Sleeping beauty: awakening urothelium from its slumber


REVIEW

Sleeping beauty: awakening urothelium from its slumber

Zarine R. Balsara1,2 and Xue Li1,2
1Department of Urology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts; and 2Department
of Surgery, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts

Submitted 17 June 2016; accepted in final form 22 January 2017

Balsara ZR, Li X. Sleeping beauty: awakening urothelium from its slumber. Am
J Physiol Renal Physiol 312: F732–F743, 2017. First published January 25, 2017;
doi:10.1152/ajprenal.00337.2016.—The bladder urothelium is essentially quiescent
but regenerates readily upon injury. The process of urothelial regeneration harkens
back to the process of urothelial development whereby urothelial stem/progenitor
cells must proliferate and terminally differentiate to establish all three urothelial
layers. How the urothelium regulates the level of proliferation and the timing of
differentiation to ensure the precise degree of regeneration is of significant interest
in the field. Without a carefully-orchestrated process, urothelial regeneration may
be inadequate, thereby exposing the host to toxins or pathogens. Alternatively,
regeneration may be excessive, thereby setting the stage for tumor development.
This review describes our current understanding of urothelial regeneration. The
current controversies surrounding the identity and location of urothelial progenitor
cells that mediate urothelial regeneration are discussed and evidence for each model
is provided. We emphasize the factors that have been shown to be crucial for
urothelial regeneration, including local growth factors that stimulate repair, and
epithelial-mesenchymal cross talk, which ensures feedback regulation. Also high-
lighted is the emerging concept of epigenetic regulation of urothelial regeneration,
which additionally fine tunes the process through transcriptional regulation of cell
cycle genes and growth and differentiation factors. Finally, we emphasize how
several of these pathways and/or programs are often dysregulated during malignant
transformation, further corroborating their importance in directing normal urothe-
lial regeneration. Together, evidence in the field suggests that any attempt to exploit
regenerative programs for the purposes of enhanced urothelial repair or replace-
ment must take into account this delicate balance.

urothelium; regeneration; progenitor cells; superficial cells; label retention; lineage
tracing; epithelial-mesenchymal cross talk; epigenetics

THE UROTHELIUM IS A UNIQUE EPITHELIAL SURFACE that lines most
of the genitourinary tract, including the renal pelvis, ureters,
bladder, and proximal urethra. Urothelium consists of multiple
layers of epithelial cells that can change size and shape to
accommodate fluctuating volumes of urine. This mucosal ep-
ithelial surface also serves as a barrier to prevent absorption of
toxic substances like acid and urea from the urine and to defend
against entry of pathogens from the external environment (30).
Implicit in this latter function is the ability of urothelium to
“sense” and respond to the presence of pathogens within the
genitourinary tract (88). Coordinating other cues from the
external environment, such as chemical, thermal, and mechan-
ical stimuli, requires an additional layer of sophistication (38).
Besides direct expression of neuronal sensory receptors and ion
channels on urothelial cells and their ability to release chemi-

cals and neurotransmitters, afferent nerves also innervate the
detrusor muscle and extend into the urothelial layer to help the
bladder respond to external stimuli (8, 9, 21, 64, 107).

Urothelium Comprises Three Major Cell Types

Despite this wide range of functions, the urothelium has a
relatively simple structure comprising three main cell types
that are distinguished by their location, size, and expression of
molecular markers. Directly facing the luminal surface are
large (50 –120 �m in diameter) multinucleated hexagonal cells
known as superficial or umbrella cells, which are principally
responsible for the barrier function of the urothelium (103).
Adjacent superficial cells are connected by tight junction pro-
teins including claudin-8 and zona occludens 1 (ZO-1) that
restrict exchange of ions and solutes between cells and between
urine and blood (1, 32, 75). Superficial cells are covered by a
crystalline lattice comprising four major uroplakin proteins that
together form asymmetric unit membrane (AUM) plaques.
These plaques further restrict permeability to water, solutes,
and toxins (37, 90, 105, 106). Superficial cells also contribute

Address for reprint requests and other correspondence: X. Li, Depts. of
Urology and Surgery, Boston Children’s Hospital, Harvard Medical School,
300 Longwood Avenue, Boston, MA 02115 (e-mail: sean.li@childrens.harvard.
edu).

Am J Physiol Renal Physiol 312: F732–F743, 2017.
First published January 25, 2017; doi:10.1152/ajprenal.00337.2016.

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http://doi.org/10.1152/ajprenal.00337.2016.
mailto:sean.li@childrens.harvard.edu
mailto:sean.li@childrens.harvard.edu


to the plasticity in urothelial cell surface area through a
regulated process of endocytosis or exocytosis of discoidal/
fusiform-shaped vesicles (DFVs) containing uroplakins (47,
50, 109). Underlying the superficial cell layer is a layer of
intermediate cells that are significantly smaller (20 �m in
diameter) than superficial cells. Finally, along the basement
membrane is a layer of basal cells. Despite being the smallest
population in size (5–10 �m in diameter), basal cells constitute
the most abundant population of cells in adult urothelium.
Given the substantial size discrepancy between superficial and
either basal or intermediate cells, it is no surprise that histo-
logical analysis of whole-mount adult mouse bladders has
revealed that one superficial cell spans the area of ~40 under-
lying intermediate/basal cells (36). Depending on the species,
there can be as few as three discrete layers of urothelial cells
in the mouse bladder and up to seven layers in the human
bladder, with the additional layers contributed by interme-
diate cells (11).

