mm. (F) Graph shows quantification of the percentage of the Ki-67-positive populations that labeled with
antibodies to PH3-Ser10. The difference in the percentage of PH3-Ser10-positive/Ki-67-positive nuclei did not reach statistical significance (p.0.05;
Student’s t-test). The numbers above the gray bars indicate the mean number of PH3-Ser10-labeled nuclei per sensory epithelium. There were
significantly more PH3-Ser10-positive nuclei in E17.5 utricles (p,0.05; Student’s t-test).
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(Figs. 3C, 6B), we sought to determine whether the BrdU-labeled
cells detected at 10 DPV were cells that had just recently entered
S-phase or whether they replicated their DNA early in the culture
and then survived. When we cultured Ad.MT58A-infected utricles
(16109TU/mL) with BrdU for the first 5 DPV, washed it out, and
then cultured for 5 more days (i.e. to 10 DPV) in its absence, the
mean number of BrdU-labeled nuclei per sensory epithelium was
lower than in utricles subjected to continuous BrdU labeling for all
10 DPV (Fig. 7A–D; mean BrdU-positive nuclei per sensory
epithelium at 10 DPV after 1–5 DPV BrdU pulse =194621;
n=4 utricles); however, this difference failed to reach significance
(p.0.05, Student’s t-test). Similar trends were observed when we
used the same pulse-labeling regimen on cultures co-infected with
Ad.O (56107TU/mL), Ad.K (26108TU/mL), Ad.S (56107
TU/mL), and Ad.MT58A (26108TU/mL) or infected with the
lower concentration of just Ad.MT58A (26108TU/mL; Fig. 7D).
The percent difference between the number of BrdU-labeled
cells in utricles fixed at 5 DPV after continuous BrdU labeling and
in utricles fixed at 10 DPV after the 1–5 DPV BrdU pulse was
44%, which indicates that 66% of cells entering S-phase prior to 5
DPV are able to survive out to 10 DPV. Furthermore, some of the
surviving cells persisted for several weeks since we still detected
BrdU-labeled nuclei in the sensory epithelium when we pulse
labeled with BrdU from 1–5 DPV and waited to fix the cultures
until 21 DPV (Fig. 7E).
To determine whether supporting cells in Ad.MT58A-infected
utricles retained the ability to enter S-phase after extended culture,
we pulse labeled with BrdU from 18–21 DPV and fixed the
cultures at 21 DPV. Labeling with antibodies to BrdU and Ki-67
revealed that supporting cells were still actively cycling and
entering S-phase during this period (Fig. 7F). Together, the results
suggest that a portion of the cells that reenter the cell cycle after
Ad.MT58A infection remain viable, and some retain the ability to
enter S-phase for at least 3 weeks in culture.
Figure 7. Some supporting cells in Ad.MT58A-infected utricles survive for weeks in culture after reentering the cell cycle. (A)
Diagram depicting the BrdU labeling paradigm. BrdU was either included in the culture medium for the entire culture period after infection with
adenovirus, or it was washed out at 5 days post-virus (DPV) and utricles were cultured for an additional 5 d in its absence. (B–C) Confocal images
show Ad.MT58A-infected utricles (16109TU/mL) fixed at 10 DPV after being cultured with BrdU from 1–10 DPV (B) or 1–5 DPV (C). Scale bar for B–C,
100 mm. (D) Graph shows quantification of the number of BrdU-labeled cells in the sensory epithelium from utricles cultured as depicted in A (gray
and white bars). Quantification of 5 DPV BrdU labeling (same as in Fig. 5G) is shown to visualize the decline in BrdU-labeled cells from 5 DPV (black
bars) to 10 DPV. Data shown is from co-infection experiments (OKSM), infection with 26108TU/mL Ad.MT58A (16M), and infection with 16109TU/
mL Ad.MT58A (56M). (E) Confocal image of an Ad.MT58A-infected utricle (16109TU/mL) fixed at 21 DPV after being cultured with BrdU from 1–5
DPV. Antibody labeling for BrdU and Ki-67 is shown in green and red, respectively. Scale bar, 100 mm. (F) Confocal image of an Ad.MT58A-infected
utricle (16109TU/mL) fixed at 21 DPV after being cultured with BrdU from 18–21 DPV. Antibody labeling for BrdU and myosin VIIA is shown in green
and magenta, respectively. Scale bar, 100 mm. (G) Confocal image of an Ad.MT58A-infected utricle fixed at 10 DPV after switching from growth
medium to differentiation medium at 5 DPV. BrdU (green) was included in the medium throughout. Scale bar, 100 mm. (H) Graph shows the mean
number of BrdU-positive nuclei per sensory epithelium at 5, 10, and 14 DPV for the experiment described in G. White dashed lines demarcate the
borders of the sensory epithelium in all panels.
