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Identification and characterization of adult stem cells in the oral cavity
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Identification and characterization of adult stem cells in the oral cavity
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Content
IDENTIFICATION AND CHARACTERIZATION OF ADULT STEM CELLS
IN THE ORAL CAVITY
BY
KEERTHI BODDUPALLY
A Dissertation presented to the
FACULTY OF USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial fulfillment of the requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, CELLULAR AND MOLECULAR BIOLOGY)
DECEMBER 2015
Copyright © 2015 Keerthi Boddupally
TABLE OF CONTENTS
LIST OF FIGURES .......................................................................................................... 1
LIST OF TABLES ........................................................................................................... 3
LIST OF ABBREVIATIONS ......................................................................................... 4
ACKNOWLEDGEMENTS ............................................................................................. 8
ABSTRACT .................................................................................................................... 10
CHAPTER 1: INTRODUCTION .................................................................................. 12
1.1
Adult
stem
cells
...........................................................................................................................................
12
1.2
Salivary
gland-‐
Structure
and
function
.............................................................................................
16
1.3
Tongue
............................................................................................................................................................
19
1.4
Cancer
Stem
Cells
.......................................................................................................................................
22
1.5
Head
and
neck
squamous
cell
carcinoma
........................................................................................
26
1.6
Epithelial
mesenchymal
interactions
in
the
tumor
microenvironment
.............................
30
1.7
Significance
...................................................................................................................................................
33
CHAPTER 2: DEFINING THE LOCALIZATION AND MOLECULAR
CHARACTERISTIC OF MINOR SALIVARY GLAND LABEL RETAINING
CELLS ............................................................................................................................. 34
2.1
Abstract
..........................................................................................................................................................
34
2.2
Introduction
.................................................................................................................................................
35
2.3
Results
.............................................................................................................................................................
38
2.3A
Label
retaining
cells
of
minor
salivary
glands
localize
predominantly
in
the
basal
layer
of
the
lower
excretory
duct
and
the
myoepithelial
layer
of
acini.
...............................
38
2.3B
Isolation
and
regenerative
potential
of
label
retaining
cells
of
minor
salivary
gland
..............................................................................................................................................................................
41
2.3C
Defining
the
molecular
characteristic
of
minor
salivary
gland
label
retaining
cells
..............................................................................................................................................................................
45
2.3D
Activation
of
TGFβ
signaling
pathway
in
minor
salivary
glands
LRCs
and
BMP
pathway
in
early
progenitors
..................................................................................................................
49
2.3E
Minor
salivary
glands
are
sensitive
to
a
carcinogenic
tobacco
compound
and
give
rise
to
a
low-‐grade
adenoma
...................................................................................................................
52
2.4
Discussion
.....................................................................................................................................................
55
CHAPTER 3: LGR5 MARKS NEURAL CREST DERIVED MULTIPOTENT
ORAL STROMAL STEM CELLS ............................................................................... 60
3.1
Abstract
..........................................................................................................................................................
60
3.2
Introduction
.................................................................................................................................................
61
3.3
Results
.............................................................................................................................................................
64
3.3A
Lgr5
marks
novel
population
of
stromal
cells
in
the
tongue
and
oral
mucosa.
......
64
3.3B
Lgr5
positive
stromal
cells
in
the
tongue
are
neural
crest
derived.
............................
70
3.3C
Lgr5
is
expressed
in
neural
crest
cells
during
embryonic
development.
..................
73
3.3D
Lgr5+
tongue
stromal
cells
can
be
propagated
in
vitro.
....................................................
75
3.3E
RNA
sequencing
revealed
that
Lgr5+
oral
stromal
cells
exhibit
characteristics
of
embryonic
neural
crest
cells.
..................................................................................................................
77
3.3F
Lgr5+
oral
stromal
cells
differentiate
into
neural
crest
lineages.
.................................
81
3.3G
Lgr5+
oral
stromal
cells
assume
mesenchymal
fate
in
de
novo
reconstitution
assay
and
participate
in
tongue
wound
healing
..........................................................................................
83
3.4
Discussion
.....................................................................................................................................................
85
CHAPTER 4: CONCLUDING REMARKS ................................................................ 88
CHAPTER 5: MATERIALS AND METHODS .......................................................... 92
5.1
Mice
..................................................................................................................................................................
92
5.2
Genotyping
....................................................................................................................................................
94
5.3
Immunohistochemistry
and
Immunofluorescence
Staining
...................................................
95
5.4
Isolation
of
minor
SG
LRCs
.....................................................................................................................
96
5.5
Fluorescence
Activated
Cell
Sorting
(FACS)
...................................................................................
97
5.6
Glandular
Injections
..................................................................................................................................
98
5.7
RNA
Isolation,
Microarray,
RT-‐PCR,
and
qPCR
.............................................................................
99
5.8
In
vitro
expansion,
growth
curve
analysis
and
colony
formation
assay
..........................
100
5.9
In
vitro
Differentiation
Assay
..............................................................................................................
101
5.10
Chamber
graft
transplantation
and
wound
healing
assays
................................................
102
5.11
Immunocytochemistry
........................................................................................................................
103
5.12
RNA
Seq
.....................................................................................................................................................
104
REFERENCES .............................................................................................................. 105
1
LIST OF FIGURES
Figure 1: Adult epithelial stem cells……………………………………………………. 15
Figure 2: Salivary gland structure………………………………………………………. 17
Figure 3: Anatomy of the tongue……………………………………………………….. 20
Figure 4: Cancer stem cell model………………………………………………………. 23
Figure 5: Hallmarks of cancer-associated fibroblasts…………………………………... 31
Figure 6: Optimization for LRC identification in salivary gland………………………. 39
Figure 7: Preferential localization of LRC in the basal layer of the salivary gland…...... 40
Figure 8: LRCs are clonogenic and have regenerative potential……………………...... 43
Figure 9: Tissue pieces and pure population engrafted in donor salivary glands were
actively proliferative……………………………………………………………………. 44
Figure 10: Transcriptional profiling of minor salivary gland LRCs……………………. 47
Figure 11: Wnt pathway is not active in minor salivary gland LRCs whereas sonic
hedgehog signals predominantly to the stromal cells…………………………………... 48
Figure 12: Activities of TGF B signaling pathway in minor salivary gland LRCs and
BMP pathway in early progenitors……………………………………………………... 51
Figure 13: Tumorigenic potential of minor salivary gland basal layer cells…………… 53
Figure 14: Lgr5 is a transmembrane receptor…………………………………………... 62
Figure 15: Lgr5 marks both hair follicle stem cells and intestinal stem cells…………... 66
Figure 16: Lgr5 OSCCs localize at specific regions in the buccal mucosa…………….. 67
Figure 17: Lgr5 positive oral stromal stem cells observed at the frenulum in the stromal
layer……………………………………………………………………………………... 69
Figure 18: Lgr5 positive oral stromal stem cells are neural crest in origin…………….. 72
Figure 19: Lgr5 is expressed in the neural crest cells at embryonic day 9.5 (E9.5)……. 74
Figure 20: Isolation and characterization of Lgr5 OSCC cells…………………………. 76
Figure 21: RNA Seq analysis reveals similarities between Lgr5 OSCC and cranial neural
crest cells………………………………………………………………………………... 79
2
Figure 22: Lgr5 OSCC cells are multipotential and give rise to cells in both ectodermal
and mesodermal lineages……………………………………………………………….. 82
Figure 23: Lgr5 OSCC cells participate in regeneration and reconstitution of the stroma
in wound healing experiments………………………………………………………….. 84
3
LIST
OF
TABLES
Table 1: PCR primer list…………………………………………………………………94
Table 2: Immunohistochemistry antibody list…………………………………………...95
4
LIST OF ABBREVIATIONS
4 NQO - 4-nitroquinoline-oxide
AJ - Adherens Junction
Ba - Branchial arch
BCC - Basal Cell Carcinoma
Bgn - Biglycan
Bm - Basement Membrane
BSA - Bovine Serum Albumin
Col - Collagen
DAPI - 4’, 6’-diamidino-2-phenylindole
DMEM – Dulbecco’s Modified Eagle’s Medium
Dox - Doxycycline
DS – Donkey Serum
ECM - Extra Cellular Matrix
EMT- Epithelial to Mesenchymal Transition
Ep - Epithelium
FACS - Fluorescent Activated Cell Sorting
FBS - Fetal Bovine Serum
5
Fov - Field of view
Fr - Frenulum
GFAP - Glial Fibrillary Acidic Protein H & E – hematoxylin and eosin
H2BGFP - Histone 2B Green Fluorescence Protein
HF – Hair Follicle
HP - Hard palate
IACUC - International Animal Care and Use Committee
IF – Immuno Fluorescence
IHC - Immuno Histo Chemistry
IPA - Ingenuity Pathway Analysis
K5 - Keratin 5
K8 - Keratin 8
Lam - Laminins
LOSSC – Lgr5 Oral Stromal Stem Cells
m- Muscle
M - molars
MEF - Mouse Embryonic Fibroblasts
6
mRNA – messenger Ribo Nucleic Acid
Nc - Neural crest
NF - Neural Filament
PCR - Polymerase Chain Reaction
RT- PCR – Reverse Transcription Polymerase Chain Reaction
RT - Room Temperature
SC - Stem Cells
SCC - Squamous Cell Carcinoma
SG – Salivary Gland
Shh – Sonic Hedge Hog
SMA - Smooth Muscle Actin
SMG - Sub Mandibular Gland
SP - soft palate
St - Stroma
T - Tongue
T- tumor
TGFβ Transforming Growth Factor Beta
7
Tnc - tenascin (Tnc)
Tnc –tenascin
Tspan6 - tetraspanin
W - wound
WM - Whole Mount
WT - Wild Type
8
ACKNOWLEDGEMENTS
There are so many people that have played a significant role in the completion of this
project, without thanking them this thesis would be incomplete. First of all I would like to
thank my mentor Dr. Agnieszka Kobielak. Agnes, you were not only one of the best
mentors that a student could ask for but you are also my strongest professional support.
Thank you for taking the time and making the effort to understand me as a person. Thank
you for giving me the opportunity to fail several times as a part of my learning process. I
would not be the person I am today if not for your guidance. Not only have you taught
me about the importance of research, you have taught me to be professional, to
understand priorities in life and the importance of maintaining a work life balance. I
would also sincerely like to thank Dr. Krzysztof Kobielak. Kris, you have been a guide to
me almost as much as Agnes. Thank you for all your guidance and help these last four
years. Thank you for helping me out with the mice when I needed it. You have been a
fantastic support to me throughout this process. I would also like to thank the rest of my
committee members, Dr. Gregor Adams and Dr. Young Hong. You both have been
strong supporters of my research throughout this process. You both have been available
to me when I would need any guidance, and for that I sincerely thank you. None of this
would have been complete without the support from my family and friends. I would like
to specially thank my parents who have stood by me through this entire process. You
both have been my pillars of support. Everything I am today is a reflection of your
affection and love for me. Thank you for believing in me when I doubted myself. I would
like to thank my sister, Akka, who is my biggest cheerleader. In all my times of doubt,
you have always nudged me on and helped me move past all the hurdles in my graduate
9
life. I would also like to thank my brother in law, Shane who has never failed to make me
smile in a bad situation. I cannot leave out a very special person in my life, Kapil. You
have always been there for me through it all and for that I can never repay you. You are
my rock and I love you for that. Ryan, I couldn’t have done this without you. Thank you
for holding my hand all the times that I was scared. Sanjeev, I cannot imagine my
graduate life without you. Thank you for all the “gandi chai” conversations. You were my
were strength, my inspiration and my home. This thesis would not be complete without
mentioning some of my friends who have been of immense support, who have answered
my late night phone calls and listened to me, rant and vent about graduate life. Reddy,
Venky, Swati, Neha, Sapna and Jessica, you are all my anchors. You have held me back
from drowning when I felt like I was overwhelmed by the workload; you lifted my spirits
high when I felt low, you wiped my tears when I was sad and celebrated my successes
when I was happy. I could not have completed my work without you all in my life. Lastly,
I would like to thank all my lab mates, Vicky, Guangfang, David and James. You all have
been a pleasure to work with and I am proud to be a part of this team. I am lucky to call
you all my colleagues and friends. You have all created a wonderful work environment
where I looked forward to coming to work each day. A big heartfelt thank you to
everyone mentioned in this acknowledgement and all the others who were not. This is as
much your victory as mine. I am eternally grateful to have such wonderful people in my
life that I can call my own. Thank you all.
10
ABSTRACT
Tongue and salivary glands are the main sites of head and neck squamous cell carcinoma.
However, little is known about the origin and progression of such tumors. In this study,
we investigated the role of stem cells in tumor initiation, and their cross talk with the
surrounding stroma. Using a pulse chase strategy, we identify keratin 5 expressing (K5+)
basal cells as slow-cycling bipotent cellular population in the minor salivary gland that
self renew and differentiate into keratin 8 expressing luminal cells. Serial cell
transplantation assays unraveled K5+ basal cells as tumor-initiating cells in vivo. To gain
further insights into the phenomenon of tumor progression and wound healing in the head
and neck region, we interrogated the stromal-epithelial interactions in the oral mucosa.
