International Journal of
Molecular Sciences
Review
Lingual Epithelial Stem Cells and Organoid Culture
of Them
Hiroko Hisha 1 , Toshihiro Tanaka 1,2 and Hiroo Ueno 1, *
1
2
*
Department of Stem Cell Pathology, Kansai Medical University, 5-1, 2-chome, Shin-machi, Hirakata-shi,
Osaka 573-1010, Japan; [email protected] (H.H.); [email protected] (T.T.)
Third Department of Internal Medicine, Kansai Medical University, 5-1, 2-chome, Shin-machi, Hirakata-shi,
Osaka 573-1010, Japan
Correspondence: [email protected]; Tel.: +81-72-804-2360; Fax: +81-72-804-2369
Academic Editor: Miroslav Blumenberg
Received: 23 November 2015; Accepted: 18 January 2016; Published: 28 January 2016
Abstract: As tongue cancer is one of the major malignant cancers in the world, understanding the
mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue
cancer, is unquestionably important. However, the actual stem cells that are responsible for the
long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and
convenient culture method for lingual epithelial stem cells has not yet been established. Recently,
we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell
layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized
epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial
stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using
a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification
of lingual stem cells and future applications of the lingual culture system for studying the regulatory
mechanisms of the lingual epithelium and for regenerative medicine.
Keywords: lingual epithelium; stem cells; Bmi1; organoid
1. Introduction
The mammalian tongue is an important digestive and sensory organ that has multiple functions
such as food intake, sensing taste and touch, and serving as an articulatory organ. The surface of the
tongue is covered with stratified squamous epithelial cell layers. Lingual dorsal epithelium contains
four kinds of papillae: filiform, fungiform, foliate, and circumvallate papillae. Stratum corneum is
seen in filiform papillae, but not in fungiform, foliate, or circumvallate papillae. In contrast, taste
buds are seen in fungiform, foliate, and circumvallate papillae but not in filiform papillae. Lingual
epithelium is replaced continually throughout the life of mammals, and the turnover of mouse lingual
epithelium, at a rate of 6–7 days, is 4–5-fold higher than that of dorsal skin [1], suggesting the existence
of stem cells in the papillae. A label-retaining assay using 3 H-TdR revealed that lingual epithelial stem
cells (LESCs) are located in the basal layer of the lingual epithelium as in other epithelial tissues [2].
Several research groups have proposed candidates for LESCs and progenitors. However, the stem cell
markers used in these studies, keratin 5 and keratin 14, are not specific to stem cells since they are also
detected in some of the mature epithelial cells. Therefore, the actual stem cells that are responsible
for the long-term maintenance of the lingual epithelium have not been identified. In recent years,
a powerful technique, in vivo lineage tracing, has been applied for the identification of LESCs. In this
review, we introduce and discuss current progress in the identification of LESCs.
To identify tissue-specific stem cells in the adult, a primary culture system that can reproduce
the physiological environment in vitro and allow the differentiation of stem cells into various kinds of
Int. J. Mol. Sci. 2016, 17, 168; doi:10.3390/ijms17020168
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 168
2 of 12
mature cells needs to be established. Using this system, we can precisely examine the pluripotency
and the growth factor requirements of the stem cells. Recently, a three-dimensional (3D) organoid
culture technique using extracellular matrix has been developed for the small intestine [3], stomach [4],
and colon [5]. This technique allows the generation of organoids containing multilayered epithelial
structures from crypts and even from single stem cells isolated from adult animals. In this review,
we introduce a new lingual epithelial organoid culture system as well as early lingual epithelial cell
culture systems.
2. Lingual Stem Cell Markers
2.1. Keratin 5 and Keratin 14
Keratin 5 (K5) and keratin 14 (K14), intermediate filament proteins, are known to be expressed
in basal keratinocytes of stratified epithelium in the skin, and the mutation or absence of both
proteins makes the cellular architecture in basal keratinocytes vulnerable [6]. Similar to the skin,
immunohistochemistry analyses of mouse tongue revealed that both proteins are expressed at the
highest level in the basal layer of the tongue epithelium. The expression level decreases in each layer
closer to the surface epithelial layer [7,8] (Table 1). Luo et al. reported that K5-positive lingual epithelial
cells (LECs) obtained from K5-eGFP mice could generate a multilayered squamous keratinized
epithelium when these cells were cultured on a collagen-fibroblastic cell-matrix in the presence of
epidermal growth factor (EGF) and fibroblast growth factor 7 (FGF7) [9], supporting that K5-positive
cells include lingual stem cells and/or progenitors.
Table 1. Markers of lingual epithelial stem cells (LESCs) and the results of their lineage
tracing experiments.
Marker
Keratin 14
NTPDase2
Bmi1
Tcf3
Position of
Maker-Positive Cells in
Lingual Epithelium
Basal Layer and
Mature Keratinized
Cell Layer
Basal Layer, Suprabasal
Layer and Taste Bud
The 2nd or 3rd Layer
from Basal Layer
(1 Cell per IPP)
Basal Layer
(2-5 Cells per IPP)
Differentiation to
Papillae
Yes (Filiform and
Fungiform Papillae)
Yes (Filiform,
Fungiform and
Circumvallate Papillae)
Yes (Filiform
Papillae Only)
Yes (Filiform
Papillae Only)
Differentiation to Taste
Buds
Yes
Yes
No
(Not Described)
Period of Chase
30 days
150 days
336 days
180 days
Reference
[7]
[10]
[8]
[11]
IPP: interpapillary pit.
Using an in vivo lineage tracing assay with K14-Cre ER:Rosa26-LacZ mice, Okubo et al.
demonstrated that K14-positive cells give rise to mature keratinocytes in both filiform and fungiform
papillae and in taste bud cells [7]. However, K14 is detected in not only Ki67-positive proliferating
cells but also many of the mature keratinized epithelial cells in the interpapillary pit (IPP). Moreover,
in the study, the fate of the K14-positive cell-derived cells were followed for only 30 days; therefore, it
is not clear whether K14-positive cells are actual long-term stem cells. Based on both the expression
patterns (immunostaining results) of several proteins (K5, K14, Sox2 [SRY {Sex Determining Region
Y}-Box 2], and Trp63 [p63, a member of the p53 transcription factor family]) in the lingual epithelium
and taste buds and the K-14 lineage tracing analyses, they proposed a model in which K5-, K14-,
and Trp63-positive, but Sox2-low-positive basal cells are long-term progenitor cells that generate
keratinocyte lineage cells. Moreover, they considered that K5-, K14-, Trp63-, and Sox2-positive cells
adjacent to taste buds can give rise to both keratinocytes and taste bud cells.