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BrdU-labeled cells with hair-cell-like characteristics
appear after culturing Ad.MT58A-infected utricles in
serum free medium
Exchanging growth medium for differentiation medium can
promote the differentiation of otic progenitors into hair cells
[31,63,64]. Differentiation medium is serum-free, however, and c-
Myc-expressing cells can enter apoptosis after withdrawal of
growth factors . Thus, we were surprised when we saw an
increase in the number of BrdU-labeled cells in Ad.MT58A-
inected utricles (16109TU/mL) after we exchanged growth
medium for differentiation medium at 5 DPV and fixed the
cultures at 10 DPV (Fig. 7G–H; mean BrdU-positive nuclei per
sensory epithelium at 10 DPV =5886121; n=4 utricles). This
could possibly be attributed to the T58A mutant, which reduces
the sensitivity of c-MycT58A-expressing cells to apoptosis induced
by serum deprivation [42,43,45]. The increase in the number of
BrdU-labeled cells was only temporary, however, as the levels of
labeled cells decreased 68% between 10 and 14 DPV (Fig. 7H;
mean BrdU-positive nuclei per sensory epithelium at 14 DPV
=186647 BrdU-positive nuclei; n=4 utricles). The reasons for
this temporary delay in cell death remain unclear, but serum
deprivation lengthens G1 in murine fibroblasts overexpressing c-
Myc, so switching to serum-free differentiation medium could
have slowed cell cycle progression and the onset of apoptosis .
Since serum removal temporarily delayed cell death and led to
an increase in the number of BrdU-labeled nuclei, we wondered
whether the accumulation of cells that were potentially dividing
would be accompanied by expansion of the sensory epithelium.
Using myosin VIIA labeling to delineate the sensory epithelium,
we made measurements of the macular area in Ad.MT58A- or
Ad.GFP-infected utricles (16109TU/mL) cultured in differenti-
ation medium from 5–10 DPV. Infection with Ad.MT58A led to
very modest, but significant expansion of the sensory epithelium
0.1960.01 mm2, meanmaculararea ofAd.GFP-infectedutricles =
0.1760.01 mm2; p=0.007, Student’s t-test; n=11 utricles),
which further supported the hypothesis that significant numbers
of supporting cells in Ad.MT58A-infected utricles are able to
progress to M phase and divide.
Although we never detected BrdU-labeled cells that also labeled
with hair cell markers such as myosin VIIA in Ad.MT58A-infected
utricles cultured with growth medium, Ad.MT58-infected utricles
cultured with differentiation medium from 5–10 DPV contained a
small number of BrdU-positive/myosin VIIA-positive cells (mul-
tiple examples shown in Fig. 8; range of 1–4 BrdU-positive/
myosin VIIA-positive cells per utricle; n=4 utricles). These cells
had a chalice shape typical of hair cells, lacked connections with
the basal lamina, and extended from the apical surface to the hair
cell nuclear layer. Some were rounded and did not extend to the
apical surface, suggesting they were damaged or dead (arrowhead
in Fig. 8A). Most were paired with a BrdU-positive nucleus that
did not label for myosin VIIA (arrows in Fig. 8A, B), suggesting a
supporting cell had divided and one of the progeny had become a
new hair cell. None displayed a prototypical, F-actin-rich hair
bundle as determined with fluorescent phalloidin labeling,
indicating these cells were not fully differentiated hair cells with
functional mechanotransduction apparatuses. Myosin VIIA is
present in stereocilia, however, and small projections from the
apical surface could be seen with myosin VIIA-labeling, suggesting
that hair bundles may have been in the very early phases of
formation (Fig. 8A, B). Since we observed a small number of GFP-
expressing hair cells after infection with Ad.GFP (Fig. 1G–H), we
cannot rule out that the BrdU-positive/myosin VIIA-positive cells
were preexisting hair cells that reentered the cell cycle after being
transduced with Ad.MT58A. However, this appears unlikely since
we never observed such cells in Ad.MT58-infected utricles
cultured with just growth medium. Also, we observed 1–3 BrdU-
positive nuclei in the Ad.GFP-infected control utricles that we
cultured with differentiation medium from 5–10 DPV, but none of
these cells were myosin VIIA-positive. The results suggest that at
least a small portion of supporting cells within intact vestibular
organs may retain competency for differentiating into hair-cell-like
cells after Ad.MT58A-induced cell cycle reentry.