After injury, the oral mucosal epithelium rapidly heals with lack of scar formation, and is
a site of relatively aggressive epithelial malignancy compared to the salivary gland
epithelia. Multipotent stem cell population of putative neural crest origin that persists into
adulthood resides in the stroma of the oral cavity. The precise location, and their role in
normal tissue homeostasis and tumor progression is not known. Herein, we identify Lgr5
expressing (Lgr5+) cellular population in the cranial neural crest during embryonic
development that persists as a distinct stem cell population in the stromal compartment of
the adult tongue and oral mucosa. The Lgr5+ oral stromal stem cells (LOSSCs) display
properties of neural crest stem cells, including clonal growth, and multipotent
differentiation into smooth muscle, neuronal, and osteogenic lineages. RNA-seq analysis
of LOSSCs revealed significant enrichment of neural crest markers and lineage-tracing
studies demonstrate that these cells give rise to stromal progeny and persist over a year in
the tongue and buccal mucosa. In vivo transplantation experiments demonstrated that
these cells preferentially reconstitute the stromal layer after injury. Our studies identify
11
for the first time the persistence of Lgr5+ embryonic neural crest cells into adulthood that
contributes to the homeostasis of the stromal compartment of the oral mucosa, and
participate in wound healing after injury. Taken together, our studies highlight the role of
adult stem cell populations in the head and neck region with respect to homeostasis,
injury-repair, and tumorigenesis.
12
CHAPTER 1: INTRODUCTION
1.1 Adult stem cells
Homeostasis is an important feature in the maintenance and sustenance of a healthy
organism (Bergmann and Steller, 2010; van der Kooy and Weiss, 2000). The body is
constantly exposed to several insults though out its lifetime that requires constant repair
and regeneration of lost tissue (Simons and Clevers, 2011; Watt and Hogan, 2000). In the
adult, the repair is associated with certain specialized group of cells that perform these
specific functions termed as adult stem cells. Adult stem cells are self-renewing and are
multipotent in nature (Fuchs and Chen, 2013; Watt and Hogan, 2000). They reside in a
specialized microenvironment termed the stem cell niche that provides the necessary
signals required to regulate their cell fate (Clements and Traver, 2013; Clevers and Nusse,
2012; Guruharsha et al., 2012; Singh et al., 2015). The differentiation potential of most
adult stem cells is restricted to two or more cell types typically within the organ that they
are residing in (Laplante and sabatini, 2012). Adult stem cells can be further classified
based on their location like hematopoitic stem cells, mammary gland stem cells, hair
follicle stem cells etc. Each of these categories of stem cells have the ability to give rise
to almost all the cells required for the normal functioning of that organ system. For
example, the hematopoitic stem cells can give rise to all the progenitor cells that
eventually give rise to transit amplifying cells of both the lymphoid and myeloid lineages
(Bollerot et al., 2005; Dzierzak et al., 1998; Sherwood et al., 2004), the hair follicle stem
cells can give rise to the epithelium, new hair follicles and sweat glands (Mesa et al.,
2015; Rogers, 2004) while the mammary gland stem cells can give rise to both the
13
myoepithelial and luminal cells that form the functional duct (Prater et al., 2014;
Shackleton et al., 2006). Understanding the role of the niche has been of key interest to
many research groups. Several groups have identified the signals provided by the niche to
the cells. Most often than not these signals arise from the cells adjacent to them, the cells
that make up the niche compartment. These cells provide them with either a gradient of
factor information or direct signals that can be used to either self-renew or activate
proliferation. One of the best-defined niches is of the hematopoitic system where CXC 12
plays a key role in the homing, maintenance and activation of the long-term hematopoitic
stem cells (Calvi, 2006; Kiel and Morrison, 2008; Zhang et al., 2003).
Embryonic stem cells on the other hand are cells isolated from the inner cell mass of the
developing blastocyst (Puri and Nagy, 2012; Thomson et al., 1998). These cells are
pluripotent and have the ability to give rise to cells of all three lineages including
embryonic and extra embryonic lineages (Carpenter et al., 2003; Lee et al., 2005a) (Amit
et al., 2000). Embryonic stem cells could be the potential source to derive a number of
cell types that can be used to replace lost tissue or restore function upon injury or trauma
(Amit and Itskovitz-Eldor, 2002; Prelle et al., 2002). However, there are several concerns
with the use of embryonic stem cells for regenerative therapies. Certain groups make it a
challenge to collect sufficient material as they ethically reprimand their source of origin
(Aguilar-Gallardo et al., 2010; Unger et al., 2008). The other major concern is the
formation of teratomas in vivo (Chen et al., 2014; Villa-Diaz et al., 2013). The lack of
proper characterization of these cells makes them a less desirable source for human
therapeutic purposes (Villa-Diaz et al., 2013). One of the leading strategies now is to
differentiate embryonic stem cells in vitro prior to transplantation (Moore et al., 2015;
14
Street et al., 2004). Advances in adult stem cell differentiation in vitro such as
differentiating embryonic stem cells in vitro to functional pancreatic beta islet cells that
can be transplanted (Blyszczuk and Wobus, 2004; Urban et al., 2006) is a promising .
One of the sub groups of adult stem cells are epithelial stem cells that includes mammary
stem cells, intestinal stem cells, hair follicle stem cells, corneal limbus stem cells
(Blanpain et al., 2007) and salivary gland stem cells (Zhang et al., 2014). The main
function of epithelial stem cells is to maintain and repair the epithelium. Epithelium is a
sheet of cells connected via tight junctions that envelope all the major organ systems
(Kobielak and Boddupally, 2014). It serves as a protective barrier from the external
environment and provides the structural framework support for the organ system.
Epithelium can be single layered (simple) or multilayered (stratified) (Blanpain and
Fuchs, 2006). There are specialized epithelial stem cell niches present in each of these
organs that maintain their homeostasis (Petersson and Niemann, 2012). It has been
identified that in skin, the hair follicle stem cells that reside in the bulge are the main
players that are activated during repair and regeneration (Mesa et al., 2015; Mokos and
Mosler, 2014). The usually quiescent population is reactivated to provide for the repair
and regeneration. It has also been recently shown that the maintenance of these cells is
regulated by the interplay between Wnt and BMP signaling (Kandyba et al., 2014;
Kandyba and Kobielak, 2014; Kandyba et al., 2013; Lin et al., 2015). One of the other
very established epithelial stem cell models is the intestinal system. Clevers group using
elegant lineage tracing experiments have shown that the crypts of the intestines have
specialized Lgr5 positive cells that are responsible for the maintenance of these structures
(Koo and Clevers, 2014; Ritsma et al., 2014; Yin et al., 2014). In a more recent
15
publication they have also shown that the BMI1 positive cells can replace and take the
position of the acting stem cell population under stress and injury (Zhu et al., 2013).
Unlike the other epithelial stem cells, intestinal stem cells are extremely proliferative
with a quick turn over rate (Carulli et al., 2015; Tian et al., 2015). These several different
groups of epithelial stem cell groups demonstrate that not all stem cells behave the same
way. There are several factors that control the fate of the stem cell and their potential to
divide.
One of the least characterized epithelial stem cells are the stem cells of the oral cavity
(Ren et al., 2014). The oral cavity is constantly exposed to several pathophysiological
insults that result in damage. The epithelium of the oral mucosa requires constant repair
and regeneration. The cells that are mainly responsible for this repair have not been well
characterized. There is an immediate need to understand the repair and regeneration
process of the epithelium of the oral cavity. The two main stem cell types focused in this
research are the minor salivary gland stem cells and the stem cells of the tongue.
Fig 1: Adult Epithelial Stem
Cells
Examples of putative stem
cells residing in their
specialized niches: Intestinal
stem cells are located at the
bottom of the crypt, the hair
follicle stem cells are in the
bulge region while the
mammary gland stem cells are
localized at the terminal end
bud. Adapted from Blanplain
C et al, 2007
16
1.2 Salivary gland- Structure and function
Salivary gland is an excocrine secretary organ that plays an important role in the
maintenance of oral health (Liang et al., 2015; Zhang et al., 2015). The main secretion by
the salivary gland is saliva, which is required for maintaining the normal oral health by
washing out the inner surfaces (Pringle et al., 2013). Saliva is also required to keep the
mouth hydrated that assists in proper speaking and swallowing (Hand et al., 1999;
Matsuo, 2000). The normal functions of the tongue cannot be performed without the help
of salivary secretions. Saliva also contains digestive enzymes such as amylase that helps
start the digestion process before it reaches the stomach. Saliva has been recently shown
to play an important role in enhancing taste sensitivity (Matsuo, 2000; Schmale et al.,
1990). In humans there are three predominant locations of salivary glands namely the
parotid, submandibular and sublingual. As their names suggest they are located in the
mandibular ramus, underneath the jaw and underneath the tongue respectively (illustrated
anatomy of head and neck, fehrenbach,and herring, elsiever, 2012, p 157). Although all
three locations produce saliva there are slight variations in the role they all play. The
parotid salivary gland is the largest of the three and it enters the oral cavity via the parotid
duct. It produces twenty percent of all saliva produced along with ptyalin. It also secretes
amylase that helps in starting the digestion process (Schwarz and Rotter, 2012). The
submandibular gland is much smaller than the parotid but it produces about sixty percent
of the saliva in the oral cavity. It also however produces mucous along with the saliva
that helps in easier bolus formation and swallowing there after. The sublingual however
contributes the least in terms of saliva production. It only contributes to about six percent
of the total saliva produced in the oral cavity. The major component produced by this is
17
mucous (Coppes and Stokman, 2011). Apart from these three main locations there are
several thousands of smaller salivary glands that are present in the oral cavity called the
minor salivary glands. The reside all over the mucosal lining of the oral cavity including
but not restricted the buccal lining, soft palate, hard palate and lingual mucosa. They are
extremely porous and have several hundred ducts that secrete saliva through its lifetime
to keep the inner lining hydrated. The main function of these minor glands is to maintain
oral hygiene by keeping a constant flow of saliva in the oral cavity (Zhang et al., 2014).
One other specified location of minor salivary glands is around the Ebner gland on the
tongue. It is seen located around the circumvallate papillae tastebuds on the dorsal
surface of the tongue (Aure et al., 2015). All salivary glands major or minor share a
common histology. They all have acini, the secretory compartment of the gland that is the
main location of saliva production. These cells then drain the secretion into an intricate
pattern of ducts that finally secrete it out into the oral cavity. Mice have a remarkably
similar pattern of salivary gland distribution, which makes them the perfect model for
studying the function and structure. Salivary gland is also a site for tumor formation in
Fig 2: Salivary Gland
Structure
The salivary gland is a
highly organized and
branched structure where
the secretions from the
alveoli drain into the main
duct. Depicted here the is
schematic representation
of the major and minor
salivary glands in humans
Adapted from
www.rci.rutgers.edu/uzwi
ak/AnatPhys/Digestive
18
head and neck squamous cell carcinoma. Very little is known about the stem cell
population that maintains the salivary gland (Schwarz and Rotter, 2012). It is important
to understand the regeneration mechanism of this gland as its function is often lost in
patients who undergo chemotherapy or radiation leading to dry mouth syndrome called
xerostomia. Salivary glands can also themselves be sites for tumor formation for head
and neck squamous cell carcinoma (Gaikwad et al., 2015; Huang et al., 2015; Peravali et
al., 2015). There is a pressing need to understand the regenerative mechanisms, cells of
cancer origin to prepare for better-targeted therapy.
19
1.3 Tongue
The tongue is a muscular organ present on the floor of the mouth where one side is
attached called the base via the frenulum, while the other is detached called the oral
tongue, which allows for its free movement that aids in mastication, speaking and
swallowing (Kubin et al., 2015). Incomplete detachment of the tongue to the floor of the
mouth leads to a condition called ankyloglossia or tongue-tied (Dodds and Neiger, 2014;
Hasan and Cousin, 2015). Lgr5 null mice readily develop this condition indicating the
direct impact of lgr5 and its role detachment (Acevedo et al., 2010; Morita et al., 2004).
In humans, this condition can be easily treated surgically to release the oral tongue free
(Cawse-Lucas et al., 2015; Junqueira et al., 2014; Power, 2014). Everyday normal
functions would not be possible if not for the synchronized movement of the tongue
along with the other muscles such as the jaw muscle to help speak or swallow. The
tongue is the main organ for taste recognition and also allows for the ability to form a
bolus required for proper swallowing (Calvo and Egan, 2015). The anatomy of the tongue
is well characterized; it has two distinct surfaces namely the dorsal and ventral surfaces.
The dorsal surface of the tongue is uneven due to the presence of numerous hair like
protrusions called papillae (Ananian et al., 2015). The ventral surface however is smooth.
The dorsal surface is lined by numerous taste buds that contain taste receptors (Barlow
and Klein, 2015; Liu et al., 2013). Lineage tracing studies have demonstrated that Lgr5
positive cells are capable of giving rise to all cell types required for the formation of a
functional taste bud (Thirumangalathu and Barlow, 2009; Yee et al., 2013). However,
there is very little known about the stem cell population in the tongue itself. There is no
evidence yet about any specialized adult stem cell population that plays a significant role
20
in the repair or regenerative processes. One of the characteristic features of the tongue is
its ability to heal at a rapid rate without a scar (Sabharwal et al., 2014). This amazing
ability raises several questions about the healing process and how it differs from other
epithelial systems where the formation of a scar is almost always associated with repair.
There are three major physiological components to the tongue – the epithelium, the
mucosa and the muscle. The muscles form about fifty percent of the tongue while the
epithelial lining is about five to six cells layer thick. Between the epithelium and the
muscle lies the stromal compartment, which contains the supportive fibroblasts, the nerve
innervations, blood vessels and extracellular matrix.
Every epithelial system is associated with an underlying stromal compartment. These two
compartments are in constant interaction that dictates cell fate and proliferation capacity
(Kobayashi et al., 2015; Li et al., 2015; Peng and Joyner, 2015; Yoshida et al., 2002).