In addition to the size discrepancy among urothelial cells in
the different layers, urothelial cells can also be distinguished
by molecular differentiation markers, which begin to be ex-
pressed at different stages of embryogenesis (Fig. 1). Superfi-
cial cells represent terminally differentiated cells and are the
only cell layer in the bladder to express the low-molecular-
weight cytokeratin 20 (Krt20) (29, 68, 82). Superficial cells
also express several uroplakins (Upk) but lack expression of
the high-molecular-weight cytokeratin Krt5, the transcriptional
factor p63, and signaling molecule Sonic Hedgehog (Shh) (25).
Similar to superficial cells, most intermediate cells are Upk�

and Krt5�, but in contrast, intermediate cells also express p63.
Diverging from superficial and intermediate cells, basal cells
distinguish themselves by expression of high levels of Krt5 and
p63 but are negative for Upk and Krt20. Notably, despite the
previous assumption that each of the three urothelial layers
comprises a homogenous population of cells based on these
five markers, our recent findings suggest that there is, in fact,
significant urothelial cell heterogeneity. For example, variable
levels of histone H3 lysine 27 trimethylation (H3K27me3), an
epigenetic modification often associated with gene silencing,
are apparent among urothelial cells within the Krt5� basal as
well as Krt5� intermediate and superficial cells (26a). In
addition, ~14% of Krt5� basal cells also express Krt14. While
the significance of urothelial cell heterogeneity remains to be
determined, the Krt5�/Krt14� subpopulation of basal cells

may play an important role in urothelial regeneration and
tumorigenesis (to be discussed in subsequent sections) (55, 77,
81). Emerging techniques like single-cell RNA sequencing
(scRNA-seq) may further stratify cells within each discrete
layer and potentially identify functional differences among
cells in each layer.

Normally Quiescent Urothelium Rapidly Regenerates in
Response to Injury

Unlike the epithelium of the skin and intestine, mature
urothelium has a very low mitotic index and turnover rate.
Pulse-labeling of unstimulated rat bladders with tritiated thy-
midine revealed a labeling index of ~0.2– 0.5% (14, 30, 61, 65)
while uninjured mouse bladders had an even lower labeling
index of 0.11% (36, 58). Similarly, bladder biopsies from
normal human patients that were cultured with tritiated thymi-
dine in vitro had a labeling index of 0.12% (28). Based on these
low labeling indexes, turnover rates of quiescent rodent urothe-
lium have been estimated to be approximately once every 200
days (14, 30). The prevailing quiescence of the urothelium
makes its ability to awaken rapidly in response to damage even
more remarkable. Within hours of chemical injury with cyclo-
phosphamide (CPP) or protamine sulfate (PS) or biological
insult with uropathogenic Escherichia coli (UPEC), the urothe-
lium begins to proliferate and initiate the process of regener-
ation (Fig. 2) (25, 71, 84). One can imagine that urothelial
regeneration needs to be carefully controlled. Incomplete re-
generation results in potential breaches in barrier function (Fig.
3) whereby toxic substances or pathogens in the urine can gain
access to the bloodstream, stimulate local tissue inflammation,
and/or depolarize afferent nerve fibers. In fact, this last process
has been hypothesized as being a potential cause of bladder
pain syndrome or interstitial cystitis (44, 83, 94). Conversely,
unrestrained regeneration can lead to urothelial hyperplasia and
possible malignant transformation (Fig. 3). An understanding
of the molecular mechanisms responsible for maintaining the
delicate balance between urothelial quiescence and regenera-
tion is critical for devising new clinical strategies to prevent or
treat diseases of the urothelium.

Given the priority of maintaining a protective barrier, it is
not surprising that one of the first steps in urothelial regener-

SC

IC

BC

(Shh- p63- Upk+  Krt5- Krt14- Krt20+)     

(Shh+  p63+ Upk+ Krt5- Krt14- Krt20-)     

(Shh+  p63+ Upk- Krt5+  Krt14-/+  Krt20-)     

A

B

<E10

Marker

Onset 
(Embryonic day, E) 

~E11 E14 E16.5 ~E17

Shh p63 Upk Krt5 Krt14 Krt20

Adult urothelium

Fig. 1. Urothelium is stratified into three major cell types. A: temporal
expression of several key urothelial cell markers is depicted. Shh is the earliest
of these markers to be expressed in the urothelium, while the cytokeratins are
expressed much later in embryogenesis. B: adult urothelium comprises basal
cells (BC), intermediate cells (IC), and superficial cells (SC), each of which
have discrete patterns of cell marker expression.

Time after injury

U
ro

th
el

ia
l c

el
l p

ro
lif

er
at

io
n

Injury

0h 12h 24h 48h36h 72h

Fig. 2. Adult urothelium is normally quiescent but rapidly responds and
proliferates upon urothelial injury. At baseline, mature urothelium remains in
a quiescent state, with extremely slow turnover. However, in response to
injury, the urothelium rapidly awakens and undergoes proliferation and dif-
ferentiation to restore the damaged epithelium. Maximal proliferation occurs
within 12–36 h, depending on the stimulus, followed by differentiation and a
return to the dormant state.

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ation is re-establishment of tight junctions between the remain-
ing and regenerated superficial cells (54, 56). Ultrastructural
analysis reveals that the de novo superficial cells undergo
successive stages of differentiation, first involving expression
of microvilli, then formation of cells with rounded microridges
that begin to express uroplakins, and finally terminal differen-
tiation in which superficial cells enlarge, adopt a rigid-appear-
ing plasma membrane, and robustly express Upk and Krt20. In
contrast to superficial urothelial injuries, full-thickness wounds
such as partial cystectomy or bladder augmentation presum-
ably require a more elaborate process to repair all layers of the
urothelium and/or smooth muscle. Using primary explant cul-
tures of mouse bladder to examine full-thickness wounds,
Kreft et al. demonstrated that basal cells at the wound edge first
proliferate (54). Through concentration of their actin skeleton
in lamellipodia and at the tip of filopodia, basal cells at the
leading edge of the wound migrate and cover the basolateral
exposed surface. At the same time, overlying superficial cells
flatten and stretch out to cover these new urothelial cells before
reestablishing tight junctions. Within 48 h of injury, cells at the
leading edge begin to express the junctional proteins ZO-1,
claudin-4, occludin, and E-cadherin as well as the superficial
differentiation marker Krt20.