Here we examined how adenoviral delivery of the four iPSC
transcription factors to adult mouse utricles affects regenerative
potential within the sensory epithelium in vitro. We found that
adenovectors encoding the T58A variant of c-Myc can initiate cell
cycle reentry of postmitotic supporting cells. A portion of the cells
that reenter the cell cycle survive for weeks in culture, proceed to
mitosis, and appear to express the hair cell marker myosin VIIA
under differentiating culture conditions. Cell cycle reentry also
corresponded with a very modest, but significant expansion of the
sensory epithelium, suggesting that ectopic expression of MYC
genes may be capable of stimulating regrowth of the sensory
epithelium after cells have been lost to damage.
Suppression of MYC may limit the proliferative potential
of supporting cells in adult mammals
Although we observed low numbers of supporting cells
ectopically expressing c-MycT58A, infection with Ad.MT58A
led to robust supporting cell S-phase entry, suggesting that c-Myc
levels may be finely balanced within the utricle to suppress
proliferation and maintain the postmitotic state. The half-life of c-
MycT58A is shorter than that of Oct3/4 and Klf4 in some cell
types (Klf4, 120 min in human esophageal cancer cells [67,68];
Oct3/4, 90 min in mouse embryonic carcinoma cells ; and c-
MycT58A, 51–63 min in NIH3T3 and REF52 fibroblasts
[43,45]), but the differences in stability do not appear great
enough to explain the large differences in the number of
supporting cells expressing detectable levels of Oct3/4 and Klf4
compared to c-MycT58A (Fig. 4). This suggests that cells within
the sensory epithelium of the adult mouse utricle may have active
mechanisms for suppressing c-Myc protein levels, most likely at the
post-transcriptional level since the CMV promoter efficiently
drives gene expression in supporting cells (Figs. 1,4).
Because there were few cells that labeled with antibodies to c-
Myc and HA, we were not able to determine whether
Ad.MT58A infection induced proliferation through autonomous
or non-autonomous effects. Given c-Myc’s well-documented role
in directly regulating cell cycle machinery , it seems unlikely
that supporting cell proliferation was stimulated by paracrine
signaling from neighboring cells that were transduced by
Ad.MT58A, especially since we did not detect any c-Myc-
labeled nuclei in the sensory epithelium of Ad.GFP-infected
controls. However, c-Myc overexpression has recently been
shown to down-regulate the secretion of proteins that inhibit
proliferation in a nontransformed epithelial cell line .
Therefore, uncovering the mechanism by which Ad.MT58A
drives proliferation could reveal novel insights into how the
sensory epithelium maintains the postmitotic state, and it will be
supporting cells into otic progenitors.
Upstream signals that might limit c-Myc translation or promote
its degradation in supporting cells have not been identified, but F-
actin and the tumor suppressor E-cadherin accumulate at
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mammalian supporting-cell-supporting-cell junctions in coordina-
tion with the postnatal decline in regenerative capacity, and it has
been posited that the signals and molecules responsible for
[4,17,18,72]. In epithelial cells, c-Myc specifically activates or
represses E-cadherin depending on the expression levels of the two
proteins encoded by MYC, c-Myc1 and c-Myc2, and increased E-
cadherin expression in response to increased c-Myc levels can
prevent cellular transformation [73,74]. This sensitive feedback
loop could potentially explain the concomitant decline in
proliferation and accumulation of E-cadherin in mammalian
supporting cells. c-Myc is a component of many signaling
networks, having been estimated to regulate ,30% of genes
, so potential repressors of c-Myc are numerous.
MYC gene family members are potential targets for
stimulating cell replacement in the mammalian inner ear
The supporting cell proliferation we observed in adult mouse
utricles after Ad.MT58A infection suggests that targeted upregula-
tion of c-MycT58A or possibly even wild-type c-Myc may be a
viable strategy for stimulating cell replacement in mammalian
inner ear sensory epithelia. The MYC gene family members, which
in mammals include the oncogenes MYC (c-Myc), MYCL (L-Myc),
and MYCN (N-Myc), are basic Helix-Loop-Helix Leucine Zipper
transcription factors that play a prominent role in regulating cell
proliferation, growth, apoptosis, metabolism, and differentiation
[65,70,76]. Despite the robust proliferative response we observed
after Ad.MT58A infection, conditional deletion of c-Myc in the
embryonic mouse inner ear has no phenotype, while deletion of N-
Myc reduces proliferative growth and disturbs morphogenesis
[77,78]. Some proliferation was still detected in N-Myc mouse
mutants, and it remains unclear whether this was due to
compensation by another MYC family member like c-Myc. These
results suggest N-Myc could play a more prominent role in
controlling proliferative regeneration in the mammalian inner ear,
and overexpression of N-Myc in adults could be more efficient at
inducing cell proliferation and promoting survival and differenti-
ation into the correct cell types.