Int. J. Mol. Sci. 2016, 17, 168
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2.2. NTPDase2
Ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is a plasma membrane
nucleoside triphosphate that has the ability to hydrolyze extracellular nucleoside triphosphates to
diphosphates. The enzyme is expressed on neural stem cells of the subventricular zone [12]. Bartel et al.
found that NTPDase2 colocalized with the glial glutamate/aspartate transporter (GLAST), which
is regarded as a marker of type I cells in taste buds, by using immunohistochemical and enzyme
histochemical staining methods [13]. In contrast, Li et al. demonstrated that LECs in basal and
suprabasal cell layers as well as taste bud cells in fungiform and circumvallate papillae express
NTPDase2 by using in situ hybridization with an NTPDase2 probe [14] (Table 1). Moreover, a genetic
tracing study of NTPDase2-positive cells (doxycycline inducible, NTPDase2-rtTA/TeTO-Cre; RosaLacZ
reporter system) revealed that descendant cells derived from the NTPDase2-positive cells generated
filiform, fungiform, and circumvallate papillae as well as taste bud cells in fungiform papillae and
circumvallate papillae. From the results, they propose the existence of common progenitor cells that
contribute to both taste bud cells and LECs. However, by the single-color lineage tracing method
using the Rosa26 reporter mouse in this study, the proof for the bipotency of K14 positive lingual
stem/progenitor cells was not sufficient, because the different clones next to each other could show
the same color.
2.3. Multicolor Lineage Tracing Method
To precisely examine the fate of each stem cell, the multicolor lineage tracing method is now
considered as one of the most powerful techniques. The multicolor lineage tracing method was
originally developed to analyze lineage relationships between blood and endothelial cells within yolk
sac blood islands of mice [15]. However, in the original method, multicolor chimeras were generated
by injecting multiple kinds of colored ES cells into blastocysts, which were then transplanted back
to the uterus of pseudopregnant mice. By this method, the timing of the generation of multicolor
chimeras was fixed at an early stage of development; therefore, application of the method to study
adult tissue-specific stem cells was limited. To overcome the problem, we developed a mouse
model for inducible multicolor lineage tracing, Rosa26-rainbow mice. In the Rosa26-rainbow mice,
Cre-mediated excision of floxed cassettes is induced by tamoxifen in all of the cells in the body
(including tissue-specific stem cells), causing the fluorescent label of every cell to change from green to
one of three colors (red, orange, or blue) (Figure 1a) [16,17]. The descendant cells of the red, orange, or
blue stem cells retain the color of the parent stem cells, and, therefore, this system is convenient to
examine the fate and dynamics of individual normal and cancer stem cells [18]. We used this system
to follow the fate of individual LESCs in vivo and found that clonal expansion of single-color cells
occurred in each IPP. In addition, each mature pit contained a single-color cell cluster of LECs (red,
orange, or blue) (Figure 1b) [8]. This finding indicates that single LESCs in each IPP have the ability to
generate LECs in the whole pit by 28 days after tamoxifen administration.
2.4. Bmi1
Bmi1 belongs to the polycomb group family and has an important role in cell cycle, self-renewal,
and maintenance of hemopoietic and neural stem cells [19,20]. In situ hybridization for Bmi1
(B cell-specific Moloney murine leukemia virus integration site 1) showed that Bmi1-positive cells
reside in the second or the third layer above the basal layer of mouse epithelium; we never observed
more than two Bmi1-positive cells in one IPP [8] (Table 1). From multicolor lineage tracing using
tamoxifen-inducible Bmi1-CreER; Rosa26-rainbow mice, it was shown that one labeled cell was detected
per IPP two days after tamoxifen injection. The descendants of Bmi1-positive cells expanded and
occupied the entire IPP by four to 12 weeks after tamoxifen induction (Figure 1c). Finally, the
descendant cells generated filiform papillae, and the single-color area generated by single stem
cells was maintained for at least 336 days (Figure 1c), indicating that Bmi1-positive cells in the IPP are
Int. J. Mol. Sci. 2016, 17, 168
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long-term LESCs. It was also shown that a single Bmi1-positive stem cell supplied the LECs for three
(or
less
Int. J.
Mol.frequently
Sci. 2016, 17,four)
168 neighboring filiform papillae (Figure 1d).
4 of 12
Figure 1.
1. (a)
(a) Cre-mediated
Cre-mediated fluorescent
fluorescent color
color change.
change. Fluorescent
Fluorescent labels
labels of
of cells
cells change
change from
from green
green to
to
Figure
either red,
red, orange,
orange, or
or blue
blue upon
upon tamoxifen-induced
tamoxifen-induced Cre-mediated
Cre-mediated excision
excision of
of floxed
floxed cassettes;
cassettes; their
their
either
descendant cells
cells retain
retain the
the new
new color;
color; (b)
(b) Individual
Individual cells
cells of
of Rosa26CreERT2;
Rosa26CreERT2; Rosa26-rainbow
Rosa26-rainbow mice
mice
descendant
injected with
with tamoxifen
tamoxifen were
were labeled
labeled and
and analyzed
analyzed at
at the
the indicated
indicated time
time points.
points. Magnified
Magnified images
images
injected
are
shown
at
the
right
upper
corner
of
the
images;
(c)
Fate
of
cells
derived
from
Bmi1-positive
are shown at the right upper corner of the images; (c) Fate of cells derived from Bmi1-positive cells.
cells.
Bmi1-CreER; Rosa26-rainbow
indicated time
time
Bmi1-CreER;
Rosa26-rainbow mice were injected with tamoxifen and analyzed at the indicated
points; (d)
(d)Areas
Areasmaintained
maintained
single
Bmi1-positive
(and
less frequently
four)
points;
by by
single
Bmi1-positive
stemstem
cells. cells.
ThreeThree
(and less
frequently
four) filiform
filiform papillae
displayed
colors, suggesting
that Bmi1-positive
are
in the
papillae
displayed
different different
colors, suggesting
that Bmi1-positive
LESCs areLESCs
present
in present
the middle
of
middle
of
the
pit.
This
figure
is
a
modified
version
of
figures
presented
in
our
previous
report
[8].
the pit. This figure is a modified version of figures presented in our previous report [8].
Using the lineage tracing experiments, we have shown that Bmi1-positive cells usually were
Using the lineage tracing experiments, we have shown that Bmi1-positive cells usually were
resting or slow-growing. However, after irradiation-induced injury, the cells rapidly entered the
resting or slow-growing. However, after irradiation-induced injury, the cells rapidly entered the
cell cycle and regenerated tongue epithelium. These results suggest that Bmi1-positive LESCs are
cell cycle and regenerated tongue epithelium. These results suggest that Bmi1-positive LESCs are
important for the tissue maintenance of lingual epithelium in the physiological state as well as for
important for the tissue maintenance of lingual epithelium in the physiological state as well as for
rapid regeneration after injury.
rapid regeneration after injury.