Identifying the Urothelial Progenitor Cells Responsible for
Regeneration Has Been Challenging

In recent years, much emphasis has been placed on identi-
fying a putative population of resident urothelial stem and/or
progenitor cells as these cells can potentially be harnessed to

repair chronically damaged or transformed urothelium, aug-
ment bladders, or repair ureteral or urethral strictures. While
the existence of this population is generally accepted, a major
debate in the field centers on the identity and location of these
progenitor cells within urothelium. Also disputed is whether
different populations of progenitor cells are responsible for
urothelial development and differentiation in embryogenesis
vs. regeneration and repair of injured adult urothelium.

Label retention studies. Two major methods have been used
to characterize these cells, namely DNA label retention studies
and lineage tracing analysis (Table 1). During development
and early post-natal life, proliferating cells can incorporate
nucleoside analogs such as bromodeoxyuridine (BrdU), 5-
ethynyl-2�-deoxyuridine (EdU), or tritiated thymidine. DNA
label retention studies rely on the concept that stem cells are
the slow-dividing cells and thereby retain the nucleoside ana-
logs for extended periods of time. These label retention studies
do not rely on prior knowledge of specific stem/progenitor cell
markers which is advantageous, given the paucity of known
markers of urothelial progenitor cells. Initial DNA label reten-
tion studies by Kurzrock et al. demonstrated that, after BrdU
labeling of 6-wk-old rats, ~9% of urothelial cells retained label
after 1 yr and these cells resided exclusively in the basal cell
layer (55). Subsequent studies uncovered that label retention
cells (LRCs) could actually be found in all three layers of the
urothelium provided that the label was administered between
embryonic day (e)15 and postnatal day (P)1 (13, 91, 110). The
later one administers the label, the more restricted the LRCs
become, ultimately becoming restricted to the basal cell layer.

Normal resolution 

0

-1 +1

0

-1 +1

Hyperplasia 

0

-1 +1

Interstitial cystitis, UTI

Fig. 3. A fine balance is necessary to ensure normal urothelial regeneration after injury. Following injury, several outcomes are possible. Most commonly,
regeneration results in restoration of the urothelium to its original state (designated as “0”). However, failure to fully regenerate the urothelium (designated as
“�1”) results in potential breaches in barrier function that may increase susceptibility to infection or increase sensory fiber stimulation and set the stage for
interstitial cystitis. Alternatively, unrestrained regeneration (designated as “�1”) can lead to urothelial hyperplasia that may ultimately lead to bladder tumor
formation.

Table 1. Comparison of label retention and lineage tracing methods

Advantages Limitations

Label retention Does not require prior knowledge of stem cells Assumes that label retention is an intrinsic property
of stem cells (which is not always true)

Pulse label replicating DNA with BrdU,
EdU, or 3H-TdR, then chase and
observe washout

Technically simple to perform Does not allow one to track the fate of labeled cells
if label gets diluted over time

Genetic lineage tracing Allows one to track the fate of labeled cells and
their progeny over long periods of time

Requires preexisting knowledge of a putative stem
cell marker

Uses genetic tools (e.g., Cre/LoxP) to
indelibly label specific cells and then
track their progeny over time

Labeled cells can be extensively analyzed using
advanced genomic technologies

Requires sophisticated genetic tools to perform

BrdU, bromodeoxyuridine; EdU, 5-ethynyl-2�-deoxyuridine; 3H-TdR, tritiated thymidine.

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Consistent with the quiescent nature of urothelium, the base-
line proliferation of urothelial cells dramatically decreases and
becomes limited to a small (~1%) population of basal cells as
the animal ages. While basal cells appear to be important in
maintaining urothelial homeostasis, Colopy et al. observed that
upon injury with UPEC, proliferation occurs within both basal
and intermediate cell layers (13). In fact, the percentage of
proliferating intermediate cells was reportedly twice that of
basal cells. These findings raised the possibility that adult
regenerating urothelium may recapitulate embryogenesis,
whereby progenitor cells can be activated in both basal and
intermediate cell layers. However, contrary to the assumption
that LRCs represent the definitive urothelial progenitors, LRCs
in both layers comprised only a small proportion of prolifer-
ating cells after injury.

Although label retention studies were previously considered
the gold standard for identifying epithelial stem cells, the
universal reliability of this method has been called into ques-
tion. One significant criticism of using this technique is that
while LRCs in adult urothelium may include the slowly divid-
ing population of progenitor cells, they may also simply
represent labeled, terminally differentiated cells. From the
existing literature it appears that LRC analysis may be more
helpful in predicting the “birth date” of cells within urothelium
than in distinguishing progenitor cells.

Lineage tracing studies. Perhaps a more powerful and reli-
able method for identifying urothelial stem/progenitor cells is
lineage tracing. Unlike label retention studies, lineage tracing
does rely on existing knowledge of putative stem cell markers.
By placing Cre recombinase expression under control of a cell/
tissue-specific promoter, one can indelibly label a cell and all of its
progeny by using a gene reporter and determine whether a
particular cell is truly pluripotent. However, like LRC studies,
lineage tracing does carry a few caveats (Table 1). One caveat is
that lineage tracing, which relies on the use of a Cre transgene,
can suffer from positional effects depending on the site of Cre
insertion within the genome. An additional caveat is that
lineage tracing using a constitutive promoter does not allow
one to distinguish specifically whether labeled cells represent
the progeny of a single multipotent progenitor cell or the
progeny of multiple unipotent progenitor cells. Nevertheless,
through this method, Pignon et al. were able to demonstrate
that urothelial stem cells express the transcription factor p63
(78). P63 encodes for two distinct isoforms, transactivating
(TA) p63 and NH2-terminal truncated (�N) p63, which are
generated by alternative promoters (108). Urothelial cells ex-
pressing the �Np63 isoform in embryogenesis were shown to
give rise to all urothelial cell lineages. However, over time,
terminally differentiated superficial cells lose �Np63 expres-
sion. Cheng et al. additionally highlighted a specific antiapo-
ptotic role for p63 in development of the ventral bladder
urothelium (12). Deletion of p63 leads to absence of the ventral
abdominal and bladder walls in association with markedly
enhanced apoptosis. Furthermore, urothelial cells along the
ventral bladder remain in a state of limbo whereby they remain
undifferentiated and uncommitted. In contrast, the dorsal
urothelium exhibits reduced thickness but superficial cells still
develop, implying that p63 exerts a predominant role in ventral
epithelium during bladder development. Nevertheless, while
p63 may not be essential for differentiation of the dorsal

bladder urothelium, it still appears to play a role in promoting
stem cell survival and proliferation along this axis.