Most epithelial-stromal interactions are involved in the maintenance of normal
homeostasis and also in disease progression such as cancer (Bezdenezhnykh et al., 2014;
Fig 3: Anatomy of the tongue
The dorsal and ventral
surfaces of the tongue are
anatomically quite different
where the dorsal surface has
numerous papillae and taste
buds while the ventral surface
is relatively smoother in
appearance. Schematic
representation of the various
taste buds on the tongue
surface and electron
microscope image of the
dorsal surface. Adapted from
www.studyblue.com
21
Buhrmann et al., 2014; Podduturi and Guileyardo, 2015). Understanding these
components separately and together is extremely important in disease models. Head and
neck squamous cell carcinoma is a highly aggressive metastatic cancer that often forms
tumors on the tongue (Kalfert et al., 2015; Yang et al., 2015). Even though the tumors
maybe epithelial in origin, the underlying stroma is believed to play a significant role in
disease progression and metastasis (De Wever et al., 2014; Folgueira et al., 2013; Pula et
al., 2011; Tommelein et al., 2015). Current therapy includes chemotherapy followed by
surgical removal of the tumor.
22
1.4 Cancer Stem Cells
The human body is a very dynamic system where there is constant loss and gain of
cellular layers. This maintenance often requires cell proliferation: a highly regulated set
of instructions provided by several key players (Krajewska et al., 2015; Norbury and
Nurse, 1992). The proliferation rate of cells is dependent on several factors such as the
need to repair or regenerate, the activation signals received and location of the cells
(Murray, 2004; Weinberg, 1995). The location of the cells determines the need to repair
and replace the lost tissue at a much faster rate like the skin or intestinal system while
some other organs might require very little replacement like the heart (Karantalis and
Hare, 2015; Trivedi et al., 2010; Williams and Hare, 2011) or brain tissue (Chu et al.,
2004; Lee et al., 2005b; Wang et al., 2015). The proliferation rate is regulated in a very
stringent manner by a set of checkpoints at every step of cell division (Sherr and Roberts,
2004). It is believed that when these check points are disturbed, it could lead to the
uncontrolled proliferation of cells leading to cancer (Malumbres and Barbacid, 2009;
Santamaria and Ortega, 2006). There are several factors that can lead to a checkpoint
disruption including but not limited to environmental exposures, chemical exposures,
viral infections and genetic mutations (Frolov and Dyson, 2004; Pavletich, 1999). Cancer
is thus defined as uncontrolled proliferation of cells that eventually leads to the death of
the organism. The hallmark of cancer is uncontrolled proliferation associated with tumor
formation, angiogenesis, nutrition depletion and hypoxia (Frolov and Dyson, 2004;
Hanahan and Weinberg, 2011). The accepted dogma for many decades considered that
tumors were a homogenous population where all cells had the same potential to
proliferate and metastasize. More recently, the theory has now gradually shifted to
23
accepting that the tumor environment is heterogeneous where there is a specific sub
group of cells that have the ability to escape the chemotherapeutic treatments and persist
(Kreso and Dick, 2014; Weinberg, 2014). This specific sub population has been termed
as cancer stem cells. The hallmarks of cancer stem cells are its ability to self renew and
initiate tumor formation (Klein et al., 2007; Sabet et al., 2014). One of the initial evidence
for the existence of the cancer stem cell theory was the development of leukemia
initiating cell assays where it was demonstrated for the first time the ability of a single
cell to reinitiate leukemia in a xenograft model (Bruce and Van Der Gaag, 1963;
Clarkson et al., 1967; Hamburger and Salmon, 1977; Testa, 2011). Flow sort analysis
helped identify that the CD34+CD38- fraction, termed as leukemia-initiating cells had the
potential to initiate leukemia in vivo (Dick, 2005; Guan et al., 2003; Hope et al., 2004).
Similar studies were carried out after this initial discovery in other cancer models.
CD44+CD24- fraction was identified to be the cancer stem cell fraction in breast cancer
(Al-Hajj and Clarke, 2004; Chekhun et al., 2015; Gudadze et al., 2013; Honeth et al.,
2015).
Fig 4: Cancer stem cell
model
The model illustrates the
potential of a single
cancer stem cell as a
tumor initiating cell that
can self-renew and divide
to give rise to transit
amplifying cells at the
same time. Adapted from
www.eurostemcell.com /
cancerstemcell
24
The other aspect that can contribute towards the evolution of a cancer stem cell is its
ability to adapt with the given genetic and epigenetic clues. In a single tumor there can be
parallel development of clones and subclones that can have different genetic mutations
(Gerlinger et al., 2012a; Gerlinger et al., 2012b). All the clones might carry one common
mutation along with a specific secondary mutation characteristic to itself (Fisher et al.,
2013; Yachida et al., 2012). Next generation sequencing techniques have enabled
researchers to genetically map these specific mutations to better characterize the
subclones. New lineage tracing studies have provided answers to partly understand the
evolution of cancer where 21 different breast cancer cases were used to trace the origin of
cancer. This study revealed that in about eighty five percent of the cases, there was a
single subclone that acted as the cell of origin for tumor formation (Nik-Zainal et al.,
2012; Stephens et al., 2012). Solid tumors on the other hand appear to be homogeneous,
but in reality they also follow this sense of hierarchy in their organization. Using a
chemically induced head and neck cancer model, it has been identified that a specific
subpopulation of cells within the solid tumor cab be serially transplanted in vivo to
generate new tumors. Using a inducible doxycycline controlled tet off system, minor
salivary gland cancer stem cells have been shown to display properties of tumor initiation
in immunocompromised mice (Zhang et al., 2014).
Understanding the cancer stem cell model is essential to answer the basic questions such
as origin of cancer, relapse potency, survival potency of some subclones over others and
proliferation rate. Some of the common traits among cancer stem cells are that they are
slow cycling in nature contain drug efflux transporters and express markers to evade the
immune system. These traits make them particularly challenging to eliminate using the
25
current traditional therapeutics. This new idea of cancer stem cell subpopulation has a lot
of implications on the treatment design. The focus of treatment has now moved to
eliminating these cells rather than focusing only on shrinking the tumor size.
26
1.5 Head and neck squamous cell carcinoma
Head and neck squamous cell carcinoma (HNSCC) is a highly metastatic cancer with
poor prognosis. Head and neck cancer is a collective term for the cancers that arise in the
head and neck region including but not limited to the oral cavity, the tongue, pharynx,
larynx and salivary gland (Worrall et al., 2015). Even though the locations might vary,
they all share a common feature of origin within the squamous cell lining of the mucosa
and hence are classified as a subtype of squamous cell carcinomas. HNSCC is the sixth
most commonly diagnosed cancer in the United States and is the eighth most common
cause of death. According to the National Cancer Institute (NCI) there are 500,000 cases
reported annually in the world of which more than 250,000 cases end in death. Not only
is the rate of diagnosis alarming but also the rate of death due to this cancer is unusually
high. This unusually high rate of death can be attributed to several factors such as the
lack of diagnostic tools for early detection, proximity to the lymph nodes, aggressive
metastatic cancer type, poorly characterized symptom recognition that leads to less than
10% survival rate post diagnosis (Dimery and Hong, 1993). The median age of diagnosis
is at <40 years of age where men are affected twice as much compared to their female
counterparts. The NCI head and neck cancer research investment in the year 2013 was
about 50 million dollars. The average cost of treatment in the United States per annum as
reported in 2012 is $80,000 per patient. Not only can this be a financial burden on any
individual but also the rate of relapse creates additional continued expenditure. Most
patients who undergo treatment for HNSCC are also in need of reconstructive surgery
that adds to the financial burden. This may also not be always convenient as the loss of
tissue while surgical removal may not allow for it. One of the most challenging
27
difficulties associated post treatment is the probability of relapse. A study conducted on
relapse patterns revealed that an estimated 51% of the patients presented with either loco
regional or distant or sometimes both sites upon relapse.
(http://www.ncbi.nlm.nih.gov/pubmed/4027864).
The most common causes have been identified as lifestyle choices such as smoking,
chewing tobacco, alcohol consumption, exposure to asbestos or certain other chemicals at
work and most recently human papilloma virus (HPV) infection (Dal Maso et al., 2015;
Inki et al., 1991; Pelucchi et al., 2011; Polesel et al., 2008). If the exposure occurs as a
combination then the probability of acquiring HNSCC is greater. Chewing tobacco has
been demonstrated to be a more potent cause than smoking as the inner mucosal lining is
directly exposed to the carcinogens in the tobacco (Metgud et al., 2015; Punyani and
Sathawane, 2013). The constant exposure to these carcinogens over extended periods of
time causes damage to the inner lining rendering the need for continuous repair. One of
the first symptoms associated with repair is the infiltration of the immune system
(Mantovani et al., 2010; Porta et al., 2009). Inflammation and cancer have long been
suggested to play synergistic roles in the initiation and progression of cancer (Coussens
and Werb, 2002; Koizumi et al., 2007; Rollins, 2006). Inflammation has also been
recognized recently as the seventh hallmark of cancer (Colotta et al., 2009). The normal
role of inflammation is to recruit the required cells to start the repair process by secreting
signaling cytokines and to help close the wound site (Coussens and Werb, 2002). The
recent correlation of HPV infection raises questions about prolonged inflammation
leading to dysplasia (Kuper et al., 2000; Schottenfeld and Beebe-Dimmer, 2006). On the
other hand there is also a lot of evidence pointing towards the role of the immune system
28
with anti-cancer proliferation properties (Dunn et al., 2004). Some of the important
consequences of prolonged inflammation are maintenance of high cytokine levels
(Dvorak, 1986) and the production of free radicals to combat infections (Hussain et al.,
2003). Many carcinogens such as 4 Nitro-Quinoline-Oxide, found in tobacco also has
similar downstream effects of free radical production. Persistent exposure to free radicals
can cause DNA damage leading to point mutations or frame shift deletions (Hussain et al.,
2003; Prasanna and Harish, 2010; Prasanna et al., 2009). Even though cells have an
intrinsic mechanism to eliminate cells with mutations, continuous repair can lead to one
or more of these cells “escaping” the surveillance to become tumor initiating cells
(Beachy et al., 2004).
The most common symptoms are persistent sore throat or cough, a slow or non-healing
ulcer. These ulcerative wounds eventually pave the way for tumor formation. A typical
tumor can vary from the size of a grape to a baseball (Gregoire et al., 2010; Licitra et al.,
2009) (WHO classification of tumors, pathology and genetics of head and neck cancer,
NCCN clinical practice guidelines in oncology). Depending on the location and stage,
there can be several tumors or a single ulcerative tumor (Gregoire et al., 2010). This
creates enormous challenges for a proper treatment plan due to difficulty in accessibility.
The current therapy available is chemotherapy or radiation followed by surgery (Wolff et
al., 2015). Combination therapies have shown a lot of potential in the recent past, where
randomized clinical trials have indicated that cetuximab in conjunction with cisplatin
when administered to patients demonstrated better survival rate (Adelstein et al., 2003;
Bonner et al., 2010; Gibson et al., 2005; Hitt et al., 2005). Therefore it is of extreme
importance to identify a new prognostic marker that can be used to detect the onset of
29
HNSCC before tumor formation. Epidermal Growth Factor (EGF) and its receptor EGFR
have been shown to play an important role in deregulated autocrine signaling leading to
uncontrolled proliferation in breast cancer and HNSCC (Palayekar and Herzog, 2008;
Ratushny et al., 2009; Tiseo et al., 2004). EGF ligand binding leads to the dimerization of
EGFR followed by phosphorylation activation of Tyrosine resides. This can then activate
several downstream pathways such as JAK/STAT, PI3 Kinase and ERK pathway. All
these pathways are involved in cell survival and proliferation (Ding et al., 2015; Earp et
al., 1995; Kalyankrishna and Grandis, 2006; Sharafinski et al., 2010). These statistics
reiterate the immediate need for a better prognostic marker for early detection and
effectively targeted therapy post diagnosis. The most promising treatment would be the
complete elimination of cancer stem cells so that the bulk of the non-cancer stem cell
tumor cells are naturally eliminated by the immune system.
30
1.6 Epithelial mesenchymal interactions in the tumor microenvironment
A tumor is a highly complex heterogeneous structure (Almendro and Fuster, 2011;
Campbell and Polyak, 2007; Janiszewska and Polyak, 2015; Lindeman and Visvader,
2010). Epithelial tumors are composed of the epithelial tumor mass, the mesenchymal
stromal compartment, new blood vessels, immune cells infiltration, extracellular matrix
and fibroblasts (Mroz et al., 2013; Rocco, 2015; Staff, 2015; Sun and Califano, 2014).