Immunohistochemistry staining of human normal oral mucosal tissues showed that
Immunohistochemistry staining of human normal oral mucosal tissues showed that Bmi1-positive
Bmi1-positive cells were weakly detected in the basal layer of epithelium, but not in the upper
cells were weakly detected in the basal layer of epithelium, but not in the upper layers [21]. Bmi1
layers [21]. Bmi1 expression was also observed in the basal layer of the lingual epithelium [22].
expression was also observed in the basal layer of the lingual epithelium [22]. These results suggest
These results suggest the possibility that Bmi1 is a marker of stem cells in human oral mucosa and
the possibility that Bmi1 is a marker of stem cells in human oral mucosa and lingual epithelium as
lingual epithelium as well as in mouse lingual epithelium.
well as in mouse lingual epithelium.
Bmi1 is now considered to be a ubiquitous stem cell biomarker expressed on various stem cells
Bmi1 is now considered to be a ubiquitous stem cell biomarker expressed on various stem
such as hemopoietic [19], neural [20] and intestinal [23] stem cells. Our more recent study using the
cells such as hemopoietic [19], neural [20] and intestinal [23] stem cells. Our more recent study
multicolor lineage tracing technique revealed that Bmi1-positive cells residing in the inner periphery
using the multicolor lineage tracing technique revealed that Bmi1-positive cells residing in the inner
of seminiferous tubules were long-term germ stem cells that could produce sperm for at least
periphery of seminiferous tubules were long-term germ stem cells that could produce sperm for at least
48 weeks [24], and that Bmi1-positive cells residing in sweat glands of the acral epithelium (does not
48 weeks [24], and that Bmi1-positive cells residing in sweat glands of the acral epithelium (does not
contain hair follicles or sebaceous glands, such as on the palms and soles) were reserver stem cells
that were slow-cycling but occasionally entered the cell cycle and then generated the acral
epithelium [25].
Int. J. Mol. Sci. 2016, 17, 168
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contain hair follicles or sebaceous glands, such as on the palms and soles) were reserver stem cells that
were slow-cycling but occasionally entered the cell cycle and then generated the acral epithelium [25].
2.5. Tcf3
Tcf3 (a member of the Lef/Tcf family) is a transcription factor important for regulating embryonic
cell function and adult normal/malignant stem cell function [26]. It is also expressed in hair follicle
bulges (stem cell niche in the skin) [25] (Table 1). Howard et al. recently demonstrated that Tcf3 is
expressed on overall cells of the basal layer and in some cells of the suprabasal layer in the mouse
lingual epithelium [11]. Lineage tracing experiments using Tcf-GC; Rosa26-mTmG mice detected
2–4 labeled cells in the basal layer of the IPP 3 days after tamoxifen administration; the basal clones
continued to expand, finally occupying the entire interpapillary epithelium and filiform papillae. After
the chase of six months, the neighboring 3–4 filiform papillae were labeled green, indicating that the
Tcf3-positive clones supplied epithelial cells for 3–4 filiform papillae. This result is consistent with our
previous observation (Figure 1d) [8]. Immunostaining for Tcf3 protein revealed that the majority of
Tcf3-positive cells in the basal layer were Bmi1-negative, but one or two Tcf3- and Bmi1-double positive
cells were detected per IPP. Bmi1-positive/Tcf3-negative cells were observed, leading Howard et al.
to conclude that the expression of these proteins is not directly linked. They further demonstrated
that there is no direct relationship between Tcf3 expression and proliferation status (examined by
immunostaining of Ki67). Overall, it was suggested that Tcf3 expression is not restricted to actual
LESCs but is expressed in more differentiated LECs (LESCs, transit-amplifying cells, and keratinized
epithelial cells).
3. Culture of Lingual Epithelial Cells
3.1. Organ Culture
According to previous studies on tongue development, the tongue epithelium remains
histologically immature until the 13th day of gestation, and pre-papilla placodes and fungiform
papillae appear on the 14th day and 15th day, respectively. Organ culture of rat embryonic tongues
was performed to examine the morphogenesis of lingual epithelium. Entire tongues were taken from
rat embryos on the 13th day of gestation and incubated at the air/liquid interface in the presence
of medium containing fetal bovine serum (FBS) and B-27. After two days of culture, fungiform
papillae were observed in the epithelium, a stage of development equivalent to the tongue of an intact
embryo on the 15th day of gestation [27] (Table 2). In the same laboratory, it was shown that the
addition of noggin to the organ culture system increased the number of fungiform papillae, whereas
bone morphogenic protein (BMP) 2, 4, or 7 inhibited papilla formation, suggesting that noggin and
BMPs, both localized within papilla placodes, have opposite effects on the morphogenesis of lingual
epithelium [28] (Table 2).
A rolling organ culture system also has been used for mouse embryonic tongues on day 12.5 or
13.5 of gestation [29,30] (Table 2). In this system, the tongues were cultured in medium containing
various cytokines, antibodies, or enzyme inhibitors; thus, the effects of the molecules on the developing
tongues could be precisely examined.
3.2. Culture on Extracellular Matrix
The application of the organ culture system for adult tongue is difficult because the proliferation
and differentiation potential of adult tissues is much lower than embryonic tissues. Lingual epithelial
cell culture of adult LECs on extracellular matrix was reported by Ookura et al. They obtained integrin
β1 (a keratinocyte stem cell marker)-positive LECs from adult mouse tongues by using a magnetic cell
separation system and cultured them on a collagen gel/Matrigel-coated dish in the presence of EGF
and basic fibroblast growth factor 2 (FGF-2) [31] (Table 2). The integrin β1-positive LECs proliferated on
Int. J. Mol. Sci. 2016, 17, 168
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the extracellular matrix and generated a monolayer with epithelial morphology. However, a stratified
keratinized epithelial layer was not generated in this culture system.
Int. J. Mol.
Sci.
2016,
16817, 168
Int. J.
Mol.
Sci.17,
2016,
Int. J.J. Mol.
Mol. Sci.
Sci. 2016,
2016, 17,
17, 168
168Table
Int.
Culture System
Table Table
2. List2.
ofList
lingual
epithelial
cell (LEC)
cultures.
of lingual
epithelial
cell (LEC)
cultures.
Table 2.
2. List
List of
of lingual
lingual epithelial
epithelial cell
cell (LEC)
(LEC) cultures.
cultures.
Table
Culture Setup (Day
0) Imaged
Advantage
Culture
SystemSystem Culture
Setup (Day
Imaged
Culture
Culture
Setup0)(Day
0) Imaged
Culture System
System
Culture Setup
Setup (Day
(Day 0)
0) Imaged
Imaged
Culture
Culture
Organ Culture
Culture
Organ
Culture on
Culture on
Culture on
Culture on
on
Culture
Extracellular
2D
Extracellular
Extracellular
2D2D
Extracellular
2D
Extracellular
2D
Matrix
Matrix Matrix
Matrix
Matrix
Organotypic
Organotypic
3D
Organotypic
3D
Organotypic
Organotypic
Raft Culture
3D
Raft Culture
3D3D
Raft Culture
Culture
Raft Culture
Raft
Organoid
Organoid 3D
Advantage
Advantage
Advantage
Advantage
Limitation
Limitation
Limitation
Limitation
Limitation
Ref.