Lineage tracing, combined with urothelial organoid cultures,
have likewise revealed that Shh is a marker for urothelial stem
cells in both embryogenesis and adulthood. Using ShhCreER;
R26mTmG mice injured with multiple rounds of UPEC, Shin et
al. demonstrated that Shh� cells have a long-standing capacity
for self-renewal in adult regenerating urothelium and can give
rise to all three urothelial cell lineages (84). Based on apparent
exclusive localization of mG-labeled reporter cells in the basal
layer prior to injury, followed by regeneration of all urothelial
layers with these mG-labeled cells after injury, this group
argued that urothelial stem/progenitor cells derive from a
subpopulation of basal cells that are specifically Shh�. These
data seem to corroborate earlier studies by Mysorekar et al.,
who demonstrated that UPEC infection of wild-type mice
results in BrdU labeling of basally located cells only (71).
These results also seem consistent with the prevailing notion
that, like epidermis, urothelial differentiation and regeneration
occur in a linear fashion, with basal stem cells progressively
differentiating into the suprabasal cell layers (Fig. 4A) (23, 31).
A more recent report by Papafatiou et al. expands upon this
notion and further suggests that a minor population of Krt5�/
Krt14� basal cells serves as the urothelial progenitors respon-
sible for superficial cell regeneration both during normal ho-
meostasis and after injury (77).

Notably, a separate report by Gandhi et al. challenges this
linear model of regeneration (Fig. 4B) (25). Arguing that Krt5�

Embryonic Adult

A   Linear Model

B   Non-linear Model

Basal Intermediate Superficial

P-cell Intermediate Superficial

Basal SuperficialUnknown progenitor?

Fig. 4. Proposed models of urothelial formation and regeneration. A: the linear
model of urothelial formation and regeneration suggests that the urothelial
stem/progenitor cells resides within the basal cell population, likely the
Krt5�/Krt14� basal cells. These cells are capable of self renewal but also give
rise to intermediate cells, which subsequently give rise to superficial cells. B:
in the nonlinear model, the transient population of P cells, distinguished by
Foxa2 expression, gives rise to intermediate and superficial cells during
embryogenesis. In contrast to the linear model, the nonlinear model argues that
a separate progenitor cell (unidentified at present) initially gives rise to basal
cells during development. In adults, stem cells are thought to reside within both
the intermediate and the basal cell populations and are capable of self-renewal
and/or differentiation into superficial cells. It is unclear, however, whether
stem cells in the basal layer need to go through an intermediate step, such as
transient amplifying cells, before they differentiate into superficial cells.

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cells are detectable only after intermediate and superficial cells
are first formed (Fig. 1A), the authors hypothesized that an
alternative population of cells is likely to be the progenitor for
superficial cells during bladder development. Using a combi-
nation of strategies, including in situ hybridization analysis of
Shh mRNA, lineage tracing with ShhCreERT2;R26mTmG em-
bryos, and marker analysis of ShhGFP/Cre bladders, this group
was able to demonstrate that Shh� cells can actually be found
in both basal and intermediate cell layers, as well as in P cells,
a transient population of Foxa2� cells that are abundant only
during discrete stages in bladder development before basal cell
formation. To test directly whether Krt5� basal cells are the
definitive progenitor cell for all embryonic urothelial layers,
Gandhi et al. performed lineage tracing in Krt5CreER;R26mTmG

mice during embryogenesis. They observed that Krt5� cells
exclusively give rise to basal cells and not to intermediate or
superficial cells. Instead, they characterized a discrete popula-
tion of P cells, which they define as progenitors for interme-
diate and superficial cells during embryogenesis. Notably, the
authors do highlight that P cells are only a transient population
of cells, and, while important during bladder development,
they are no longer detected in mature bladders. The authors
also performed lineage tracing in adult Krt5CreER;R26mTmG

mice after chemical injury with CPP. Consistent with their
findings in embryogenesis that Krt5� cells are not progenitors
for terminally differentiated superficial cells, mG-labeled
Krt5� genetic lineages never gave rise to either intermediate or
superficial cells in the adult urothelium. Rather, lineage tracing
with Upk3aGCE;mCherry adult mice injured with CPP rein-
forced the concept that in mature urothelium intermediate cells
become the source of progenitors that give rise to superficial
cells.

While these latter results provide compelling evidence
against a linear model of urothelial regeneration that begins
with basal progenitor cells giving rise to intermediate cells that
then differentiate into superficial cells (Fig. 4A), one needs to
consider the potential reasons for this discrepancy among the
various groups. One possible explanation is the different injury
models used (i.e., UPEC vs. CPP). This notion that distinct
injuries may result in activation of different pools of urothelial
progenitor cells was described by Mysorekar et al., who noted
that UPEC-induced injury was associated with basal cell pro-
liferation whereas protamine sulfate-based injury awakened the
intermediate cell compartment (71). One obvious question that
is raised from these studies is whether the degree of inflam-
mation induced by the urothelial injury influences which subset
of urothelial progenitor cells is stimulated. Regardless, it is
possible to ascribe Gandhi’s results to the use of chemical
injury with CPP. To further clarify this point, it would be
informative to repeat lineage tracing results in Krt5CreER;
R26mTmG or Upk3aGCE;mCherry mice infected with UPEC.
Additionally, it would be instructive to examine which cells
serve as progenitors for regeneration in a surgical model of
injury such as bladder resection and/or augmentation. Given
the increased appreciation of the heterogeneity of urothelial
cells (26a), it is possible that genetic lineage tracing strategies
have caught only a glimpse of the complete repertoire of
urothelial progenitors, thus accounting for the discrepancy
among these studies. For example, Krt14� progenitor cells
only make up less than 1% of the total urothelial cells in the
adult bladder (77). Of course, this argument is less plausible

during development, since we have observed that ~14% of
murine urothelial cells are Krt14� at e18.5 (26a). Moreover,
the observation by Papafatiou et al. that Krt14� cells are first
observed at e16.5 (77) casts some doubt on these cells being
progenitors for superficial and intermediate cells during em-
bryogenesis, as these latter cells emerge several days earlier.
Could a nonlinear model account for urothelial formation in
embryogenesis (Fig. 4B) while a linear model explains urothe-
lial regeneration in adulthood (Fig. 4A)? The chapter on this
fascinating topic has yet to be closed.