The defined roles of these components have recently gained importance in the field of
cancer progression and metastasis (Huang et al., 2010; Orimo et al., 2005; Orimo and
Weinberg, 2006). The hallmarks of the tumor microenvironment are low pH, hypoxia and
nutrition deprivation (Anastasiadis et al., 2003; Chan et al., 2007; Huang et al., 2007). It
has been demonstrated that the survival rate of the tumor mass depends on the underlying
stroma for nutrition, survival and proliferation signals (Chiarugi et al., 2014; Condon and
Bosland, 1999; Lee et al., 2014; Olumi et al., 1999; Peng and Joyner, 2015). The stroma
of a tumor makes up more than half the tumor mass while the epithelial component is
usually at the surface of the tumor and at the invasive front (Orimo et al., 2005; Orimo
and Weinberg, 2006; Tommelein et al., 2015). The new emerging field that is gaining
importance is to understand the role of cancer-associated fibroblasts (CAF) (Bhowmick
et al., 2004; Chang et al., 2002; Elenbaas and Weinberg, 2001; Junttila and de Sauvage,
2013; Kalluri and Zeisberg, 2006; Mueller and Fusenig, 2004). Cancer associated
fibroblasts have been shown to play a significant role in tumor progression and are now
no longer considered being passive cells lying in the stroma (Basset et al., 1990; Dvorak,
1986; Mackie et al., 1987; Olumi et al., 1999). The underlying fibroblasts in the tumor
undergo changes that leads to the formation of a highly proliferative active cell that
31
continuously provides signals that are conducive for growth and metastasis (Durning et
al., 1984; Fukino et al., 2004; Schedin and Elias, 2004). Using SNP array platform,
studies have indicated that the CAF can also have certain specific mutation patterns that
allow them to transform and survive independently in the tumor, although the pattern was
not consistent and statistically relevant (Qiu et al., 2008; Weber et al., 2006). The concept
of epithelial mesenchymal interactions is well characterized in the embryological studies
where the primitive epithelium receives instructions from the underlying mesenchymal
cells (Cunha et al., 1992; Dassule and McMahon, 1998; Sheppard, 2015; van Genderen et
al., 1994). Similar instructive cues are imitated in the tumor microenvironment by the
CAF to provide instructive signals for metastasis.
HNSCC is a particularly highly aggressive and metastatic cancer. A recent clinical study
has demonstrated that the aggressiveness in HNSCC may be due to the presence of an
activated stroma (Leef and Thomas, 2013; Sicoli et al., 2014). The study revealed that the
stroma not only assisted in cell migration, invasion and proliferation but also provided
Fig 5: The hallmarks of
cancer-associated fibroblasts
Survival signals, tumor
promoting factors, replicative
immortality are some of the
key attributes that enable
cancer associated fibroblasts
to play an active dynamic role
in tumor progression and
metastasis. Adapted from
Tommelien J et al, 2015
32
signals for tumor growth and metastasis (Junes-Gill et al., 2014). The stroma of the
tongue or the buccal mucosa is significantly different from the stroma of the skin. One of
the major differences is that wound healing in the oral cavity is accelerated and often scar
less while on the other hand, repair in the skin is almost always associated with scar
formation. This significant difference may be due to the difference in the stromal
compartment of these highly organized epithelial systems. Therefore it is extremely
important to understand these two components with respect to each other. Understanding
the epithelial mesenchymal interactions in a tumor is necessary to better understand
metastasis. One of the aims of my dissertation was to define the molecular profile and
the role of the stroma in the tongue. The tongue displays the features of rapid wound
healing, scar less repair and is also one of the major sites for tumor formation in HNSCC.
Therefore it was of utmost importance to define this specialized compartment under
homeostasis so as to define its role under disease conditions.
33
1.7 Significance
As the adult stem cells of the oral cavity are poorly defined, this dissertation attempts to
define the role of adult stem cells in the salivary gland and the tongue. Identification of
these stem cells and defining their molecular characteristic will provide insight into
understanding the homeostasis of these organs. The tongue and salivary gland have been
clinically observed to be sites for tumor formation in head and neck squamous cell
carcinoma patients hence additionally we also aim to define the role of these cells in both
tumor initiation and progression. Epithelial tumors always work in conjunction with the
underlying stroma. It would also be interesting to identify the role of the activated stroma
in the tongue or the salivary gland to promote metastasis.
34
CHAPTER 2: DEFINING THE LOCALIZATION AND MOLECULAR
CHARACTERISTIC OF MINOR SALIVARY GLAND LABEL RETAINING
CELLS
2.1 Abstract
Adult stem cells (SCs) are important to maintain homeostasis of tissues including several
mini-organs like hair follicles and sweat glands. However, the existence of stem cells in
minor salivary glands (SGs) is largely unexplored. In vivo histone2B GFP (H2BGFP)
pulse chase strategy has allowed us to identify slow cycling, label retaining cells (LRC)
of minor salivary glands that preferentially localize in the basal layer of the lower
excretory duct with a few in the acini. Engraftment of isolated SG LRC in vivo
demonstrated their potential to differentiate into keratin 5 (basal layer marker) and keratin
8 (luminal layer marker) positive structures. Transcriptional analysis revealed activation
of TGFβ1 target genes in SG LRC and BMP signaling in SG progenitors. We also
provide evidence that minor SGSCs are sensitive to tobacco-derived tumor inducing
agent and give rise to tumors resembling low-grade adenoma. Our data highlight for the
first time the existence of minor salivary gland LRCs with stem cells characteristic and
emphasize the role of TGFβ pathway in their maintenance.
35
2.2 Introduction
The ability to speak, swallow, taste food, and maintain a healthy oral cavity is heavily
reliant on the presence of saliva, an important effect of which on our everyday lives is
often unappreciated. Salivary glands (SGs) have an important role not only in
maintaining oral health but also for the general well-being. Hyposalivation is the most
common condition underlying xerostomia, the subjective feeling of dry mouth along with
50% reduction in salivary flow. It is highly common especially in patients undergoing
radiotherapy treatment for head and neck cancer, drug usage and patients suffering from
Sjogren’s syndrome. Considering the severe impact that xerostomia may have on the
patient’s quality of life, there is an unmet clinical need for an efficient treatment. Stem
cell therapy could provide an option to prevent and repair damage of tissues induced by
degenerative processes due to autoimmune responses, radiation-side effects or other
cytotoxic events of the salivary glands.
The salivary gland system consists of the major and minor salivary glands. While the
major glands secrete their fluids fully only upon stimulation, the minor mucosal glands
function more or less continuously providing ongoing protection to the oral tissues (Hand
et al., 1999) (Riva et al., 2000). There are estimated to be 600–1,000 minor salivary
glands in humans and these are located in the buccal, labial, distal palatal, and lingual
regions of the oral mucosal membrane, although they are occasionally found at other oral
sites (Hand et al., 1999).
Cells with stem/progenitor properties have been detected in major salivary glands, but till
date, no data has described their presence within the minor salivary glands (Pringle et al.,
2012) (Schwarz and Rotter, 2012) (Lombaert et al., 2011) (Nanduri et al., 2011).
36
Additionally, very little is known about the molecular regulators of the development,
stem cell activity and the regenerative processes of the minor salivary glands. The
progenitor cell population isolated from major salivary gland based on the cell surface
marker c-Kit has been used to regenerate salivary glands after irradiation. Remarkably,
post irradiation stem cell treatment with c-Kit+ adult salivary gland stem cells restored
radiation-induced dysfunction (Lombaert et al., 2008). Other groups have used cell
surface markers such as Sca-1, CD133, CD24 and CD49f to enrich the epithelial
progenitor cells in the major salivary glands but a combination of definitive cell markers
for salivary progenitor cells remains to be determined (David et al., 2008). There have
been a few studies that have used genetic lineage tracing experiments in mice to identify
progenitor cells in the developing salivary glands. In one such study, an Ascl3+
progenitor population was identified in the ductal compartment of the submandibular
gland (SMG), which gave rise to both ductal and acinar cells. Importantly, not all SMG
cells were derived from the Ascl3 cells, but only a subpopulation of acinar and ductal
cells. The Ascl3+ cells were therefore considered to be progenitor cells (Bullard et al.,
2008).
In this report, using previously developed strategy to fluorescently label slow-cycling
cells (Tumbar et al., 2004) we were able to, for the first time localize and isolate label
retaining cells (LRCs) of the minor salivary gland (SGs) in the lower part of its excretory
duct. However some myoepithelial LRCs were also detected in the acini. The LRCs in
the duct and the acini localized within the keratin 5 positive basal layer and not the
keratin 8 positive luminal layers. When these cells were sorted and injected into the
salivary glands of the NOD.Cg donor mice, they regenerated some structures containing
37
both the basal and the luminal types of cells. By determining the gene expression profiles
of the isolated minor SG LRCs and the non-LRCs in vivo, we have identified that TGFβ1
pathway is active and dominant in the minor SG LRCs. On the other hand, non-LRCs
showed high expression of BMP dependent expression of P-SMAD 1, 5, 8. We have also
provided evidence that minor SGSCs are sensitive to tobacco derived tumor inducing
agent and give rise to tumors resembling low grade adenoma (Iyer et al., 2010). Our data
highlight for the first time the existence of label retaining minor SG stem cells and their
primary importance in SG homeostasis. It also emphasizes the role of TGFβ pathway in
SGSCs maintenance.
38
2.3 Results
2.3A Label retaining cells of minor salivary glands localize predominantly in the
basal layer of the lower excretory duct and the myoepithelial layer of acini.
To localize LRCs in the minor salivary glands we used a previously developed system for
in vivo detection of infrequently dividing cells (Fig. 7A) (Tumbar et al., 2004). During 4
weeks of “chase”, slow cycling cells retain H2BGFP expression (label retaining cells),
whereas rapidly dividing transit amplifying cells dilute out the H2BGFP label upon each
division. In the animals before “chase”, H2BGFP expression was uniformly detected in
all cells of the oral epithelium including soft palate where high number of minor salivary
glands are localized (Fig. 7B). All the cells including cells in the ducts and acini were
uniformly labeled with H2BGFP (Fig. 7E). After 4 weeks of “chase” by switching off
H2BGFP expression with Doxy treatment beginning at 3 to 4 weeks of age, we
demonstrated the presence of infrequently dividing LRCs in minor SGs (Fig. 7C, F and
O). The pattern of LRC at 4 weeks of chase was similar to pattern seen after 7 weeks of
chase (Figure 6A and B) indicating that there is a slow turnover of minor salivary gland
epithelial cells. The H2BGFP expression of LRCs was enriched in the lower excretory
duct (Fig. 7F-I), however some LRCs were also observed in the acini (Fig. 7 F-I, arrows).
In contrast no GFP positive cells were observed in the soft palate epithelium (Fig 7F-I).
All of the LRCs were slow cycling excluding immunofluorescence staining for
proliferation marker Ki67 (Fig. 7G and Figure 6A). Histological localization of LRCs in
SGs was confirmed by visualizing serial sections of the soft palate region by hematoxylin
and eosin staining (H&E) (Fig. 7D). Immunofluorescence staining with a number of
39
different markers was performed to further define the precise location of SG LRCs. We
have demonstrated that the minor SG LRCs are all positive for β4 integrin indicating that
they are attached to the basement membrane (Fig. 7L and L’). In addition, these SG
LRCs also co-expressed the basal layer marker keratin 5 (K5) (Fig. 7J-J’), whereas the
luminal layer marker, keratin 8 (K8) did not overlap with SG LRCs (Fig. 7K and K’). All
the epithelial cells, including LRCs were also positive for CD44 (Fig. 7M and M’).
However only LRCs within the acini expressed smooth muscle actin (SMA) indicating
their myoepithelial characteristic with basal layer localization (Fig. 7N and N’).
Fig 6: Optimization for LRC identification in the salivary gland
View of the salivary gland transverse section after 4 week chase and 7 week chase
pattern. Slow cycling H2BGFP label retaining cells are located in the lower ductal
region indicated by arrows (A). The cells are slow cycling and are also seen in the
ductal region indicated by arrows (B). Rare label retaining cells also observed in the
acini as indicated by arrows (A and B). Abbreviations: ep- epithelium
40
Fig 7: Preferential localization of LRC in basal layer of minor SG
Chart illustrating the Doxy treatment of pTreH2BGFP/K5tTA mice at p21 for 4 weeks
chase (A). View of soft palate of pTreH2BGFP/K5tTA mice before (B) and after (C) 4
weeks Doxy chase, with the slow cycling cells retaining GFP label. Shown are side
view sections of soft palate of mice before (E) and after 4-week chase (F-N’). Shown
are H&E staining (D) and epifluorescence of H2B-GFP (green) and 4’,6’-diamidino-
2-phenylindole (DAPI) (blue), and indirect immunofluorescence with antibodies (Abs)
indicated (Red) (E-N’). Schematic representation of minor salivary gland with LRCs.
Majority of LRCs is localized within the basal layer (K5) of excretory duct and few
within myoepithelial layer of acini (O). Arrows denote GFP positive LRC, asterix
indicate acinar part of the gland. Arrowheads in (G) denote Ki67 positive epithelial
cells in the gland and soft palate epithelium. Scale bar: D-F 100µm; G-I 50µm; J-N’
20µm. Abbreviations: HP, hard palate; SP, soft palate; M, molars; ep, epithelium;
Doxy, doxycycline
41
2.3B Isolation and regenerative potential of label retaining cells of minor salivary
gland
To isolate the minor SG LRCs, we used a combination of surgical dissection with
subsequent enzymatic digestions followed by flourescence-activated cell sorting (FACS).