3D
3D
3D3D
Ref.
Ref.
Ref.
Ref.
Impossible
to
Impossible
to
Impossible
to to apply to
Impossible
to
Impossible
apply to
adult
apply
to adult
apply
to
adult
apply to adult
tongues
adult
tongues
tongues
Close Close
to
physiological
to
physiological
tongues
Close
to physiological
tongues
to
Difficult
to maintain
Closeconditions
to physiological
physiological Difficult
Difficult
to [27–30]
[27–30]
Close
to
conditions
Difficult
to
[27–30]
conditions
Difficult
to
[27–30]
maintain
long-term
growth
conditions
maintain
conditions
maintain
maintain
long-term
growth
(maximum
6 days)
long-term
growth
long-term
growth
long-term
(maximum
6 days)growth
(maximum
6 days)
(maximum 66 days)
days)
(maximum
Organ
Culture
Organ
Culture
Organ
Culture
Organoid
Organoid
Organoid
Culture
Culture
Culture
Culture Culture
6 of 12 6 of 12
of 12
12
66 of
2. List of lingual epithelial cell (LEC) cultures.
[27–30]
Impossible
to
Impossible
to
Possible
to generate
Impossible
to to generate
Possible
to generategenerate stratified
Impossible
to
Impossible
Possible
to generate
generate
generate
stratified [31]
Possible
to
Possible
to
generate
epithelial
epithelial
cell
monolayer
generate
stratified
epithelial cell monolayer keratinized
[31]
generatestratified
stratifiedkeratinized
epithelial
cell
monolayer
[31]
epithelial
cell
monolayer
[31]
cell
monolayer
from
single cells keratinized
from
single
keratinized
from cells
single
cells
keratinized
epithelial
cell
layer
from single
single cells
cellsepithelial
epithelial
cell layer cell layer
from
epithelial cell
cell layer
layer
epithelial
[31]
Possible
to generate
Possible
to generatePrior preparation
Prior preparation
Possible
to generate
generate
Prior preparation
preparation
Possible
to
Prior
stratified
keratinized
of the feeder
layer
Possible
to generate
stratified
stratified
keratinized
of the feeder
layer
stratified
keratinized
of the
thePrior
feederpreparation
layer
of the
stratified
keratinized
of
feeder
layer
epithelial
cell layer
is required
keratinized
epithelial
cell layer
epithelial
cellfrom
layer from
is required
epithelial
cell
layer
from
is
required
feeder
epithelial
cell layer from
is requiredlayer is required
single
cells
Results
of
culture
single
cellscells
Results of culture
from
single
[9,32,33]
single
cells
Results
of
culture
[9,32,33]
Results
of culture
single
cells
Results
of
culture
Also possible
to
observe
experiments
are
[9,32,33]
Also possible
possible
to
experiments
are
Also
toobserve
observe
[9,32,33]
Also
possible
to
observe generally
experiments
are
experiments
are
Also
possible
to
observe
experiments
are
inversion
activity
and
its
inversion activity
and
itsits
generally
inversion
activity
and
inversion
activity
and its
its
generally
generally
influenced by
inversion
activity
and
generally
process
into
collagen
gel
influenced
by
process
into
collagen
gel
influenced by
process
into
collagen
gel
layer
of
process
intoLECs
collagen gel
gel
influenced
by
feeder
cells
process
into
collagen
influenced
by
layer oflayer
malignant
cells cells
of malignant
LECs feeder feeder
malignant
LECs
layer of
of malignant
malignant LECs
LECs
feeder cells
cells
layer
feeder
[9,32,33]
SimpleSimple
and easy
ineasy in
and
Simple
and easy
easy in
in
and
cultureSimple
technique
culture
technique
culture
technique
culture
technique
Simple
and
easy in
Possible
to
generate
High High
Possible
to generate
Possible
to generate
generateconcentrationHigh
High
Possible
to
culture
technique
stratified
keratinized
of
stratified
keratinized
concentration
of [34]
High concentration
stratified
keratinized
concentration
of
[34] of
stratified
keratinized
concentration
of
Possible
generate
epithelial
celltolayer
growthgrowth
factorsfactors
is
[34]
epithelial
cellfrom
layer stratified
from
is factors
[34]
growth
epithelial
cell
layer
from
growth
factors
is
epithelial
cell layer from
growth factors is
keratinized
epithelial
cell
layer
single
cells
required
single cells
required is required
single
cells
required
single
cells
frompossible
single
cells
Also
possible required
Also
to
trace
Also
possible
to trace
Also
possible
toeach
trace
Also
possible
to
trace
the
of the
each
tofate
trace
fate
of
the
fate
ofLEC
each
LEC LEC
the fate
fate of
of each
each LEC
LEC
the
[34]
3.3. Organotypic
Raft Culture
3.3.
3.3.Organotypic
OrganotypicRaft
RaftCulture
Culture
3.3.
Organotypic
Raft
Culture
3.3. Organotypic
Raft
Culture
To achieve
a
more
suitable
environment for the
proliferation
and maturation
of LECs,LECs,
an
To
achieve
a
more
To achieve
achieve aa more
more suitable
suitable environment
environment for
for the
the proliferation
proliferation and
and maturation
maturation of
of LECs,
LECs, an
an
To
suitable
environment
for
the
proliferation
and
maturation
of
an
organotypic
raft
culture
system
was
developed
by
Luo
et
al.
[9]
(Table
2).
K5-positive
cells,
obtained
organotypic
raft
culture
system
developed
al.
(Table
cells,
obtained
To
achieve
a
more
suitable
environment
for
proliferation
and
maturation
organotypic
raft
culture
systemwas
was
developedby
byLuo
Luoet
etthe
al.[9]
[9]
(Table2).
2).K5-positive
K5-positive
cells,
obtained of LECs,
organotypic
raft
culture
system
was
developed
by
Luo
et
al.
[9]
(Table
2).
K5-positive
cells,
obtained
by cell
sorting
LECs LECs
from K5-eGFP
mice, mice,
were placed
on the
top
oftop
collagen
gel containing
3T3
by
K5-eGFP
on
the
of
collagen
gel
containing
by cell
cell sorting
sorting
LECs from
from
K5-eGFP
mice, were
were placed
placed
onet
the
top
of
collagen
gel
containing 3T3
3T3 obtained
by
cell
sorting
LECs
from
K5-eGFP
mice,
were
placed
on
the
of
collagen
gel
containing
3T3
an organotypic
raftcollagen
culture
system
was
developed
bycultured
Luo
al.top
[9]
(Table
2).
K5-positive
feederfeeder
cells.
The
gel
ingel
a cell
culture
insert
was
at
the
air-liquid
interface
in thein cells,
cells.
The
collagen
in
a
cell
culture
insert
was
cultured
at
the
air-liquid
interface
feeder cells.
cells. The
The collagen
collagen gel
gel in
in aa cell
cell culture
culture insert
insert was
was cultured
cultured at
at the
the air-liquid
air-liquid interface
interface in
in the
the
feeder
the
presence
of LECs
EGF EGF
and
This procedure
generated
keratinized
by cell
sorting
fromFGF7.