A Presumptive Anatomical Urothelial Stem Cell Niche Has
Yet To Be Identified

One challenging point to reconcile in these models of
urothelial regeneration is the absence of a clear anatomical
stem/progenitor cell “niche” within the urothelium. For in-
stance, intestinal stem cells reside at the base of intestinal
crypts and are supported by soluble factors and cell-associated
ligands from the surrounding Paneth and stromal cells (92).
Likewise, within hair follicles, the bulge area serves as a niche
for stem cells (31). In contrast, there is no obvious structural
niche within the urothelium. Attempts have been made to
define the proliferation unit within the bladder urothelium by
using either X chromosome inactivation analysis (98) or mi-
tochondrial mutation-based analysis (24) of human bladder
specimens. These studies revealed variably sized patches of
urothelial cells carrying the same genetic signature and con-
tinuously extending from the basal layer toward the luminal
surface. Similarly, in the setting of UPEC-induced injury, Shin
et al. similarly demonstrated that regeneration occurs in clonal
units that are vertically oriented and include all three layers of
urothelium (84). Collectively, these findings reinforce one
existing theory that the urothelial stem cell niche may simply
represent cells in contact with the basement membrane. Nota-
bly, this finding does not necessarily discount the argument of
Gandhi et al. (25) that intermediate cells, rather than basal
cells, are progenitors for superficial cells, as there is evidence
that intermediate cells do send downward projections that
contact the basement membrane (30, 62). Whether there is a
specialized population of supporting stromal cells that are
anatomically restricted to specific regions within the lamina
propria remains to be determined.

Epithelial-Mesenchymal Cross Talk Is Critical for
Urothelial Regeneration

Regardless of the debate surrounding the identity of urothe-
lial progenitors, several studies have underscored the impor-
tance of epithelial-mesenchymal interactions in directing pro-
cesses of both urothelial formation and regeneration. One
prime example is the cross talk between urothelium and the
underlying stroma through the retinoid signaling pathway (Fig.
5). In situ hybridization analysis of both embryonic and adult
bladders identified the suburothelial stroma as the source of
retinaldehyde dehydrogenase 2 (Raldh2), while P cells, inter-
mediate cells, and superficial cells were identified as RA-
responsive cells (25). Using ShhCre;RaraDN�/� mice, which
are deficient in retinoic acid signaling within Shh� progenitor
cells, Ghandi et al. further illustrated that differentiation of
intermediate and superficial cells in embryogenesis, as well as
regeneration of the superficial cell layer after injury, depend on

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an intact RA signaling pathway. In a separate experiment, Shin
et al. demonstrated that mesenchymal cells also undergo Gli1-
mediated upregulation of Wnt2 and Wnt4 expression in re-
sponse to increased soluble Shh ligand that is released from
injured urothelium (84). A paracrine feedback loop is estab-
lished in which Wnt signals back to urothelial progenitor cells
to promote their proliferation and differentiation with restora-
tion of urothelial integrity (Fig. 5). Without Gli-mediated
proliferation after injury, the urothelial barrier remains com-
promised and mice are more susceptible to ascending infec-
tions of their kidneys. Whether this increased risk of upper
tract infection is simply due to reduced urothelial regeneration
or whether additional Gli-mediated signaling pathways are
responsible for limiting the spread of infection remains uncer-
tain. Similarly, Mysorekar et al. illustrated the importance of
epithelial-mesenchymal cross talk through the BMP signaling
pathway (Fig. 5) in ensuring proper urothelial regeneration
(71). Deletion of Bmpr1a on urothelial cells abrogates BMP4
signaling and leads to reduced urothelial cell proliferation in
response to UPEC infection. More remarkably, disruption of
this signaling pathway results in failure of regenerating super-
ficial cells to undergo terminal differentiation and to enter a
state of quiescence.

Local Growth Factors Promote Urothelial Regeneration
and Repair

In addition to receiving signals from mesenchymal cells,
urothelial cells also respond to local growth factors that pro-
mote regeneration and repair. Glycosaminoglycans, including
heparan sulfate, in the extracellular matrix of the urothelium
help to restrain diffusion of growth factors and provide a
concentrated pool of factors that can be temporally released
(33). One conspicuous growth factor axis involved in this

process is the epidermal growth factor receptor (EGFR) and its
ligands (Fig. 5). EGF produced by injured urothelial cells
functions in an autocrine fashion to promote urothelial cell
proliferation and regeneration (22, 100). EGF achieves this
function, in part, through induction of the transcription factor
Sox9. Despite high levels of Sox9 expression in the developing
urothelium, Sox9 becomes undetectable in quiescent adult
urothelium (60). However, with different types of urothelial
injury, Sox9 is transiently upregulated in all three urothelial
layers, coinciding with the timing of urothelial repair. Other
growth factors noted to enhance urothelial regeneration are the
EGFR ligands transforming growth factor-� (TGF�), keratin-
ocyte growth factor (KGF), heparan-binding EGF (HB-EGF),
and amphiregulin, as well as fibroblast growth factor 2 (FGF2)
(3, 7, 15). Several groups have begun to take advantage of this
knowledge to design better bladder scaffolds impregnated with
individual or combination growth factors to facilitate bladder
regeneration and ingrowth of urothelial cells in bladder aug-
mentation procedures (reviewed in Ref. 63). Kanematsu et al.
illustrated that sustained release of FGF-2 from a bladder
acellular matrix could promote VEGF production and improve
angiogenesis within the graft (39). By combining nerve growth
factor (NGF) with VEGF in a rat model of spinal cord injury,
Kikuno et al. were able to enhance smooth muscle and nerve
fiber content in the regenerating bladder while significantly
improving bladder capacity and compliance (48). It is possible
that additional incorporation of immunomodulatory factors
into scaffolds may further enhance bladder repair.