MYC can be a potent oncogene, so MYC gene therapy may not
be a realistic approach for stimulating regeneration in humans
. In addition, c-MycT58A is more efficient than wild-type c-
Myc at inducing immortalization and transformation [80,81],
which increases the probability that some cells in our cultures were
being immortalized or transformed. In support of this possibility,
we observed cells actively in the cell cycle and entering S-phase
after 3 weeks in culture (Fig. 7). Consequently, small molecules
that target MYC and its binding partners may be more useful for a
therapy that can be used in humans.
Similarities and differences between proliferation
induced by cyclin D1 and c-MycT58A
Adenovector-mediated expression of cyclin D1 (Ad.CD1) in
cultured utricles from adult mice robustly induces supporting cell
S-phase entry [39,41]. At 7 d after Ad.CD1 infection, 0.6% of the
actively cycling cells (i.e. Ki-67-positive) were in M-phase (i.e PH3-
Ser10-positive). This percentage was ,4-times lower than in P9
mouse utricles infected with Ad.CD1, and it was ,3-times lower
than the percentage we observed in Ad.MT58A-infected utricles
Figure 8. Supporting cells that reenter the cell cycle in Ad.MT58A-infected utricles may be capable of differentiating towards a
hair-cell-like fate. Shown are confocal images taken from Ad.MT58A-infected utricles (16109TU/mL) fixed at 10 days post-virus (DPV) and labeled
with antibodies to BrdU (green) and myosin VIIA (magenta). Growth medium was exchanged for differentiation medium at 5 DPV. (A–B) Confocal
images show views parallel to the long axis of the hair cells. The arrowhead points to a BrdU-positive/myosin-VIIA-positive cell that does not extend
to the apical surface and is probably damaged or dying. The other BrdU-positive/myosin-VIIA-positive cells extend from the hair nuclear layer up to
the apical surface, where they appear to display tiny, bundle-like projections. BrdU-positive/myosin-VIIA-positive cells are located within close
proximity to these hair-cell-like cells (arrows), suggesting a divisional pair. Scale bars, 5 mm. (C–E) Confocal images of three hair-cell-like cells at the
level of the hair cell nuclear layer. Arrow in C indicates a neighboring cell that is BrdU-positive/myosin-VIIA-negative. Scale bars, 5 mm.
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from adults (Fig. 6). A thorough analysis, which showed DNA
damage in Ki-67-positive cells, minimal Aurora B kinase antibody
labeling, few cells in cytokinesis, few cells completing multiple
rounds of division, and no hyperplasia of the sensory epithelium
led to the conclusion that ectopic cyclin D1 expression in
supporting cells was inefficient at inducing cell cycle progression
Determining the exact percentage of cells capable of proceeding
to M-phase may be difficult, especially if the proliferating
population is not cell cycle synchronized. We detected similar
numbers of PH3-Ser10-positive cells at 5, 6, 7, and 8 DPV, which
suggests S-phase entry after Ad.MT58A infection is stochastic.
Mitosis is typically the shortest phase of the cell cycle, making cells
in M-phase the smallest fraction of the cycling population, and we
detected low numbers of mitotic cells in both embryonic utricles
from developing mice and Ad.MT58A-infected utricles from adult
mice. While the percentage of cells in M phase was higher in
Ad.CD1-infected utricles, the percentages in all cases were
relatively low, underscoring the need for further characterization
of cell cycle progression under all these conditions. It should be
noted that many supporting cells are exiting the cell cycle around
E17.5 in the utricle , and in the rat retina, cell cycle length
increases as more cells become postmitotic . Therefore, it is
possible that a significantly higher percentage of M phase cells may
be present in the utricle at timepoints earlier than E17.5. In
addition, ectopic c-Myc expression shortens the duration of G1
phase in murine fibroblasts and reduces the synchronization of cell
cycle entry, consistent with our findings here . The shortened
G1 phase duration could increase the percentage of cells in M
phase, potentially explaining why we found a higher percentage of
M phase cells in utricles infected with Ad.MT58A compared to
Can ectopic c-MycT58A expression be used to reprogram
supporting cells into multipotent otic progenitors?