Since minor SG LRCs were attached to the basement membrane (Fig. 7H, L, L’) and all
the cells in the minor salivary gland were expressing epithelial marker CD44 (Fig. 7 I, M,
M’), we stained these cells with a FACS specific antibody against α6 integrin and CD44
to achieve a high purity of sorted live LRCs and adjacent non-LRC basal cells. FACS
analysis revealed that minor SG LRCs accounted for approximately 3% of the epithelial
cells enriched by CD44 staining fraction. H2BGFP+ LRCs were highly enriched in the
colonies, some of which contained ~400 cells (Fig. 8B, C and D), consistent with the
high proliferative capacity documented for hair follicle LRCs (Tumbar et al., 2004). On
the other hand non-LRCs (H2BGFP-) had limited potential in colony formation (Fig. 8B
and C). To test the potential of H2BGFP+ minor SG LRCs in the reconstitution of the
salivary gland, we performed intra-glandular transplantation of the sorted cells as well as
dissected pieces of 4 weeks chased minor SG tissue into the salivary glands of NOD.Cg
mice (Fig. 8E-J and Fig. 9A). Eight weeks after the transplantation dominant structures
that formed from dissected pieces of minor SG tissue were resembling ducts (Fig. 8 E-
G’). A pure population of sorted H2BGFP+ cells that were initially positive only for K5
and negative for K8, after transplantation gave rise to both K5 and K8 positive structures
indicating its differentiation potential (Fig. 8H- J). In contrast, we did not observe any
structures formed by the non-LRCs (H2BGFP-) in the transplanted salivary glands in
three independent experiments. At week eight after initial transplantation the cells within
42
the transplant were still expanding as indicated by positive staining for proliferation
marker Ki67 (Fig. 9B and C). Together the in vitro cell culture and in vivo transplantation
experiments indicate that the minor SG LRCs possess stem cell characteristics such as
differentiation potential and can self-renew.
Fig 8: LRC are clonogenic and have regenerative potential.
Minor salivary glands obtained from pTreH2BGFP/K5tTA mice, 4 weeks after chase were
digested and used to sort for LRCs. Epithelial marker CD44, basement membrane marker
integrin α6 were used to isolate slow cycling GFP+ CD44+ α6+ LRC population and
CD44+ α6+ progenitor population (A). The FACS sorted cells (LRC and progenitors)
were placed in culture for clonogenic potential assessment. Colonies were counted at day
10 post sort in two independent experiments (B). The epithelial morphology of the
colonies that formed from LRC and progenitors are shown (C, D). Epifluorescence of
H2BGFP (green) and indirect immunofluorescence with antibodies as indicated of
sections of ductal structures that formed after transplantation of dissected minor salivary
glands from soft palate (E-G’) and sorted GFP+ α6+ LRC (H-J) are shown. Scale bar: E-
G 100µm; E’-J 20µm. Arrows denote GFP positive cells that formed ductal like structures
after transplantation.
43
44
Fig 9: Tissue pieces and pure population engraftment in donor salivary
glands were actively proliferative
Brightfield image of intra-glandular transplantation of the sorted cells as well
as dissected pieces of 4 weeks chased minor SG tissue into the salivary glands
of NOD.Cg mice (A). Eight weeks post transplantation; the implants were
proliferative as indicated by co localization of positive Ki67
immunohistochemistry staining in red and nuclear H2BGFP in green (B, C)
marked by arrows. Abbreviations: SG- salivary gland
45
2.3C Defining the molecular characteristic of minor salivary gland label retaining
cells
Using microarray analyses, we obtained the transcriptional profiles for the two
populations: GFP+/α6+/CD44+ (minor SG LRCs) and GFP-/α6+/CD44+ adjacent basal
layer cells (minor SG non-LRCs), and high-stringency analyses uncovered the
distinguishing features of the minor SG LRCs. There were 4834 mRNAs up regulated
and 1889 mRNAs down-regulated by at least 2.5 – fold. Functional annotations revealed
up-regulation of a number of mRNAs in LRCs that are involved in cell adhesion,
signaling and transcription (Fig. 10A). Some of the genes were validated through real-
time PCR using separately isolated biological samples and immunofluorescence staining
(Fig. 10B and 3C-E). Preferential ductal localization of Ncadherin (Cdh2), tenascin (Tnc)
and tetraspanin 6 (Tspan6), which are up regulated in the array data for LRCs indicates
that the sorted fraction was enriched in ductal minor SG LRCs (Fig.10 C-E). Up-
regulated LRC mRNAs encoded members of the transforming growth factor beta (TGFβ)
signaling pathway known to inhibit epithelial cells proliferation, like Tgfb2, Tgfb3 and
Ltbp1 necessary for latent TGFβ activation. Although LRCs expressed some of the
cyclins involved in cell cycle progression, they also expressed high levels of cell cycle
inhibitors (Cdkn1c and Cdk2ap1). Inhibitors of Wnt pathway including Sfrp2, Dab2,
Dact1, Tcf3, and Wif1, and the Wnt receptors Fzd3, Fzd7, and Fzd2 were also up
regulated (Fig. 10A). Consistent with the array data the Wnt signaling appeared to be
repressed as we didn’t observe activation of β-catenin-dependent signaling, using
transgenic reporter mice (Fig. 11A and B) (Ferrer-Vaquer et al., 2010). We also observed
that genes of Sonic hedgehog (Shh) pathway (Shh, Ptch1, Gli3 (Fig. 10A) were up
46
regulated in the LRC, which is associated with maintenance of various adult epithelial
stem cells (Ahn and Joyner, 2005), (Brownell et al., 2011; Wang et al., 2000). To
determine which cells respond to Shh signaling we used Gli1-CreERT2; ROSA26LoxP-
STOP-LoxP-ZsGreen1 mice (Gli1-CreER; Zsgreen), injected with tamoxifen at 4–6
weeks of age. 1 month after Tamoxifen induction we detected ZsGreen+ cells near the
minor salivary gland ducts (Fig. 11C). Analysis of the sections of the upper palate
revealed sporadic presence of Shh responsive cells in the most upper duct of the minor
SG, however majority of the cells responding to Shh are present in the stroma
surrounding the minor SG (Fig. 11D). Interestingly this observation is consistent with the
model proposed by A.L. Joyner group (Peng et al., 2013) where Shh from basal epithelial
cells of adult prostate signals to the surrounding stroma. Many of the up-regulated
mRNAs in the SG LRCs encode for secretory or integral membrane proteins (Fig. 10A),
which suggests the ability of these LRCs to organize their niche and respond to their
special environment. Many of the extracellular matrix and cell adhesion proteins involved
in niche formation/interaction like tenascin (Tnc), biglycan (Bgn), collagens (Col) and
laminins (Lam) were also up regulated. Interestingly when minor SG LRCs were
compared with the signature gene list of LRC of quiescent hair follicle (HF) stem cells
(Tumbar et al., 2004) (Greco et al., 2009), minor SG LRC were found to express almost
30% of mRNAs present in hair follicle LRCs (Fig. 10F).
47
Fig 10: Transcriptional profiling of minor SG LRCs.
Functional annotation of genes up-regulated (>2.5x) in minor SG LRCs (A). Real-time
PCR confirmation of selected signature genes of the minor SG LRCs obtained in array
analysis (B). Side view sections of soft palate of mice after 4-week chase with
H2BGFP (green) and 4’,6’-diamidino-2-phenylindole (DAPI) (blue), and indirect
immunofluorescence with antibodies (Abs) for Cdh2, TnC, Tspan6 as indicated (Red)
(C-D). Comparisons of minor SG LRCs and hair follicle LRCs profiles. Venn diagram
showing similarities between the minor SG LRCs and hair follicle LRCs molecular
signature (F). Table in (F) highlights several of the key similarly expressed genes in
minor SG LRCs and hair follicle LRCs. Scale bar: C-E 50µm.
48
Fig 11: Wnt pathway is not active in minor SG LRCs, whereas Sonic hedgehog
signals predominantly to stromal cells
View of soft palate of Tcf/Lef:H2B-GFP reporter mice (A) and
Gli1CreER/RosaZsGreen mice 1 month after Tamoxifen induction (C). Side view
sections of soft palate of Tcf/Lef:H2B-GFP reporter mice (B) and
Gli1CreER/RosaZsGreen mice 1 month after Tamoxifen induction (D). Shown are
epifluorescence of H2B-GFP (green) or ZsGreen and 4’,6’-diamidino-2-
phenylindole (DAPI) (blue), and indirect immunofluorescence with antibody as
indicated (Red). Arrows in A and B denote Tcf/Lef1:H2BGFP positive cells
localized in the upper duct of minor salivary gland and epithelium of soft palate.
Arrows in C and D denote Shh responsive cells in the stroma surrounding the
minor SG, whereas arrow head indicate Shh responsive cells localized in the
upper duct of minor SG. Scale bar: B and D 50µm. Abbreviations: ep, epithelium;
SP, soft patale; HP, hard palate.
49
2.3D Activation of TGFβ signaling pathway in minor salivary glands LRCs and
BMP pathway in early progenitors
Consistent with their slow-cycling properties, minor SG LRCs expressed elevated
transcripts encoding cell cycle inhibitors: Cdkn1c (p57) and Cdk2Ap1 (p12) that are
known targets of TGFβ signaling pathway (Scandura et al., 2004) (Hu et al., 2004).
TGFβ-was previously shown to induce cell cycle arrest in human hematopoietic stem
cells through the up-regulation of p57/KIP2 (Scandura et al., 2004). It is well established
that at high levels, TGFβ often inhibits cell proliferation in a reversible manner
(Massague, 2008). Increasing evidence suggests that the TGFβ constitutes as an integral
component in the intercellular crosstalk between stem cells and their microenvironment
along with maintaining the balance between active proliferation and reversible cell cycle
exit in reservoirs of stem cells (Oshimori and Fuchs, 2012). Therefore we further tested if
TGFβ pathway is activated preferentially in the minor SG LRCs. Indeed we observed that
the prevalent TGFβ receptor activation by nuclear phospho-Smad2 immunoreactivity in
the H2BGFP LRCs when compared to the adjacent progeny (Fig. 12B-F). We used
Upstream Regulator Analysis in Ingenuity Pathway Analysis (IPA) software to identify
the cascade of upstream transcriptional regulators that can explain the observed gene
expression changes in our dataset, which can help illuminate the biological activities
occurring in the minor SG LRCs. Consistent with our initial observation TGFβ was the
top growth factor - upstream regulator (Fig. 12A). Minor SG LRC had 587 activated
TGFβ target genes within all up/down (+/- 2.5) genes including cell cycle inhibitors
Cdkn1c (p57) and Cdk2Ap1 (p12), known stem cell genes ABCA1, ALDH18A1, Hmga2
and also components of TGFβ pathway, Smad2, Smad3, Smurf2, TGFβ2, TGFβ3, TGFBI,
50
TGFBR1 and TGFBR2 and TGFBR3. Targets of TGFβ involved in niche regulation
included tenascin, biglycan and collagens (Fig. 12A). Since BMP signaling pathway was
previously shown to be required to maintain a quiescent state in hair follicle stem cells
(Kobielak et al., 2007) we also checked the status of BMP pathway activation in minor
SG LRCs. Surprisingly indicative of BMP receptor activation, nuclear phospho-Smad1,
5,8 immunoreactivity was prevalent in the non-LRC adjacent progenitor cells but not in
LRCs (Fig. 12G-K). Consistently the array data showed down-regulation of known BMP
target genes Id2 and Id4 in the LRCs when compared to the non-LRCs.
51
Fig 12: Activation of TGFβ signaling pathway in minor salivary glands LRCs and
BMP pathway in early progenitors
Upstream regulator analysis in IPA. Venn diagram showing known TGFβ targets (IPA
database) that are differentially expressed in minor SG LRCs, examples of those genes
are shown in table (A). Active TGFβ signaling is found in minor SG LRCs indicated by
positive phospho-Smad2 expression (B-F). Active BMP signaling is found in minor SG
non-LRCs indicated by positive phospho-Smad1/5/8 expression (G-K). The number of
phospho-Smad2 and phospho-Smad1/5/8 cells and overlap with GFP positive cells is
shown in F and K, respectively. Scale bar: B and G 50µm; C-E’ and H-J’ 20µm.
Arrows denote GFP positive LRC, asterisk indicate non-LRCs. Abbreviations: ep,
epithelium; fov, field of view.
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2.3E Minor salivary glands are sensitive to a carcinogenic tobacco compound and
give rise to a low-grade adenoma
The analysis of tumor cell of origin requires a better understanding in all tissue cell types
and their position in the lineage hierarchy. In particular, stem cells are often considered to
be excellent candidate cells of origin for cancer, given their inherent ability to self-renew.
To test the tumorigenic potential of K5 driven H2BGFP positive cells of the minor
salivary glands we employed a previously published mouse model of 4NQO-induced oral
tumors (Czerninski et al., 2009) on transgenic K5-tTA/pTRE-H2B-GFP mouse line. Mice
were administered for 16 weeks with 4-nitroquinoline-oxide (4NQO, synthetic water-
soluble organic compound that
forms DNA adducts, serving as a surrogate of tobacco
exposure) to induce oral cancer (Fig. 13A) (Czerninski et al., 2009). At 24 weeks
majority of the mice developed squamous cell carcinoma of the tongue as expected
(Czerninski et al., 2009). In addition, small tumors on the soft palate were also observed
(Fig. 13A’). Since the tumors were very small in size excluding possibility of chase and
FACS sorting we’ve established H2BGFP positive cell lines from dissected primary
tumors to further test their tumorigenic potential. The minor SG derived tumor cells were
highly clonogenic and could be maintained in culture for long period of time (Fig. 13 D,
E). When grown in 3D matrices the cells were prone to form tubular-like structures
resembling ducts (Fig. 13 F, G). To test the tumorigenic potential of those cells we
injected the H2BGFP positive cells into the salivary gland of NOD.Cg donor mice. After
5 weeks the cells formed well-vascularized, visible tumors (Fig. 13 H, I), which were still
GFP positive (Fig. 13 I). The morphology of the tumors that formed resembled low-grade
adenoma. Histology revealed that the tumor had different growth patterns of ductal or
53
tubular like structures (arrows in Fig. 6J and K), solid (arrows in Fig. 13 K’) and
microcystic with central lumina formed by one layer of cubic cells (asterisk in Fig. 13 J
and K’). The tumors that formed after the injection of established H2BGFP positive cell
line expressed basal layer marker beta4-integrin (Fig. 13 L and M), some of the cells also
stained positive for luminal marker keratin 8 (Fig. 13 N and O).