Urothelial Homeostasis and Regeneration Is Also Modulated
at the Epigenetic Level

Recent work from our laboratory and others’ also suggests
that progenitor cell behavior during urothelial development and

Shh 

Shh 

Shh 

Wnts 

SC

IC

BC

LP

SMC

Bmps 

Bmps 
EGF EGF 

EGF 

RA

Squamous 
differentiation 

Urothelial 
differentiation 

Wnts 

Wnts 

2 

3 

4 

Differentiation 

Proliferation 

Target genes 

U
Target genes 

1 

5 

6 

Fig. 5. Urothelial regeneration is achieved through multiple layers of regulation. The urothelium comprises three main cell types: superficial cells (SC),
intermediate cells (IC), and basal cells (BC). Below the urothelium lies the lamina propria (LP) and smooth muscle cell (SMC) layers. Significant
epithelial-mesenchymal cross talk facilitates urothelial regeneration after injury (1). In response to damage, urothelial cells upregulate and secrete the soluble
molecule Shh. Shh acts on the underlying mesenchymal cells and promotes SMC proliferation and differentiation (2). Shh signaling also leads to activation of
target genes, including the Gli family of transcription factors, and (3) increased stromal expression of Wnt molecules. Wnt signals subsequently promote urothelial
proliferation and differentiation (4). Injured urothelium also secretes local growth factors like EGF, which function in an autocrine manner to stimulate urothelial
cell proliferation (5). Likewise, BMP signaling through BMP receptors on the urothelium is important in ensuring terminal differentiation of regenerating
superficial cells (6). Finally, RA produced by stromal cells acts on RA-responsive urothelial cells to promote superficial cell regeneration and inhibit squamous
differentiation, in part through activation of transcription factors like FOXA1.

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injury-induced regeneration is additionally regulated by epige-
netic factors. Epigenetics refers to the study of changes in gene
expression that are not the result of changes in actual DNA
sequence. These changes in gene expression are, instead, the
result of chemical modifications to DNA or histone proteins
which result in local chromatin remodeling that alters accessi-
bility of the transcriptional machinery (52). While many epi-
genetic programs exist, one program that has been significantly
implicated in stem cell biology is the polycomb repressive
complex (PRC)2 (18). The PRC2-dependent H3K27me3 epi-
genetic modification is tightly associated with local chromatin
compaction and gene repression. Our laboratory made the
initial observation that core components of the PRC2 complex,
including Ezh2 and Eed, are expressed within murine bladders,
and H3K27me3 is highly enriched within the urothelium (26a).
These data suggest that PRC2 may play a role in the normal
function of urothelium. By deleting Eed, the obligatory struc-
tural component of PRC2, or Ezh2, the major enzymatic
component, in Shh� progenitor cells, we discovered that the
PRC2-mediated epigenetic program represses both the cell
cycle inhibitors p15 and p16 and Shh signaling molecules, and
it is critical for maintaining urothelial progenitor cell prolifer-
ation during development. The study also reveals that PRC2
not only ensures the proper timing of differentiation of Krt5�

basal cells and Upk3a� superficial cells, but it also prevents
aberrant induction of squamous-like differentiation programs
within the urothelium and prevents ectopic expression of Krt14
and Krt17. In adult urothelium, we have observed that PRC2
plays a similar role. In the quiescent state, PRC2 activity is
important in preventing precocious differentiation of Shh�

progenitor cells to superficial cells, thereby regulating normal
homeostasis. In the setting of repeated urothelial injury in vivo
with CPP, PRC2 is needed to ensure maximal proliferative and
regenerative potential of the progenitor cells. These results
have been corroborated in an in vitro model of UPEC-infec-
tion, where Ting et al. demonstrated that UPEC induces ex-
pression of Ezh2 in human bladder cancer cells (93). Pharma-
cological inhibition of Ezh2 resulted in reduced urothelial cell
proliferation in response to UPEC, confirming the role of
PRC2 in maintaining the early proliferative response to infec-
tion. Last but not least, we have observed that PRC2 not only
plays a role in promoting urothelial proliferation but may also
regulate the host inflammatory response to UPEC infection
(our unpublished data). Together, these emerging data point to
the importance of epigenetic regulation throughout the lifespan
of urothelial cells, i.e., in establishing the identity of urothelial
cells, in maintaining their quiescence, and in quickly respond-
ing to injury to promote proliferation and differentiation.
Whether other epigenetic programs play a role in the urothelial
regenerative process remains an active area of investigation.

Dysregulated Urothelial Regeneration Can Lead
to Carcinogenesis

From this discussion, it is clear that maintaining urothelial
homeostasis for extended periods of time while facilitating
rapid regeneration upon injury is a complicated process gov-
erned by epithelial-mesenchymal signaling, secreted growth
factors, and epigenetic mechanisms. A disruption in any one of
these processes could lead to abnormal urothelial regeneration
and malignant transformation. Not surprisingly, then, addi-

tional knowledge has been gleaned about the molecular mech-
anisms of urothelial regeneration by studying the factors that
drive urothelial carcinogenesis.