K5-eGFP
mice, were
placed aonmultilayered
the top of squamous
collagen gel
containing
3T3 feeder
presence
presence of
of EGF
EGF and
and FGF7.
FGF7. This
This procedure
procedure generated
generated aaa multilayered
multilayered squamous
squamous keratinized
keratinized
presence
of
and
FGF7.
This
procedure
generated
multilayered
squamous
keratinized
epithelium
expressing
high
levels
of
K14
and
Trp63
[9].
The
limitations
of
this
system
include
the
epithelium
expressing
high
levels
of
K14
and
Trp63
[9].
The
limitations
of
this
system
include
the
cells. Theepithelium
collagen expressing
gel
in a cell
culture
insert
was
cultured
atlimitations
the air-liquid
in the
epithelium
expressing
high
levels of
of
K14 and
and
Trp63
[9]. The
The
limitations
of this
thisinterface
system include
include
thepresence of
high
levels
K14
Trp63
[9].
of
system
the
requirement
for a for
specific
cell
separation
procedure
(namely
K5-positive
cell separation)
to induce
aa specific
separation
procedure
K5-positive
cell
to
EGF sufficient
and requirement
FGF7.
This
procedure
generated
a
multilayered
squamous
keratinized
epithelium
requirement
for
specificcell
cell
separation
procedure (namely
(namely
K5-positive
cell separation)
separation)
to induce
induce expressing
requirement
for
a
specific
cell
separation
procedure
(namely
K5-positive
cell
separation)
to
induce
epithelial
cell growth
and the
low colony-forming
efficiency
(only (only
0.78%)
of the the
sufficient
epithelial
cell
growth
and
colony-forming
efficiency
0.78%)
sufficient
epithelial
cell[9].
growth
and the
the low
lowof
colony-forming
efficiencythe
(only
0.78%) of
of the
the
sufficient
epithelial
cell
growth
and
the
low
colony-forming
efficiency
(only
0.78%)
of
highK5-positive
levels
of
K14
and
Trp63
The
limitations
this
system
include
requirement
for a specific
cells
[9].
K5-positive
K5-positivecells
cells[9].
[9].
K5-positive
cells
[9].
The organotypic
raft(namely
culture
system
has beencell
applied
in studies
carcinoma
cell invasion.
cell separation
procedure
K5-positive
separation)
toon
induce
sufficient
epithelial cell growth
The
The organotypic
organotypic raft
raft culture
culture system
system has
has been
been applied
applied in
in studies
studies on
on carcinoma
carcinoma cell
cell invasion.
invasion.
The
organotypic
raft
culture
system
has
been
applied
in
studies
on
carcinoma
cell
invasion.
Nurmenniemi
et
al. cultured
an
invasive
human
tongue
squamous
cell
carcinoma
cell [9].
line
(HSC-3)
Nurmenniemi
et
al.
cultured
an
invasive
human
tongue
squamous
cell
carcinoma
cell
line
and the
low
colony-forming
efficiency
(only
0.78%)
of
the
K5-positive
cells
Nurmenniemi et
et al.
al. cultured
cultured an
an invasive
invasive human
human tongue
tongue squamous
squamous cell
cell carcinoma
carcinoma cell
cell line
line(HSC-3)
(HSC-3)
Nurmenniemi
on collagen
gel containing
gingival
fibroblasts,
wherewhere
the gel
was mechanically
supported
on(HSC-3)
a
on
collagen
gingival
fibroblasts,
the
mechanically
supported
aa invasion.
The
organotypic
raft
culture
system
has
been
applied
in
studies
on
carcinoma
on
collagen gel
gel containing
containing
gingival
fibroblasts,
where
the gel
gel was
was
mechanically
supported on
oncell
on
collagen
gel
containing
gingival
fibroblasts,
where
the
gel
was
mechanically
supported
on
a
stainless-steel
grid [32]
(Table
2). They
measured
the invasion
depthdepth
and area
of
HSC-3
cells into
the
stainless-steel
grid
[32]
(Table
2).
They
measured
the
invasion
and
area
of
HSC-3
cells
into
the
stainless-steel
grid
[32]
(Table
2).
They
measured
the
invasion
depth
and
area
of
HSC-3
cells
into
the
stainless-steel
grid [32]
(Table
2).
They
measured
the
invasion
depth and
area
ofcarcinoma
HSC-3
cells
into
the
Nurmenniemi
et
al.
cultured
an
invasive
human
tongue
squamous
cell
cell
line
(HSC-3)
collagen
gel
layer,
thereby
assessing
the
invasion
activity
and
the
process
used
by
HSC-3
cells.
It
is
collagen
isis
collagengel
gellayer,
layer,thereby
therebyassessing
assessingthe
theinvasion
invasionactivity
activityand
andthe
theprocess
processused
usedby
byHSC-3
HSC-3cells.
cells.It
collagen
gel
layer,
thereby
assessing
the
invasion
activity
and
the
process
used
by
HSC-3
cells.
ItIt is
known
that
the
cancer
environment
is
mainly
composed
of
cancer-associated
fibroblasts.
However,
on collagen
gel
containing
gingival
fibroblasts,
where
the
gel
was
mechanically
supported
on
known
knownthat
thatthe
thecancer
cancerenvironment
environmentis
ismainly
mainlycomposed
composedof
ofcancer-associated
cancer-associatedfibroblasts.
fibroblasts.However,
However,
known
that
the
cancer
environment
is
mainly
composed
of
cancer-associated
fibroblasts.
However,
other
cells,
such
as
endothelial
cells
and
inflammatory
cells,
also
affect
cancer
progression,
invasion,
other
cells,
such
as
endothelial
cells
and
inflammatory
cells,
also
affect
cancer
progression,
invasion,
a stainless-steel
grid
[32]
(Table 2). cells
They
measured
thecells,
invasion
depth
and
area of HSC-3
cells into the
other cells,
cells,
such
as endothelial
endothelial
cells
and
inflammatory
cells,
also affect
affect
cancer
progression,
invasion,
other
such
as
and
also
cancer
progression,
invasion,
and recurrence.
Therefore,
Nurmenniemi
et al.inflammatory
used
real human
tissue,
uterine
leiomyoma,
instead
Therefore,
Nurmenniemi
etetal.
real
uterine
collagen and
gel
layer,
thereby
assessing
the invasion
activity
andtissue,
the process
used by instead
HSC-3
andrecurrence.
recurrence.
Therefore,
Nurmenniemi
al.used
used
real human
human
tissue,
uterineleiomyoma,
leiomyoma,
instead cells. It is
and
recurrence.