54
Fig 13: Tumorigenic potential of minor SG basal layer cells.
Schematic of pTreH2BGFP/K5tTA mice with 4NQO treatment (A). View of mouse soft
palate with developed tumors in the minor SG (A’). Tumor cells were placed in culture
and formed epithelial colonies (B-E), as indicated by E-cadherin presence (C). Minor
SG tumor cells formed tubular-like structures when placed in 3D matrigel (arrows in
F-G). Injected minor SG tumor cells into the gland of NOD.Cg mice formed secondary
tumors that are highly vascularized (arrow in H) and could be traced due to the GFP
label (I). Shown is H&E for tumor morphology (J, K and K’) and epifluorescence of
H2BGFP (green) and 4’,6’-diamidino-2-phenylindole (DAPI) (blue), and indirect
immunofluorescence with antibodies as indicated (Red) (L-O). Scale bar: L and N
100µm; M and O 20µm. Arrows in J, K, L, and N indicate tubular-like structures,
arrows in K’ show solid pattern and asterisk in J, K’, L and N indicate microcystic
structures. Arrows in (M) indicate basal layer localization of GFP positive cells;
arrows in (O) indicate K8 expression in GFP positive cells. Abbreviations: HP, hard
palate; SP, soft palate; SG, salivary gland; T, tumor.
55
2.4 Discussion
Hundreds of minor salivary glands are present in the lining of the mucosa in the
aerodigestive tract that provide protection to the oral tissues day and night (Hand et al.,
1999) (Riva et al., 2000). The cells with stem/progenitor properties have been detected in
major salivary glands, however their precise characterization and function has not been
addressed so far. In addition limited data is available about the existence of stem cells
within the minor salivary glands (Pringle et al., 2012) (Schwarz and Rotter, 2012)
(Lombaert et al., 2011) (Nanduri et al., 2011). Our studies provide for the first time an
insight into the stem cells biology of the minor salivary glands.
Preferential localization of LRCs in the excretory duct of the minor salivary gland
and regenerative potential
The minor salivary glands are simpler in structure than the major SGs. They consist of
small clusters of secretory cells with intercalated duct and the striated duct either less
developed or not present and shorter excretory duct that delivers the saliva product onto
the surface of the mucosa (Hand et al., 1999). Accumulating data indicates that like other
glandular tissues such as mammary gland and prostate, the cells with stem/progenitor
properties are also present in the major salivary glands, however till date, the existence of
stem cells in the minor salivary glands of soft palate has not been addressed. Here, we
demonstrate that cells with slow cycling characteristic are present in minor salivary gland
as a population localized in the basal layer of the excretory duct. The LRC were always
present in the basal layer adjacent to K8 positive luminal layer suggesting that the LRC
of minor salivary glands are only present in the excretory duct but not intercalated duct.
Some of the LRC were also present in the acinar part of the gland bottom and we
56
demonstrate their myoepithelial characteristic by SMA co-expression (Fig. 1). Previous
studies on major salivary glands imply that cells capable of proliferation and
differentiation reside only within the ducts of SGs and may represent potent stem cells
(Pringle et al., 2012). Studies have also demonstrated that acinar cells themselves display
a limited degree of proliferative ability, and the total ablation of acinar cell function in
ligation experiments suggests that acinar cell proliferation is unlikely to account for the
rescue of salivary gland function (Pringle et al., 2012). In our transplantation experiments
we used a sorted fraction of GFP+/CD44+/α6+ cells but we were not able to distinguish
between ductal versus acinar LRC. Further studies are needed to address the importance
of ductal and acinar LRC in the minor salivary glands homeostasis.
TGFβ pathway governs the gene network regulation to maintain quiescent
homeostasis in minor salivary glands
Recent discoveries suggest that the quiescent state is not just a passive state but, instead,
actively regulated by different intrinsic mechanisms. Quiescent stem cells can sense
environmental changes and respond by re-entering the cell cycle for proliferation
(Cheung and Rando, 2013). To respond to those changes rapidly, a quiescent stem cell
would maintain the expression of all necessary components that are required for
activation and proliferation (Cheung and Rando, 2013). Our results indicate that slow
cycling cells of minor salivary glands maintain the expression of some of the cyclins
involved in cell cycle progression and at the same time consistent with their slow-cycling
properties express high levels of cell cycle inhibitors Cdkn1c (p57) and Cdk2ap1 (p12).
Many cyclin-dependent kinase inhibitors, including p21, p27 and p57, are expressed in
quiescent stem cells and promote cell cycle arrest by inhibiting cyclin-dependent kinases.
57
In double-knockout mice lacking both p57 and p27, HSC quiescence is severely impaired
(Zou et al., 2011). These studies suggest that cyclin-dependent kinase inhibitors are
functionally important for the maintenance of stem cell quiescence. Some of the cyclin-
dependent kinase inhibitors were shown to be directly regulated by TGFβ that at high
levels TGFβ often inhibits cell proliferation in a reversible manner (Scandura et al., 2004)
(Massague, 2008). Our analyses suggest that TGFβ might play a crucial role in
maintaining the minor salivary stem cells in quiescent state by regulating genes important
for cell cycle of stem cells but also by maintaining the proper microenvironment to
balance active proliferation and reversible cell cycle exit in reservoirs of stem cells
(Oshimori and Fuchs, 2012). In hair follicle stem cells another member of TGF signaling
family, namely BMP was shown to be crucial to maintain the hair follicle stem cells in
quiescent state (Kobielak et al., 2007). In addition intra-stem cell antagonistic
competition, between BMP and Wnt signaling turned out to be crucial for maintaining
balance in stem cell activity (Kandyba et al., 2013). In the hair follicle SC niche, Wnt
signaling and beta-catenin stabilization transiently activates Lef1/Tcf complexes and
promotes their binding to target genes that promote transiently amplifying cell conversion
and proliferation (Lowry et al., 2005). Consistent with those observations our data
indicate that minor salivary gland LRC show activation of TGFβ pathway whereas Wnt
signaling appears to be repressed, as we didn’t observe activation of β-catenin-dependent
signaling, using transgenic reporter mice. Moreover minor SG LRCs have high
expression levels of Wnt pathway inhibitors including Sfrp2, Dab2, Dact1, Tcf3, and
Wif1. We also observed genes of Sonic hedgehog (Shh) pathway to be upregulated in
LRC, which is associated with the maintenance of various adult epithelial stem cells (Ahn
58
and Joyner, 2005), (Brownell et al., 2011; Wang et al., 2000). Interestingly using Gli1
reporter mice we have learned that cells responding to Shh signaling are present in the
stroma surrounding the minor SG. This observation is consistent with the model proposed
by A.L. Joyner group (Peng et al., 2013) where Shh from basal epithelial cells of adult
prostate signals to the surrounding stroma.
Tumorigenic potential of minor salivary gland basal cells
Minor salivary gland neoplasm represents a heterogeneous group with diverse
morphologies, tumor biology and consequently, varied clinical behavior. Tumors that
develop from these glands have the same histopathologic heterogeneity seen in those that
arise from the parotid or submandibular glands. However, in contrast to major salivary
gland tumors, which are predominantly benign, most minor salivary gland neoplasm tend
to be malignant (Guzzo et al., 2010). The etiology of these tumors remains unknown.
There is limited association with conventional risk factors associated with squamous cell
carcinoma, such as smoking and alcohol intake (Guzzo et al., 2010). Interestingly our
results showed that treatment of mice with 4NQO which is mimicking tobacco exposure
resulted in formation of tumors that resembled low-grade adenoma with mixed pattern
indicating that K5 derived cells from minor salivary glands give rise to both cribriform
areas–and double-layered ductal like structures with K5 and K8 positive cells.
Unfortunately with our current system where K5 marks both basal cell population within
the duct and myoepithelial population within the acini we can’t distinguish if one of those
populations can give rise preferentially to different structures within the tumor or they
participate equally.
59
So far the knowledge on minor salivary gland development and presence of stem cells in
those glands was relatively limited compared with what was known about major gland. In
this current report for the first time we localize and characterize label-retaining cells with
stem cell properties in the minor salivary glands. Regeneration or repair of salivary
glands requires understanding of the spatial and temporal interactions of the various cell
types within the gland as it develops and during homeostasis. The signaling systems in
the minor SG stem cells and their niche will need to be incorporated into current models
of salivary bioengineering as well as regenerative therapies for a successful outcome. Our
data serve to emphasize the importance of some of those signaling systems in the
development and maintenance of minor SG stem cells.
60
CHAPTER 3: LGR5 MARKS NEURAL CREST DERIVED MULTIPOTENT
ORAL STROMAL STEM CELLS
3.1 Abstract
It has been suggested that multipotent stem cells with neural crest (NC) origin persist into
adulthood in oral mucosa. However their exact localization and role in normal
homeostasis is unknown. In current study we discovered that Lgr5 is expressed in NC
cells during embryonic development, which gives rise to the dormant stem cells in the
adult tongue and oral mucosa. Those Lgr5 positive oral stromal stem cells (Lgr5-OSSC)
display properties of NC stem cells including clonal growth and multipotent
differentiation. RNA sequencing revealed that Lgr5-OSSC express NC related genes.
Using lineage-tracing experiments we show that those cells persist over a year in the
tongue and mucosa and give rise to stromal progeny. In vivo transplantation demonstrated
that these cells reconstitute the stroma. Our studies show for the first time the persistence
of Lgr5 positive embryonic NC cells into adulthood that participate in the maintenance of
stroma in the oral mucosa.
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3.2 Introduction
The vertebrate neural crest (NC) is a multipotent cell population derived from the lateral
ridges of the neural plate in the early embryo (Barembaum and Bronner-Fraser, 2005;
Bronner-Fraser, 1993). During embryonic development NC cells disperse from the dorsal
neural tube and migrate extensively, giving rise to a wide variety of differentiated cell
types that include neurons, glia, fibroblasts, bone, cartilage, connective tissue and dermis
of the head. Important contributions of NC cells have been demonstrated in formation of
mammalian craniofacial structures including branchial arches (Chai and Maxson, 2006).
In addition, recent reports have showed that in adult tissues, neural crest derived tissue
specific stem⁄progenitor cells are widely distributed, as they were observed in the skin,
cardiac muscle, and corneal stroma (Toma et al., 2001; Tomita et al., 2005; Wong et al.,
2006; Yoshida et al., 2006; Yu et al., 2006). These neural crest derived cells have the
capability to differentiate into multiple cell lineages originating from the neural crest.
Therefore, these cells are thought to be neural crest stem cells (Dupin et al., 2007;
Morrison et al., 1999). Oral mucosa that lines the oral cavity contains stromal cells that in
high degree originate from the embryonic neural crest (Chai and Maxson, 2006;
Kaltschmidt et al., 2012) (Crane and Trainor, 2006; Dupin et al., 2007; Shakhova and
Sommer, 2008; Teng and Labosky, 2006). Numerous recent studies suggest that stem
cells with a neural crest origin persist into adulthood, especially within the mammalian
craniofacial compartment (Davies et al., 2010; Ganz et al., 2014; Kaltschmidt et al.,
2012; Mao and Prockop, 2012; Marynka-Kalmani et al., 2010; Zhang et al., 2012).
However the number and localization of highly multipotent adult neural crest stem cells
(NCSCs) that persist long-term in oral mucosa is largely unknown. It is known that such
62
cells have a high capacity for both self-renewal and the generation of multiple kinds of
progeny under the appropriate conditions in vitro and in vivo (Kaltschmidt et al., 2012),
therefore they represent an extremely important adult stem cell population with a broad
differentiation potential, especially in regard to the generation of neuronal and glial cells,
α-smooth muscle actin positive mesodermal cells, osteogenic cells, adipocytes,
chondrocytes, melanocytes, keratinocyte-like cells and multinucleated MyoD-positive
myotubes. One of the key questions is if the multipotent stem cells with neural crest (NC)
origin persist in the stroma of oral mucosa, what is their precise localization and what
function they play in the normal homeostasis.
Fig 14: Lgr5 is a transmembrane receptor
Leucine rich repeat containing G protein coupled receptor is a transmembrane receptor
that along with R Spondin binds to the Wnt ligand to activate several downstream Wnt
targets (A). Evolutionary relationship between the several Lgr5 proteins across species
(B). Adapted from Barker et al, 2013
63
The Wnt target gene Lgr5, has been shown to mark adult stem cells in variety of adult
organs including small intestine, colon, stomach, hair follicle, kidney, ovary and tubal
epithelia, taste buds and short-term hematopoietic stem and progenitor cells (Barker et al.,
2010; Barker et al., 2012; Barker et al., 2007; Jaks et al., 2008; Liu et al., 2014; Ng et al.,
2014; Yee et al., 2013). Lgr5 was also shown to be expressed and have functional
relevance in the craniofacial development (Morita et al., 2004). Here, we employ Lgr5
reporter mice (Barker et al., 2007) to show the presence of Lgr5 positive stromal stem
cells that are derived from embryonic NC and display properties of NCSC in the tongue
and oral mucosa and evaluate their endogenous stem/progenitor cell identity using in vivo
lineage tracing.
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3.3 Results
3.3A Lgr5 marks novel population of stromal cells in the tongue and oral mucosa.