Altered epithelial-mesenchymal signaling. One popular ro-
dent model of bladder cancer involves administration of the
pro-carcinogen N-butyl-N-4-hydroxybutyl nitrosamine (BBN)
in the drinking water. BBN exposure induces urothelial hyper-
plasia and carcinoma in situ (CIS) within 3– 4 mo (reviewed in
Ref. 102). Unlike the normal hyperplastic response seen in
urothelium after acute injury with CPP or UPEC, which re-
cedes quickly after the exposure is removed, BBN-induced
hyperplasia develops progressively over time and fails to
regress. Shin et al. made the important observation that Shh�

cells, which are urothelial progenitors and are responsible for
the normal regenerative response after injury, are also respon-
sible for bladder carcinogenesis, because ablation of Shh� cells
attenuates BBN-induced bladder cancer development (85).
Remarkably, however, during progression of CIS to invasive
carcinoma, Shh expression is frequently lost. Supporting the
hypothesis that Shh secretion normally helps curb development
of advanced bladder cancer, genetic ablation of the Shh sig-
naling pathway led to accelerated progression to invasive
disease (86). This observation further highlights the role of
epithelial-mesenchymal signaling in keeping the regenerative
process in check. While Shh/Wnt signaling promotes urothelial
progenitor cell proliferation after injury (84), Shh signaling in
the mesenchyme through Smo and Gli is also critical to
feedback on the urothelium and induces upregulation of genes
such as BMP4 and BMP5, which tip the balance toward
urothelial terminal differentiation (86). The finding that en-
hanced BMP signaling can delay progression of BBN-treated
animals to invasive cancer is somewhat analogous to the
observation of Mysorekar et al. that the BMP4/Bmpr1a axis is
important in ensuring terminal differentiation of superficial
cells after UPEC injury (71). Together, these examples high-
light how specific signaling pathways can be manipulated to
maximize regeneration, but they must be carefully titrated to
avoid progression to cancer.

Abnormal growth factor signaling. In addition to the grow-
ing evidence that dysregulation of normal epithelial-mesenchy-
mal interactions may promote carcinogenesis, an imbalance in
growth factor signaling, which normally functions to promote
urothelial repair, has also been associated with increased blad-
der cancer risk. Overexpression of EGFR has been reported in
40 – 60% of bladder tumors and has been correlated with
invasive bladder cancer (5, 66, 73, 87). In dysplastic and
malignant urothelium, EGFR begins to be expressed through-
out all urothelial cell layers, rather than remaining confined to
the basal cells (67). The increased expression of EGFR on
tumor cells leads to enhanced signaling through urinary EGF
and other ligands, thereby promoting tumor cell growth and
proliferation (4, 67, 74). Additional studies have shown a
correlation between bladder cancer prognosis and the specific
cellular localization of HB-EGF, a potent growth factor ligand
for EGFR (2, 49, 76). Cleavage of proHB-EGF leads to
increased secretion of the soluble amino terminal fragment
sHB-EGF, which can function as an autocrine or paracrine
activator of EGFR and several family members. Concomitant
with this process, increased nuclear translocation of the car-
boxy terminal fragment HB-EGF-C is associated with in-
creased cyclin A expression and cell cycle progression (72).

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Not surprisingly, patients with an increase in both the secreted
and nuclear forms of HB-EGF, as opposed to the cytosolic pro
form, exhibit worse prognosis due to increased tumor cell
survival and proliferation (2, 53).

While the EGFR axis is one of the most prominent growth
factor pathways altered in bladder cancer, other growth factors
have also been shown to be dysregulated in their expression.
Both VEGF and VEGFR1 can be overexpressed in bladder
tumors, but they are more significantly upregulated in super-
ficial as opposed to invasive tumors (51). FGF signaling
pathways are also altered in a significant proportion of bladder
cancer patients (reviewed in Ref. 19). Patients with either
superficial or muscle-invasive disease may demonstrate over-
expression of FGFR1, whereas aggressive disease has been
correlated with reductions in FGFR2 (20, 96, 97). Furthermore,
mutations in FGFR3 have been reported in ~80% of low-grade
superficial tumors, and FGFR3 overexpression has been de-
tected in ~20 –50% of muscle-invasive tumors (6, 27, 95). It is
no surprise, then, that several new therapies for urothelial
cancer have centered on blockade of growth factor signaling as
a method of inhibiting tumor cell proliferation (27, 35).

Mutations in epigenetic programs. Consistent with our ob-
servation that PRC2-mediated repression controls urothelial
progenitor cell proliferation and regenerative potential, several
groups have observed that dysregulation of this epigenetic
program is associated with urothelial carcinoma in human
bladder tissue samples (57, 79, 104). Specifically, human
EZH2 is upregulated in urothelial carcinoma samples com-
pared with adjacent benign urothelium, although debate re-
mains whether degree of EZH2 elevation is predictive of
oncologic outcome. Conversely, whole exome sequencing of
human bladder cancer as part of the Cancer Genome Atlas
project found that 24% of bladder tumors studied had inacti-
vating mutations in KDM6A, the histone demethylase that
works in opposition to PRC2 (10). Moreover, we found that
urothelium-specific knockout of murine Kdm6a significantly
increases the risk of BBN-induced bladder cancer (Kaneko S
and Li X, unpublished observations). Collectively, these data
suggest that tipping the overall balance in favor of increased
H3K27me3 modification in urothelial cells may predispose to
bladder cancer. Other chromatin remodeling genes that are
frequently mutated in bladder cancer include the histone
acetyltransferase (HAT) genes CREBBP and EP300, the SWI/
SNF-related chromatin remodeling gene ARID1A, and the
H3K4 methyltransferase MLL (10, 26). However, because
epigenetic programs regulate a broad range of genes, it can be
challenging to know whether specific epigenetically-targeted
genes responsible for normal urothelial regeneration are the
same genes disrupted in urothelial cancer.

Urothelial metaplasia. While the examples above focus on
how abnormal regeneration pathways can lead to dysregulated
urothelial cell growth and urothelial cell carcinoma, it is
important to keep in mind that aberrant urothelial regeneration
can alternatively result in squamous metaplasia that has the
potential to progress to squamous cell carcinoma (SCC). It has
long been appreciated that Vitamin A deficiency in rats and
mice can lead to keratinizing squamous metaplasia that occurs
heterogeneously throughout the bladder (59, 69). This phenom-
enon suggested the importance of retinoic acid signaling in
maintaining the urothelial differentiation phenotype in mature
bladders and potentially deterring neoplastic transformation.