Therefore,
Nurmenniemi
et al.
used
real
human
tissue,
uterine
leiomyoma,
instead
known that the cancer environment is mainly composed of cancer-associated fibroblasts. However,
other cells, such as endothelial cells and inflammatory cells, also affect cancer progression, invasion,
and recurrence. Therefore, Nurmenniemi et al. used real human tissue, uterine leiomyoma, instead of
collagen gel containing gingival fibroblasts and observed a much higher invasion activity for HSC-3
Int. J. Mol. Sci. 2016, 17, 168
7 of 12
Int. J. Mol. Sci. 2016, 17, 168
7 of 12
of collagen gel containing gingival fibroblasts and observed a much higher invasion activity for
HSC-3 cells into the myoma tissues. Similar results were obtained by Bitu et al. [33] (Table 2), and,
cells
into thethe
myoma
tissues.
Similar results
were obtained
byconsidered
Bitu et al. [33]
(Table 2), and,
the
therefore,
myoma
organotypic
raft culture
method is
advantageous
fortherefore,
studies on
myoma
organotypic
raft
culture
method
is
considered
advantageous
for
studies
on
tongue
squamous
tongue squamous cell carcinoma.
cell carcinoma.
3.4. Organoid Culture
3.4. Organoid Culture
To generate a multilayered keratinized epithelium at a high efficiency from unseparated
To
a multilayered
keratinizedwe
epithelium
at aa three-dimensional
high efficiency from
unseparated
(whole)
(whole)generate
lingual epithelial
cell populations,
attempted
(3D)
lingual epithelial
lingual
epithelial
cell populations,
weEnzymatically
attempted a three-dimensional
(3D)cultured
lingual epithelial
cell in
culture
cell culture
system
[34] (Table 2).
isolated LECs were
in Matrigel
the
system
[34]
(Table
2).
Enzymatically
isolated
LECs
were
cultured
in
Matrigel
in
the
presence
of
presence of EGF, noggin, and R-spondin1. Round-shaped organoids with concentric EGF,
cell
noggin,
and R-spondin1.
Round-shaped
organoids
withof
concentric
cell arrangements
were generated
arrangements
were generated
at a seeding
efficiency
3.4% (Figure
2a). The organoids
showed
ata astratum
seeding corneum
efficiency (stained
of 3.4% (Figure
2a).
The
organoids
showed
a
stratum
corneum
(stainedand
red
red with eosin; Figure 2b). Positive staining for anti-K5
with
eosin;
Figure
2b).
Positive
staining
for
anti-K5
and
K14-antibodies
was
observed
in
the
outer
K14-antibodies was observed in the outer periphery of the organoids, indicating the localization of
periphery
of the
organoids,
indicating
the localization
of LESCs cells
and progenitor
cells. We
LESCs and
progenitor
cells.
We deduce
that differentiated
migrate toward
thededuce
center that
of
differentiated
cells
migrate
toward
the
center
of
organoids
according
to
their
maturation.
Several
organoids according to their maturation. Several Ki67-positive cells (proliferating cells) were
Ki67-positive
cellsouter
(proliferating
were
observed(indicated
in the outer
of the
observed in the
peripherycells)
of the
organoids
by periphery
arrows), and
weorganoids
speculate(indicated
that cell
by
arrows),
and we mainly
speculate
cellsites
mitosis
occurred
at these sites
(Figuremicrograph
2b). A transmission
mitosis
occurred
atthat
these
(Figure
2b).mainly
A transmission
electron
of the
electron
micrograph
of
the
organoid
confirmed
the
existence
of
a
stratum
corneum
in
the
center
with
organoid confirmed the existence of a stratum corneum in the center with flattened cells
flattened
cells the
surrounding
the stratum
corneum
(Figure
2c). On
side of the
organoid,
surrounding
stratum corneum
(Figure
2c). On
the outer
sidethe
of outer
the organoid,
a cell
with
aaggregated
cell with aggregated
chromosomes
black was
arrow)
was observed,
suggesting
that mitosis
chromosomes
(indicated(indicated
by blackby
arrow)
observed,
suggesting
that mitosis
had
had
been
actively
occurring
at
this
site.
Figure
2d,e
is
higher
magnification
photographs
of
the
areas
been actively occurring at this site. Figure 2d,e is higher magnification photographs of the areas
enclosed
and fibers
fibers specific
specific to
to cells
cells in
in the
the intermediate
intermediate
enclosed in
in the
the squares
squares in
in Figure
Figure 2c,
2c, and granules and
epithelium
InIn
addition,
thethe
presence
of desmosomes
between
the the
LECs
was
epitheliumare
areshown
shownininboth
bothfigures.
figures.
addition,
presence
of desmosomes
between
LECs
observed
(red arrow;
Figure
2e). 2e).
was observed
(red arrow;
Figure
a
Day 1
Day 3
Day 5
Day 8
Day 10
Day 13
100μm
b
Keratin 5
HE
Keratin 14
Ki67
100μm
c
d
*
e
*
10 μm
1 μm
1 μm
Figure2.2. (a)
(a) Time
Time course
course of
of lingual
lingual organoid
organoid growth.
growth. LECs
Figure
LECs were
were cultured
culturedin
inthe
theorganoid
organoidculture
culture
system,
generating
round-shape
organoids
with
a
concentric
cell
arrangement.
Phase-contrast
system, generating round-shape organoids with a concentric cell arrangement. Phase-contrastimage;
image;
(b)Histological
Histological analysis
analysis of
of organoids.
organoids. Organoids
(b)
Organoids were
were separated
separated from
from Matrigel
Matrigeland
andtheir
theirparaffin
paraffin
sectionswere
werestained
stainedwith
with
HE
reagents
and
anti-K5
antibodies.
K5 and
K14-positive
sections
HE
reagents
and
anti-K5
andand
K14K14
antibodies.
K5 and
K14-positive
cellscells
were
were observed
in theperiphery
outer periphery
of the organoids.
Staining
Ki67 showed
thatcells
some
cells
observed
in the outer
of the organoids.
Staining
for Ki67forshowed
that some
actively
actively proliferated
inperiphery
the outer(arrows);
periphery
(c) Transmission
electronofmicrograph
of
proliferated
in the outer
(c) (arrows);
Transmission
electron micrograph
organoids. The
organoids.
The
organoids
had
a
stratum
corneum
in
the
center
and
flattened
cells
surrounding
the
organoids had a stratum corneum in the center and flattened cells surrounding the stratum corneum.
stratum
corneum.ofAtthe
theorganoid,
peripheryaofcell
thewith
organoid,
a cell with
aggregated(indicated
chromosomes
(indicated
At
the periphery
aggregated
chromosomes
by arrow)
was
by
arrow)
was
detected;
(d,e)
Higher
magnification
views
of
the
boxed
areas
in
(c).
Many
detected; (d,e) Higher magnification views of the boxed areas in (c). Many keratohyaline granules
(blue
keratohyaline
(bluearrow),
arrow),and
keratin
fibersgranules
(green arrow),
and by
lamellar
granules
(indicated
arrow),
keratingranules
fibers (green
lamellar
(indicated
asterisk)
were detected
in by
the
asterisk)
were
detected
in
the
cytoplasm.