To detect Lgr5 positive cell population in oral cavity we used previously developed
system Lgr5-EGFP-ires-CreERT2 reporter mice (Barker et al., 2007). In those mice GFP
serves as a surrogate marker for Lgr5 expression. We observed expression of Lgr5 in cell
population at the lingual frenulum of the ventral tongue of adult mice (Fig. 17 a, b, d, e)
and small clusters in the oral mucosa (Fig. 16 a, c, d’, e’). Surprisingly the Lgr5+ cells
were restricted to the connective tissue of the tongue (Fig. 17 h-h2 and 1 j-k) and oral
mucosa (Fig. 16 h) subjacent to the lingual epithelium. The GFP pattern of Lgr5 observed
in other adult tissues (hair follicle and intestine) faithfully recapitulated the epithelial
pattern seen previously with this mouse model (Fig. 15 a-d).
An in vivo lineage-tracing approach was adopted to directly assess the stem cell potential
of Lgr5+ cells. We crossed the Lgr5-EGFP-ires-CreERT2 reporter mice with the Rosa26-
Tomato reporter mice. Treatment with tamoxifen (Tam) at postnatal day 40 and 41 (P40,
P41) activated the CreERT2 fusion enzyme in Lgr5-expressing cells (Fig. 17 g-i). Cre-
mediated excision of the stop codon sequence in the Rosa26-Tomato reporter irreversibly
marked Lgr5+ cells. Moreover, although potential progeny of these cells will no longer
express GFP, the activated Tomato reporter should act as a genetic marker, facilitating
lineage tracing. The mice were analyzed at different time points with the longest lasting
till one year from the initial Tam treatment. One year after tamoxifen induction, we
detected Tomato positive cells expanding in both directions from the frenulum in the
ventral tongue (Fig. 17 c and f). Detailed analysis revealed double positive (Lgr5GFP+
65
and Tom+ cells) within the stroma of frenulum (Fig. 17 i and i-1) indicating that the
Lgr5+ cells here are capable of self-renewing. In addition the Lgr5+ cells gave rise to
majority of stromal cells progeny that are Tom+ only (Fig. 17 i and i-2). It was also a
case for mucosa where Lgr5+ cells gave rise to stromal Tom+ only cells (Fig. 16 and i).
Progeny of Lgr5+ cells were also observed in other adult tissues like hair follicle (Fig.15
f and h), intestine (Fig.15 e and g) and taste buds on dorsal site of the tongue (Fig. Sup. 3),
faithfully recapitulating epithelial pattern seen previously with this mouse model (Barker
et al., 2007; Jaks et al., 2008; Yee et al., 2013). The Lgr5GFP+/Tom+ cells and their
Tom+ progeny could be detected even one year after the initial Tam induction.
66
Fig 15: Lgr5 marks both hair follicle stem cells and intestinal stem cells
Lgr5-EGFP-ires-CreERT2 x Rosa26-Tomato reporter mice without and after tamoxifen
treatment. Lgr5 expression patterns observed in the resting hair follicles and intestinal crypts
labeled by the GFP. Lgr5 marks the epithelial stem cells in the bulge region of the hair
follicle (b, d) and in the intestinal crypts (a, c). Upon tamoxifen treatment, observed are the
similar patterns as published previously, confirming that this system is specific to the Lgr5
expressing cells and its subsequent progeny.
67
Fig 16: Lgr5-OSCCs localize at specific regions in the buccal mucosa
Endogenous Lgr5-OSCCs localize at specific regions in the buccal mucosa (a, d’, e’) and give
rise to double positive Lgr5GFP+/Tom+ post tamoxifen treatment as indicated by arrows (b,
f’, g’). Immunohistochemistry proves the localization of the Lgr5-OSCC is stromal, beneath
the E-cadherin positive epithelial layer, before tamoxifen treatment (h) and after treatment
(i). Bright field image of the hard palate (c) Scale bar 100µm Abbreviations: HP: Hard
Palate, SP: Soft Palate, M: Molar teeth, ep: epithelium
68
69
Fig 17: Lgr5 positive oral stromal stem cells observed at the frenulum in the stromal
layer
Endogenous Lgr5 expressing cells are localized at the frenulum of the tongue (b, e).
Tamoxifen treatment in Lgr5-EGFP-ires-CreERT2 x Rosa26-Tomato reporter mice leads
to the activation of CRE recombinase, giving rise to the Rosa26-Tomato positive
population expressed in the ventral region of the tongue (c, f). Bright field images of the
whole tongue shown as side (a) and ventral views (d). Schematic representation of
tamoxifen treatment where two pulses were administered at P40 and chased for one year
(g). Schematic representation of the whole tongue shown before (h) and after tamoxifen
treatment (i). 1 and 2 in (h, i) represent the location of section used for
immunohistochemistry. Before tamoxifen treatment since there is no recombination,
observed are the endogenous Lgr5-OSCC (Lgr5GFP+) in the tongue stroma (h-1), which
are absent in a subsequent section representing the tongue away from the frenulum closer
to towards the tip. After tamoxifen treatment, seen are the endogenous Lgr5-OSCC
(Lgr5GFP+) that did not undergo recombination denoted by arrows yellow double
positive Lgr5-OSCC (Lgr5+/Tom+) marked by asterisks and Tomato positive only
progenitor cells marked by arrowhead (i-1). In a subsequent section representing the
tongue away from the frenulum closer to towards the tip only Tomato positive progenitor
cells marked by arrows are visible (i-2). Matched hematoxylin and eosin staining to
visualize the anatomical structures (j, k). Scale bars 100µm. Abbreviations: Fr: frenulum,
m: muscle, st: stroma, ep: epithelium
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3.3B Lgr5 positive stromal cells in the tongue are neural crest derived.
Important contributions of NC cells have been demonstrated in formation of mammalian
craniofacial structures including branchial arches, where the tongue forms (Chai and
Maxson, 2006; Cordero et al., 2011; Liu et al., 2012). Therefore to determine the origin
of Lgr5+ cells in the stroma of the tongue of adult mice, we genetically mapped the fate
of neural crest cells in the tongue using well established NC reporter mouse line Wnt1Cre,
which drives Cre expression in neural crest and dorsal domains of the developing
hindbrain and spinal cord beginning at E8 (Danielian et al., 1998). The Wnt1Cre mice
were crossed to Rosa26-Tomato and Lgr5-EGFP-ires-CreERT2 mice. In these mice GFP
serves as a surrogate marker for Lgr5 expression whereas Tomato will indicate NC
derived cells. Analysis of the tongue from adult mice revealed that Lgr5 positive cells
overlap with the Wnt1Cre derived Tomato positive progenitors (Fig. 18 a-c). In addition
the Lgr5+ cells in the frenulum of the tongue of adult mice maintain the expression of
embryonic NC marker Sox9 (Fig. 18d) (Bronner-Fraser, 1994; Cheung and Briscoe,
2003) and are actively dividing as indicated by the expression of Ki67 proliferation
marker (Fig. 18 e). This data provide the first direct evidence that Lgr5 positive tongue
stromal cells are descended from the NC lineage and might maintain NC characteristic in
adult tissue.
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Fig 18: Lgr5 positive oral stromal stem cells are neural crest in origin
Lgr5-OSCCs (Lgr5GFP+) present in the stromal layer of the tongue are
denoted by arrows (a). Wnt1Cre mice crossed with a Rosa26-Tomato reporter
marks cells of neural crest origin shown as tomato positive cells. Wnt1 positive
cells contribute towards the stroma in the tongue (b). Using a subsequent cross
of these two mice where the neural crest origin cells are marked by tomato and
the Lgr5-OSCC cells are marked by endogenous GFP expression, observed is
the overlap of the Lgr5-OSCC cells as a subset of the Wnt1 derived neural
crest cells (arrows in c). These cells are proliferative marked by proliferation
marker Ki67 (e) and also express known neural crest marker Sox9 (d) as
denoted by arrows. Scale bars 100µm. Abbreviations: m: muscle, ep:
epithelium
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3.3C Lgr5 is expressed in neural crest cells during embryonic development.
We showed that in adult tongue Lgr5+ stromal cells are derived from embryonic NC cells
however question remained whether Lgr5 is expressed in early stages of NC formation in
cranial structures. To address this question we analyzed the Lgr5-EGFP-ires-CreERT2
mice at embryonic day E9.5 when NC cells are still migrating. At this stage of
development expression of Lgr5 as marked by presence of GFP was visible in branchial
arches I, II and III as well as in midbrain, otic and optic vesicle (Fig. 19 a-c). The Lgr5+
cells were present in the migrating NC cells (Fig. 3 d and e) as well as in NC of branchial
arches (Fig. 19 f and h). Based on the fact that the oral tongue forms from branchial
arches I and II, the presence of Lgr5 positive cells in the NC of branchial arches suggest
the contribution of those cells in the future Lgr5+ population of NC derived adult stromal
cells.
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Fig 19: Lgr5 is expressed in the neural crest cells at embryonic day 9.5 (E9.5)
Lgr5 expression is observed in neural crest cells as early as E9.5 in the mouse
embryo. Lgr5 expression is observed in the optic vesicle, otic vesicle and in the
migrating neural crest cells (b). Brightfield image of E9.5 embryo shows a normal
developed mouse embryo. The dashed yellow line indicates the plane of the section
(a). Magnified image of the branchial arches I, II and III, seen are the migrating Lgr5
positive neural crest cells (c). Sagittal sections of the embryo represent the presence of
Lgr5 positive neural crest cells underneath the epithelium marked by beta-catenin (d-
g). Scale bars 100µm. Abbreviations: mb: mid brain, op: optic vessel, ot: otic vessel,
h: heart, Ne: neuroepithelium, Nc: Neural crest, Ba: Branchial arch
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3.3D Lgr5+ tongue stromal cells can be propagated in vitro.
To assess the contribution of Lgr5+ cells during adult homeostasis, we performed short
and long-term lineage tracing followed by FACS analyzes. Analysis of cells isolated
from tongues of adult Lgr5-EGFP-ires-CreERT2 x Rosa26-Tomato reporter mice without
Tam treatment revealed only the presence of Lgr5-GFP (Lgr5+) population (Fig. 20 a).
After 3 days post induction we mostly observed presence of Lgr5+ and Lgr5+/Tom+ cells
labeled by Tomato (0.08% and 0.16% respectively) (Fig. 20 a). There was only low
number of Tom+ progenitors detected (0.01%). However after two weeks post induction
the Tom+ progenitor fraction increased dramatically (0.14%) (Fig. 20 a). Lgr5+,
Lgr5+/Tom+ and Tom+ cells isolated using FACS after 2 weeks of Tam treatment were
expanded in vitro using embryonic neural crest culture conditions previously described
by Ishii and colleagues (Ishii et al., 2012) with medium conditioned by SNL feeder cells
and supplemented with bFGF and LIF. Those cells maintained sustainable growth and
showed proliferation rate greater than fibroblast used as a control stromal cells (Fig. 20 c).
In vitro, primary cultures of FACS-isolated cells, Lgr5+, Lgr5+/Tom+ and Tom+ formed
appreciable numbers of tightly packed, large colonies containing cells of small size and
relatively undifferentiated morphology (Fig 20 b, d and e). Although the Tom+ fraction
also formed holoclones the colonies often displayed irregular borders and consisted of
bigger, morphologically differentiated cells. Those colonies resembled epithelial
morphology and formed with similar efficiencies and were very distinct in appearance
from fibroblast used as a control stromal cells which didn’t form any colonies (Fig. 20 d
and e) Referred to as holoclones, such colonies are clonally derived from single stem
cell (Barrandon and Green, 1987)
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Fig 20: Isolation and Characterization of Lgr5-OSCC cells
Representative FACS plot of dissociated adult Lgr5-EGFP-ires-CreERT2 x Rosa26-
Tomato reporter mouse tongues for two-color analysis (a). Using endogenous Lgr5
driven green fluorescent protein expression, pure Lgr5 positive population was
isolated using the negative population as control (a). Post tamoxifen treatment
followed by a short chase (3 days) and a long chase (2 weeks), all four populations
unstained, Lgr5GFP, Lgr5GFP/Tomato double positive (post recombination) and
Tomato positive only cells were sorted with 99% purity (a) (n=3). The cells appeared
to grow as an adherent monolayer with tight junctions in vitro (b). Graphical
representation of the growth curve indicates a specific growth pattern of a sudden
rapid growth at day 10 (c) (n=3). Colony formation assay confirms that these cells
have the ability to form colonies in vitro indicated by crystal violet staining (d) (n=3)
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3.3E RNA sequencing revealed that Lgr5+ oral stromal cells exhibit characteristics
of embryonic neural crest cells.
Initial analyses suggested that Lgr5+ cells have features of neural crest cells because of
its origin as well as mesenchymal because of its stromal localization within the tongue.