And, in fact, in vitro cultures of normal human urothelial
(NHU) cells deprived of exogenous retinoids adopt a squamous
phenotype that can revert to a basal phenotype following
introduction of RA derivatives (89). Intersecting with RA
signaling is the peroxisome proliferator-activated receptor-�
(PPAR�) pathway, a nuclear hormone receptor that, when
bound by ligand, heterodimerizes with RXRa to form a tran-
scription factor complex that activates several genes including
the forkhead box A1 (FOXA1) (16, 34, 101). FOXA1 plays a
role in urothelial differentiation by binding to the promoters of
Upk1a, Upk2, and Upk3a and enhancing expression of these
terminal differentiation markers (101). Not only is FOXA1
expression significantly reduced in human samples of aggres-
sive bladder cancer, there is also an increased frequency of
FOXA1 loss in cases of SCC compared with transitional cell
carcinoma (TCC) (17, 80). Likewise, female mice deleted in
FOXA1 within the urothelium developed keratinizing squa-
mous metaplasia with increased Krt14 expression, further con-
firming that this axis helps maintain the terminal differentiation
state of urothelium (81). Remarkably, using lineage-tracing
analysis, Van Batavia et al. were able to show that, although
intermediate cells could give rise to noninvasive papillary
tumors, they were not responsible for SCC; rather, Krt5� basal
cells were identified as the cell of origin for SCC (99). These
results suggest that despite common themes the molecular
mechanisms governing the regenerative process in Krt5� cells
may truly be distinct from that in intermediate cells. Regard-
less, these data collectively highlight that successful regener-
ation of urothelium does not simply involve carefully regulated
cell proliferation but also progression along the correct differ-
entiation pathway.

Inadequate Urothelial Regeneration May Weaken the
Defense Barrier and Contribute to Interstitial Cystitis and/or
Increased Susceptibility to Infection

While an overabundant regenerative process has the danger
of progressing to malignancy, there can also potentially be
deleterious effects from insufficient regeneration of the urothe-
lium. Consequences may include increased exposure to urinary
toxins, inadequate clearance of microorganisms, chronic low-
level bladder inflammation, and increased sensory nerve acti-
vation leading to chronic pain. In patients diagnosed with
interstitial cystitis/painful bladder syndrome (IC/PBS), bladder
biopsies frequently reveal erosion and/or thinning of the
urothelium concerning for insufficient urothelial regeneration
(94). The discovery of antiproliferative factor (APF) in the
urine of IC/PBS patients, along with reduced levels of HB-
EGF, has led to the hypothesis that an initial insult to the
bladder epithelium can lead to upregulation of APF in a select
group of patients (40 – 43, 46). Not only does APF impair
urothelial cell proliferation, but it may also inhibit the produc-
tion of autocrine or paracrine growth factors like HB-EGF (41,
45, 46). Together, this process results in blunted reepithelial-
ization of the urothelium characterized by fewer urothelial cell
layers, altered tight junction formation, and increased paracel-
lular permeability.

One can similarly imagine that exposure of incompletely
regenerated urothelium to UPEC may result in enhanced sus-
ceptibility to and/or reduced clearance of infection. Using a
mouse model in which bladders were pretreated with PS to

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induce chemical damage of the urothelium before UPEC in-
fection, Mysorekar and Hultgren observed that UPEC was now
able to gain access and establish infection within the underly-
ing intermediate and/or basal cell layers cells (70). Further-
more, compared with vehicle-treated controls, PS-treated blad-
ders contained increased numbers of quiescent intracellular
reservoirs (QIR) within the deeper urothelial cell layers where
they are presumably protected from exfoliation and/or host
defense mechanisms. Notably, with a new stimulus, bacteria
present within these QIR could be triggered to reemerge from
these underlying compartments and seed a new acute infection.
These studies underscore the possibility that conditions leading
to an incompletely regenerated urothelial layer may set the
stage for increased susceptibility to recurrent UTI.

Conclusions

Urothelium is a dynamic epithelial surface that relies upon
its ability to maintain a state of quiescence while remaining
poised for rapid regeneration in the face of injury. Identifying
the key molecular players in this process is important to
improve our understanding of what host factors may predis-
pose to IC/PBS, recurrent UTI, and malignant transformation.
Furthermore, capitalizing on this knowledge may improve our
efforts to safely and effectively regenerate or replace the
bladder, ureters, or urethra. Work in the past few years has
focused on characterization of the urothelial progenitor cell
population responsible for regeneration. Debate still exists
regarding the precise identity and location of these progenitor
cells. While this may be related to variability in the genetic
models tested, a more intriguing possibility that merits further
investigation is that different types or severities of injury
(chemical vs. biological vs. surgical) may stimulate different
progenitor cell pools within the urothelium. Whether the de-
gree of injury and/or the degree of inflammation are key
determinants of this process remains to be determined.

Regardless, judicious progenitor cell proliferation and dif-
ferentiation are necessary to ensure proper regeneration. This is
achieved through multiple layers of regulation including
growth factor stimulation and epithelial-mesenchymal cross
talk (Fig. 5). More recently, there has been a growing appre-
ciation for the role of epigenetic programs, more specifically
the PRC2 epigenetic program, in modulating urothelial homeo-
stasis and regeneration. Additional work is warranted to exam-
ine the role of other epigenetic programs in urothelial regen-
eration. When this fine balance of regulatory networks is
disrupted, regeneration can go awry and lead to extremes of
insufficient or overabundant regeneration. Identifying methods
to restore the equilibrium between urothelial regeneration and
quiescence should be the focus of translational research in this
area.

ACKNOWLEDGMENTS

We thank all members of the Li laboratory, particularly Dr. Chunming Guo,
for helpful discussions.

GRANTS

This work was supported by American Urological Association scholar
awards (Z. R. Balsara) and National Institute of Diabetes and Digestive and
Kidney Diseases (1R01 DK-091645 and 1R01 DK-110477, X. Li) and Na-
tional Cancer Institute (1R21 CA-198544, X. Li).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Z.R.B. prepared figures; Z.R.B. drafted manuscript; Z.R.B. and X.L. edited
and revised manuscript; X.L. interpreted results of experiments; X.L. approved
final version of manuscript.

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