Moreover,
formation
of
desmosomes
between
cells
was
cytoplasm. Moreover, formation of desmosomes between cells was observed (red arrow). Modified
observed
from
[34]. (red arrow). Modified from [34].
Int. J. Mol. Sci. 2016, 17, 168
8 of 12
ToJ.examine
whether
injected
Int.
Mol. Sci. 2016,
17, 168 Bmi1-positive cells have the potential to generate organoids, we8 of
12
Bmi1-CreER; Rosa26-rainbow mice with tamoxifen. Two days later, the LECs were collected and cultured
To examine
whether
Bmi1-positive
have
potential
organoids,
injected
in our organoid
culture
system.
Figure 3a cells
shows
thethe
time
coursetoofgenerate
the formation
of awe
representative
Bmi1-CreER; Rosa26-rainbow mice with tamoxifen. Two days later, the LECs were collected and
blue-colored organoid. A phase-contrast image at day 10 shows that the organoid has a concentric cell
cultured in our organoid culture system. Figure 3a shows the time course of the formation of a
arrangement. As shown in Figure 3b, all organoids were of a single color, and organoids containing
representative blue-colored organoid. A phase-contrast image at day 10 shows that the organoid has
morea than
one color
were never observed.
this 3b,
result,
it is evident
that
colored
concentric
cell arrangement.
As shown From
in Figure
all organoids
were
of each
a single
color,organoid
and
was organoids
generatedcontaining
from a single
Bmi1-positive
cell.
The
findings
clearly
show
that
single
Bmi1-positive
more than one color were never observed. From this result, it is evident that
cellseach
havecolored
the potential
to was
generate
an organoid.
organoid
generated
from a single Bmi1-positive cell. The findings clearly show
Next,
tamoxifen
was added
to the
Bmi1-CreER;
Rosa26-rainbow
mouse organoid culture system at
that
single
Bmi1-positive
cells have
the
potential to generate
an organoid.
Next, tamoxifen
added to
the Bmi1-CreER;
Rosa26-rainbow
organoid culture
system
the beginning
of culturewas
to induce
Cre-mediated
recombination
in mouse
the Bmi1-positive
cells.
On day 7
at
the
beginning
of
culture
to
induce
Cre-mediated
recombination
in
the
Bmi1-positive
cells.
On
day
of the culture, a few orange or red cells were detected at the outer periphery of the green-colored
7
of
the
culture,
a
few
orange
or
red
cells
were
detected
at
the
outer
periphery
of
the
green-colored
organoids (Figure 3c). Thereafter, the orange and red cells proliferated rapidly, some spreading toward
organoids
(Figure
3c). Thereafter,
orangecorneum
and red cells
proliferated
rapidly,All
some
spreading
the center
of the
organoids
to form athe
stratum
(indicated
by arrows).
of the
organoids
toward the center of the organoids to form a stratum corneum (indicated by arrows). All of the
were green with either orange or red cells. This indicates that only one Bmi1-positive cell existed per
organoids were green with either orange or red cells. This indicates that only one Bmi1-positive cell
organoid at the time of tamoxifen administration.
existed per organoid at the time of tamoxifen administration.
These
findings
(Figure
3) have
confirmed
ourour
previous
study
These
findings
(Figure
3) have
confirmed
previous
study[8][8]showing
showingthat
thatBmi1-positive
Bmi1-positive cells
are LESCs.
The
above
studies
demonstrate
that
each
organoid
is
generated
from
a
single
cell and
cells are LESCs. The above studies demonstrate that each organoid is generated from a single
cell that
single
LESCs
can
differentiate
into
mature
keratinized
LECs.
Therefore,
our
organoid
culture
system
and that single LESCs can differentiate into mature keratinized LECs. Therefore, our organoid is
useful
for identifying
LESCs.
culture
system is useful
for identifying LESCs.
Figure 3. Potential of Bmi1-positive cells to generate organoids. (a) Time course of organoid growth
Figure
Potential
of Bmi1-positive
cells toTwo
generate
(a) Time course
of organoid
growth
(in 3.vivo
administration
of tamoxifen).
days organoids.
after administering
tamoxifen,
LECs from
(in vivo
administration
of
tamoxifen).
Two
days
after
administering
tamoxifen,
LECs
from
Bmi1-CreER;
Bmi1-CreER; Rosa26-rainbow mice were cultured in the organoid culture system for three days.
Rosa26-rainbow
were cultured
in cells
the organoid
culture
system
for organoids
three days.
Induction
of Cre
Induction ofmice
Cre labeled
individual
red, orange,
or blue,
and the
showed
mosaic
labeled
individual
cells
red,
orange,
or
blue,
and
the
organoids
showed
mosaic
patterns
composed
patterns composed of the colored cells on day 5 of culture. Fluorescence image; (b) Representative
of the
colored cells
on day on
5 of
culture.
Fluorescence
image; (b) of
Representative
photographs
photographs
of organoids
day
12 of culture
(in vivo administration
tamoxifen). Most
organoids of
were green,
but12
some
round-shape
organoids
were red,oforange,
or blue.Most
Overlay
of fluorescence
organoids
on day
of culture
(in vivo
administration
tamoxifen).
organoids
were green,
image round-shape
and phase-contrast
image;were
(c) Time
of or
organoid
administration
of and
but some
organoids
red, course
orange,
blue. growth
Overlay(inofvitro
fluorescence
image
tamoxifen).image;
Active (c)
form
of tamoxifen
added growth
to the LECs
fromadministration
Bmi1-CreER; Rosa26-rainbow
mice
phase-contrast
Time
course ofwas
organoid
(in vitro
of tamoxifen).
Active
start of culture.
Bmi1-positive
cellsfrom
wereBmi1-CreER;
induced by the
tamoxifen to turn
or orange
formatofthe
tamoxifen
was added
to the LECs
Rosa26-rainbow
micered
at the
start ofand
culture.
formed colored stratum corneum (indicated by arrows). Modified from [34].
Bmi1-positive cells were induced by the tamoxifen to turn red or orange and formed colored stratum
corneum (indicated by arrows). Modified from [34].
Int. J. Mol. Sci. 2016, 17, 168
Int. J. Mol. Sci. 2016, 17, 168
9 of 12
9 of 12
4. Consideration
Considerationof
ofthe
theLingual
LingualStem
StemCell
CellNiche
Niche
The stem cell niche
niche for
for lingual
lingual epithelial
epithelial stem
stem cells
cells is
is unknown.
unknown. Here, we discuss
discuss candidate
candidate
niche cells deduced from other
other stem
stem cell
cell systems.
systems. In the intestine, Paneth cells and myoepithelial
myoepithelial
cells located at crypt bottoms have been reported to function as niche cells of Lgr5-positive intestinal
stem cells [35]. Notably, Paneth cells are terminally differentiated cells generated by intestinal
intestinal stem
cells, indicating
areare
at at
least
partially
supported
by their
own own
descendant
cells.
indicating that
thatintestinal
intestinalstem
stemcells
cells
least
partially
supported
by their
descendant
On
the
hand,hand,
otherother
nicheniche
cells cells
located
at the
bottom,
defined
as myoepithelial
cells
of
cells.