The appearance of a small number of BrdU-positive/myosin-
VIIA-positive cells after culturing in differentiation medium
suggests that at least some supporting cells that reenter the cell
cycle after Ad.MT58A infection retain otic identity and
competency for differentiating into hair-cell-like cells. The
paucity of these cells could be attributed to the inability of the
culture environment to replicate induction cues that may be
present in vivo. Consistent with this notion, proliferation induced
by hair cell death in newborn mouse utricles gave rise to new
hair cells in vivo but not in vitro . Alternatively, we did observe
a small number of hair cells that were transduced with Ad.GFP,
and we have not yet ruled out that the BrdU-positive/myosin
VIIA-positive cells were pre-existing hair cells that reentered the
cell cycle after being transduced with Ad.MT58A. Conditional
deletion of the retinoblastoma protein or ectopic expression of
the HPV-16 E7 oncogene within hair cells can drive their reentry
into the cell cycle, so it is reasonable to suspect that ectopic
expression ofc-Myc mayproduce
57,59,62,83]. If the BrdU-positive/myosin VIIA-positive cells
we observed did originate from the supporting cell population
infected with Ad.MT58A, then ectopic c-Myc expression may be
capable of dedifferentiating supporting cells into cells with
characteristics of otic progenitors.
The effects of Oct3/4, Klf4, and Sox2 are not observed until
later in the iPSC reprogramming process , and these factors
did not appear to influence proliferation over the time periods we
investigated. Expression of all four factors within individual
supporting cells may have been unlikely since each was encoded
by a separate adenovirus, and it remains to be determined
whether simultaneous expression of Oct3/4, Klf4, and Sox2
would aid in directly reprogramming supporting cells into otic
Transient expression of the four iPSC transcription factors
under cardiac- or neural-promoting culture conditions directly
reprograms fibroblasts into cardiomyocytes or neural stem/
progenitor cells, respectively [24,25]. This short expression
appears to induce epigenetic activation that results in an unstable,
partially reprogrammed state that bypasses pluripotency but
remains amenable to differentiation . Similarly, we hypothe-
size that when the four iPSC factors are delivered to terminally
differentiated somatic cells in situ, the native organ environment
may be able to suppress complete reprogramming to induced
pluripotency while allowing direct reprogramming into lineage-
restricted progenitor/stem cells. Lineage-specific transcription
factors have been used to transdifferentiate cardiac fibroblasts
into cardiomyoctes within intact mouse hearts in vivo, which
demonstrates the feasibility of in situ reprogramming .
Furthermore, the bHLH transcription factor Atoh1 is both
necessary and sufficient for hair cell differentiation during
development, and ectopic expression of Atoh1 in situ appears to
directly reprogram inner ear epithelial cells into hair cells in some
instances [86–93]. Directly reprogramming postmitotic supporting
cells into hair cells could decrease the size of the supporting cell
population without some form of nonautonomous cell replacement
, which makes restoration of self-renewal capacity vital to the
reprogramming strategy. Cyclins, cyclin dependent kinase inhib-
itors (CDKIs), and pocket proteins have been shown to regulate
proliferation and cell cycle exit during development of the sensory
epithelium, and a growing list of these genes have been targeted to
force reentry of supporting cells into the cell cycle [41,54–
62,83,94–98]. MYC controls the expression and activity of various
cyclins, CDKIs, and pocket proteins [65,76], which may make it
more suitable for orchestrating the complex fluctuations of cell
cycle proteins that are necessary to drive efficient progression
through the various restriction points. If MYC alone is unable to
reprogram supporting cells into true otic progenitors, then
combining forced cell cycle reentry – either via a MYC family
member or direct targeting of cell cycle machinery – with ectopic
Atoh1 expression may be a more successful cocktail.
In summary, ectopic MYC expression shows promise for
restoring proliferative capacity to inner ear sensory epithelia and
may be used to stimulate regeneration. Further investigations
should test this potential by characterizing supporting cell
genotype, phenotype, transformation, survival, and differentiation
after ectopic c-MycT58A expression in both auditory and
vestibular organs in vivo.
Conceived and designed the experiments: JCB JJY JDJ. Performed the
experiments: JCB. Analyzed the data: JCB. Contributed reagents/
materials/analysis tools: JCB JJY AA JDJ. Wrote the paper: JCB JJY AA
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