Onother
the other
located
at crypt
the crypt
bottom,
defined
as myoepithelial
cells
mesenchymal
origin,
do do
notnot
originate
from
intestinal
stem
cells.
of mesenchymal
origin,
originate
from
intestinal
stem
cells.
In the tongue, we reported that Bmi1-positive cells were not located at the base of the epithelial
layers; instead, these cells were present most frequently in the second or third layer [8]. Therefore,
we speculate that surrounding cells generated by lingual epithelial stem cells could be candidate
candidate
niche cells.
marrow, several
several concepts
concepts for the
the hematopoietic
hematopoietic stem cell niche were proposed in the
In the bone marrow,
2000s; these include
include the osteoblastic
osteoblastic niche
perivascular niche
2000s;
niche [36],
[36], reticular
reticular cell
cell niche
niche [37], and perivascular
niche [38].
[38].
Recently, the arteriolar niche [39] and perisinusoidal niche [40] were also proposed. Yamazaki
Yamazaki et al.
demonstrated that Schwann cells were components of the perivascular niche and had the ability to
hematopoietic stem
stem cells
cells [41].
[41].
induce a dormancy state in hematopoietic
Based on the concepts of the stem cell niche proposed in various tissues, it is possible that
similar niche
nichesystems,
systems,such
suchasasthe
the
descendant
niche,
perivasucular
niche,
mesenchymal
niche,
similar
descendant
cellcell
niche,
perivasucular
niche,
mesenchymal
niche,
and
and neurogenic
niche,
function
inlingual
the lingual
epithelium
(Figure
4). Further
characterization
of
neurogenic
niche,
also also
function
in the
epithelium
(Figure
4). Further
characterization
of the
the lingual
epithelium
is needed
to uncover
niche
controlling
actual
LESCs
to determine
lingual
epithelium
is needed
to uncover
niche
cellscells
controlling
actual
LESCs
and and
to determine
the
the physiological
of niche
cells
the maintenance
and regeneration
of the epithelium.
Our
physiological
rolesroles
of niche
cells for
thefor
maintenance
and regeneration
of the epithelium.
Our lingual
lingual organoid
culture[34]
system
[34] is extremely
useful
for theofscreening
of niche
factors
and the
organoid
culture system
is extremely
useful for the
screening
niche factors
and the
identification
identification
of coculture
niche cellswith
by coculture
with isolated LESCs.
of
niche cells by
isolated LESCs.
Figure 4. Putative lingual stem cell niche deduced from other stem cell systems.
5. Applications
5.
Applicationsand
andPerspectives
Perspectives
Tongue squamous
squamouscell
cellcarcinoma
carcinoma
(TSCC)
is known
to occur
a high
incidence
relative
to
Tongue
(TSCC)
is known
to occur
withwith
a high
incidence
relative
to other
other
types
of
oral
cancer,
and
long-term
physical
and
chemical
stimulation
is
one
of
the
causes
of
types of oral cancer, and long-term physical and chemical stimulation is one of the causes of TSCC.
TSCC.
Most
TSCC
is
derived
from
LESCs
that
have
acquired
malignant
changes.
However,
the
Most TSCC is derived from LESCs that have acquired malignant changes. However, the mechanisms
mechanisms
causeschanges
of malignant
changes
have not been
elucidated.
determine
the
and
causes ofand
malignant
in LESCs
have in
notLESCs
been elucidated.
To determine
theTocauses
of cancer
causes
of
cancer
outbreak,
identification
of
LESCs
and
their
niche
is
necessary.
We
have
shown
that
outbreak, identification of LESCs and their niche is necessary. We have shown that Bmi1-positive
Bmi1-positive cells are long-term LESCs [8]. In TSCC, immunohistochemistry analyses revealed
that the number of Bmi1-positive cells increased significantly according to malignant progression.
Int. J. Mol. Sci. 2016, 17, 168
10 of 12
cells are long-term LESCs [8]. In TSCC, immunohistochemistry analyses revealed that the number
of Bmi1-positive cells increased significantly according to malignant progression. Bmi1 expression
levels were found to be correlated with advanced malignant features on TSCC, such as invasion,
metastasis, chemoresistance, and poor prognosis [20,42]. In contrast, Hayry et al. demonstrated that
negative Bmi1 expression in TSCC predicted poor prognosis [43]. Bmi1 is a direct target of c-myc,
and the c-myc-Bmi1-manganese superoxide dismutase (SOD2) pathway is related to the invasion
and metastasis of TSCC [20]. Upregulation of Bmi1 expression induces enhanced chemoresistance to
cisplatin in TSCC cells [42]. Thus, many studies have demonstrated that Bmi1 is a key molecule in
the initiation or progression of TSCC. It is not clear whether the aberrant overexpression of Bmi1 was
caused by malignant conversion of normal Bmi1-positive cells or whether Bmi1-negative cells acquired
Bmi1 overexpression through unknown mechanisms before transforming into malignant cells. From
the above-mentioned studies, it is clear that the Bmi1 molecule has a role in TSCC; however, further
studies are needed to determine the details of this role.
Rapid and complete tongue regeneration should be induced after lingual tumor excision. For this
purpose, polyglycolic acid sheets and fibrin glue sprays have been used for covering open wounds
after lingual tumor excision [44]. Using our organoid culture system, we observed that immature
organoids harvested on day 3 of culture, which did not have stratum corneum, could be engrafted
and matured on the tongues of recipient mice [34]. Technical difficulties prevented injection of the
organoids into the subepithelial area; thus, the grafted organoids expanded in the muscle layer rather
than the epithelial layer of the recipient tongues. When techniques to inject the organoids directly into
the subepithelium area can be developed, our culture system will be useful in lingual regeneration
medicine to treat injury and for tumor excision.
We also observed that organoids generated from carcinogen (4-nitroquinolin 1-oxide)-treated mice
had an abnormal morphology and a high number of Ki67-positive cells [34]. These results showed
that the organoid culture system is useful for studying the regulatory mechanisms of normal lingual
epithelium and the mechanism of carcinogenesis.
Acknowledgments: The authors acknowledge financial support from the following sources: Funding Program
for Next Generation World-Leading Researchers, Grant-in-Aid for Scientific Research(A), Grant-in-Aid for
Challenging Exploratory Research, The Mochida Memorial Foundation, The Naito Memorial Foundation, The
Cell Science Research Foundation, The Uehara Memorial Foundation, The Mitsubishi Foundation, The Yasuda
Memorial Foundation and The Takeda Science Foundation to Hiroo Ueno.
Author Contributions: All authors discussed about the content of the article. Hiroko Hisha and Hiroo Ueno
wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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