To define the transcriptome signature of Lgr5+ cells we used RNA-sequencing. We used
mRNAs isolated from primary culture of two independent biological samples, namely
Lgr5+ cells isolated by FACS on the basis of GFP expression and Lgr5+/Tom+ cells that
consist of the same cell population but in addition to GFP expression they were labeled
by Tomato. As expected both cell lines showed ~95% overlap of gene expression with
4551 and 4368 genes with RPKM>10 present in Lgr5+ and Lgr5+/Tom+ respectively
(Fig. 21 a). Gene ontology analyses revealed that many of the genes expressed in Lgr5+
adult cells were involved in developmental processes (Fig. 21 b) especially in ectoderm
and mesoderm development suggesting ectomesenchymal characteristic of Lgr5+ tongue
stromal cells (Fig. 21 c). To test the possibility that Lgr5+ cells exhibit characteristics of
neural crest cells we performed the enrichment comparison of Lgr5+ expressed genes
with the signature of embryonic cranial neural crest cells established by Ishii and
colleagues (Ishii et al., 2012) (Fig. 21 d). Markers of neural crest were increased
significantly in the Lgr5+ and Lgr5+/Tom+ cell lines, almost 60% of genes present in
embryonic NC signature gene list was present in Lgr5+ and Lgr5+/Tom+ cell lines
including Sox9, Twist1, Snai1, Myc, Ets1, Epha2 and Itgb1. Additional neural crest
markers (a total of 26) also showed high levels of expression (Fig. 21 d). With respect to
their gene expression profiles, the majority of adult NCSCs express high levels of nestin,
an intermediate filament that is essential for the self-renewal of neural stem cells (Toma
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et al., 2001). As shown by immunofluorescent staining of Lgr5+ and Lgr5+/Tom+ cell
lines they express Nestin (Fig. 21 e), which is negative in fibroblasts used as a stromal
cells control. However Lgr5+ and Lgr5+/Tom+ cell lines also express in some degree
mesenchymal marker Vimentin, which is typically present in fibroblasts (Fig. 21 e). We
also compared the Lgr5+/Tom+ gene list against an independent list of genes known to
be associated with various aspects of neural crest that was defined by LifeMap Discovery
and included 1394 genes. In this comparison 27% of all the neural crest associated genes
were present in Lgr5+/Tom+ cells (Fig. 21 f). Interestingly many of those genes were
expressed specifically during development in branchial arches, the place of migration of
cranial neural crest (Barembaum and Bronner-Fraser, 2005; Bronner-Fraser, 1994). We
used Upstream Regulator Analysis in Ingenuity Pathway Analysis (IPA) software to
identify the cascade of upstream transcriptional regulators that can explain the observed
gene expression, which can help illuminate the biological activities occurring in the
Lgr5+ stromal cells. Interestingly Myc that was highly expressed in both Lgr5+ and
Lgr5+/Tom+ (Fig. 21 d) as a neural crest specific gene was also the top growth factor -
upstream regulator (Fig. 21 g). Lgr5+/Tom+ cells contained 451 activated Myc target
genes within all genes with RPKM>10 including other neural crest markers like Sox9,
Rarg, Ccnd1, Col1a1, Col1a2. Interestingly Myc was shown to be an essential early
regulator of neural crest cell formation in Xenopus (Bellmeyer et al., 2003). Myc was
localized at the neural plate border prior to the expression of early neural crest markers,
such as slug and morpholino-mediated “knockdown” of c-Myc protein resulted in the
absence of neural crest precursor cells and a resultant loss of neural crest derivatives
(Bellmeyer et al., 2003). Expression of Myc and its targets in adult Lgr5+ stromal cells
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may indicate importance of Myc in the maintenance of neural crest stem cells
characteristics of those cells.
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Fig 21: RNA Seq analysis reveals similarities between Lgr5-OSCCs and cranial
neural crest cells
RNA was isolated from two independent early passage adult Lgr5-OSCCs (Lgr5GFP+
and Lgr5GFP+/Tom+). Using RNA Seq methodology, it was identified that the
molecular characteristics of Lgr5-OSCC is similar to cranial neural crest cells. Venn
diagram showing 95% overlap in the gene expression profile between Lgr5-OSCCs
before and after recombination with tamoxifen (Lgr5GFP+ and Lgr5GFP+/Tom+,
respectively) (a). GO analysis reveals that 11% of the markers identified were
involved in developmental processes (b). Further analysis of these developmental
processes exposed that several of them are involved in factors involving differentiation
into ectodermal and mesodermal lineages which are the key cranial neural crest cell
fates (b, c). Comparative analysis of the Lgr5-OSCCs to an established cranial neural
crest marker profile suggests that they share common markers with respect to cranial
neural crest cells, neural crest developmental processes and specific neural crest
derived cell fate markers (d). Indirect immunofluorescence of Lgr5GFP+,
Lgr5GFP+/Tom+ cells and control fibroblasts with Nestin and Vimentin antibodies
(e). LifeMap data analysis software indicated that Lgr5-OSCCs share about 23%
similarities with all established neural crest markers including markers expressed in
the branchial arches during development (f). Upstream analysis using Ingenuity
Pathway Analysis (IPA) indicated that Myc is the main up stream transcriptional
factor for 451 genes out of the total 4369 genes expressed in Lgr5+/Tom+ cells with
RPKM>10 (g).
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3.3F Lgr5+ oral stromal cells differentiate into neural crest lineages.
To test the idea that tongue derived Lgr5+ stromal cells contain multipotent cells capable
of generating neural and non-neural cell types we exposed those cells to specific culture
conditions (Ishii et al., 2012). When cultured with various differentiation media, both of
the cell lines (Lgr5+ and Lgr5+/Tom+) were capable of differentiating into several
different cell types, including smooth muscle cells, glial cells and osteoblasts (Fig. 22 a –
f). The formation of glia expressing glial fibrillary acidic protein (GFAP) (Fig. 22 c and
d) and smooth muscle actin (SMA) expressing non-neural cells (Fig. 22 a and b) was
readily detectable in both cell lines. Osteogenic potential distinguishes cranial neural
crest from other neural crest populations (Santagati and Rijli, 2003). We therefore
investigated the osteogenic differentiation of Lgr5+ and Lgr5+/Tom+ cells. We placed
cells in osteogenic conditions and monitored cellular morphology and used alizarin red to
determine the presence of calcific deposition by cells of an osteogenic lineage (Fig. 22 e
and f). The morphology of Lgr5+ and Lgr5+/Tom+ cells began changing shortly after
exposure to osteogenic media. The cells lost their fibroblastic appearance and formed
aggregates that stained positive with alizarin red. The osteogenic differentiation potential
of Lgr5+ and Lgr5+/Tom+ cells support the finding that the Lgr5+ cells that reside in
tongue stroma are derived from the cranial neural crest.
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Fig 22: Lgr5-OSCC are multi potent giving rise to cells in both ectodermal and
mesodermal lineages
Evaluation of differentiation potential in vitro, indicates that Lgr5-OSCCs have
the ability to differentiate into cells of mesodermal origin such as smooth muscle
cells indicated by smooth muscle actin (a, b) scale bars 20µm and osteogenic
progenitor cells marked by positive alizarin red staining (e, f) scale bars 500µm.
Lgr5-OSCCs can also be differentiated into cells from the ectodermal lineage such
as astrocyte like cells expressing glial fibrillary acidic protein (c, d) scale bars
20µm.
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3.3G Lgr5+ oral stromal cells assume mesenchymal fate in de novo reconstitution
assay and participate in tongue wound healing
To assess the behavior of Lgr5+ cells in vivo we used two approaches. First we utilized
chamber graft experiments where suspension of cultured mouse epithelial cells that have
potential to regenerate epithelium was mixed with suspension of Lgr5+/Tom+ cells and
transplanted into a chamber on the back skin wound of a recipient nude mouse.
Following removal of the chamber dome three weeks after engraftment, the grafts
became covered by epidermis (Fig. 23 a and c) and also showed presence of Lgr5+/Tom+
cells (Fig. 23 b) indicating survival and expansion of those cells. Graft cryosections
revealed that Lgr5+/Tom+ cells preferentially reconstituted the stromal layer (Fig. 23 d –
f). None of the labeled Lgr5+/Tom+ cells was seen localizing in the epithelium labeled
by keratin 5 (K5) (Fig. 23 d). The cells formed a multi cell layer of stroma as
Lgr5+/Tom+ cells expressed vimentin (Vim) (Fig. 23 f) and were still actively dividing
as indicated by presence of proliferation marker Ki67 (Fig. 23 e) mimicking the natural in
vivo state. We also tested how Lgr5+ cells participate in the tissue wound healing. Lgr5-
EGFP-ires-CreERT2 reporter mice crossed with Rosa26-Tomato reporter mice were
treated with Tam at postnatal day 40 and 41 (P40, P41) to label the Lgr5+ progenitors
with Tomato. After 1-week post Tam treatment the small biopsy type wound was induced
at the ventral side of the tongue. Analysis of the tongues 10 days after wounding revealed
that Lgr5+ progenitors expanded in the wound area below the epithelium that was already
regenerated (Fig. 23 g – i) and present in between the muscles that were cut during the
biopsy (Fig. 23 i). Those results suggest that Lgr5+ cells and their progenitors have
potential role in tongue wound healing.
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Fig 23: Lgr5-OSCC participate in regeneration and reconstitution of the stroma
in wound healing experiments
Using a chamber graft experiment, a sterile plastic dome was inserted on the back
of a nude mouse to keep the 2cm wide wound region from closing. Mix of mouse
epithelial cells and Lgr5-OSCCs (Lgr5GFP+/Tom+ for tracking) were plated on
the wound area to verify their role in the regeneration process. Bright field and
fluorescent images of the wound closed after the chamber was removed, arrows
indicate the area with Lgr5GFP+/Tom+ cells (a, b) scale bar 500µm.
Hematoxylin and Eosin staining reveals that the wound area repaired itself,
observed are the newly formed intact epithelium and reconstituted stroma (c).
Immunohistochemistry reveals that the Lgr5+/Tom+ double positive cells
specifically localized in the stroma and helped in the reconstitution indicated by
active proliferation marked by Ki67 positive staining (e). These cells specifically
localized under the epithelium marked by Keratin 5 (d) while maintaining their
stromal characteristics as indicated by vimentin (f) scale bars 100µm. Lgr5-
EGFP-ires-CreERT2 x Rosa26-Tomato reporter mice were treated with tamoxifen
followed by a chase to allow for recombination. Mice were then subjected to a
biopsy punch through the midline section of the tongue, creating a wound that
disrupted both the epithelial and the stromal layers. Post 14 days, it was observed
that the wound repaired to form an intact E-cadherin positive epithelium (i) scale
bar 100µm. Observed beneath the epithelium was the expanding stromal Lgr5-
OSCCs into the wound area. The wound area beneath was not completely healed
indicated by the incomplete muscle formation, but interestingly the Lgr5-OSCCs
were observed expanding into this wound area. Lgr5+/Tom+ double positive cells
are usually not located deep within the muscle region. Abbreviations: ep:
epithelium, st: stroma, w: wound, m: muscle
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3.4 Discussion
The mesenchymal components of the oral tongue are known to form primarily from the
first branchial arch. Although it has been demonstrated that NC cells contribute to
formation of mammalian craniofacial structures including branchial arches (Chai and
Maxson, 2006; Cordero et al., 2011) there is no clarity about the distribution patterns of
NC-derived cells in the mesenchyme of developing tongue. In current study we
discovered the presence of Lgr5 positive stromal cells in the tongue and oral mucosa that
display properties of neural crest stem cells (NCSC), including clonal growth and
multipotent differentiation. If provided with the right differentiation signals the Lgr5+
cells have the potential to form cells of different lineages like smooth muscle, neuronal
and osteogenic demonstrating their stem cell like nature. RNA sequencing (RNA-seq)
revealed that adult Lgr5+ oral stromal stem cells (LOSSC) express high number of neural
crest markers (Sox9, Twist1, Snai1, Myc, Ets1, Crabp1, Epha2 and Itgb1). Using lineage-
tracing experiments we showed that those cells persist over a year in the tongue and
buccal mucosa and give rise to stromal progeny. We also showed that not only Lgr5
marks the population of oral stromal stem cells in the adult tissue but is already expressed
in the cranial neural crest cells during embryonic development. This is the first report on
persistence of Lgr5 positive embryonic neural crest cells into adulthood that participate in
the maintenance of stroma in the tongue and oral mucosa.
Due to their extraordinary plasticity and their on-going presence in the adult organism,
craniofacial NCSCs may represent an ideal source of cells for regenerative medicine
(Kaltschmidt et al., 2012). Because described here Lgr5+ oral stromal cells are
multipotent and are of neural crest origin, their capacity to give rise to neural and non-
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neural cell types at clonal density reflects the broad potential inherent to NCSCs. The fact
that these cells can be easily expanded in culture, even when isolated from the adult oral
stroma, might make them a valuable source for cell replacement therapies because
sufficient cell material could be obtained for such purposes. Indeed, in a pre-clinical
study, isolated and cultivated epidermal neural crest stem cells (EPI-NCSCs) and
olfactory ensheathing cells (OECs) were shown to encourage the recovery of function
after experimental spinal cord injury or lesions with no signs of tumor formation (Hu et
al., 2010; Li et al., 1997; Sieber-Blum, 2010; Sieber-Blum et al., 2006). Ganz J. et al.
showed that astrocyte-like cells derived from human oral mucosa provide neuroprotection
in vitro and in vivo (Ganz et al., 2014). Human OECs have been successfully employed in
an experimental rat model of Parkinson’s disease (Murrell et al., 2008). In addition to
these promising reports concerning regeneration after neurological defects, several pre-
clinical reports have described the contribution of adult NCSCs to the regeneration of
mesodermal tissues, like bone repair in a tibial bone fracture model (Lavoie et al., 2009)
or repair of large mandible bone defects resulting from the extraction of third molars
(d'Aquino et al., 2009). In conclusion, adult craniofacial NCSCs represent an easily
accessible, ethically unambiguous, and highly plastic source of cells with high clinical
potential. Adult NCSCs show great promise concerning the treatment of several diseases
requiring the regeneration of multiple cell types, and may be of vital importance for the
future of regenerative medicine (Kaltschmidt et al., 2012).
Important aspect of existence of Lgr5+ neural crest derived stromal stem cells in the oral
mucosa is their potential role in wound healing and tumor progression. Although the skin
and the oral mucosa tissues are similar in nature, oral mucosa has more rapid healing and
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a lack of scar formation and more aggressive epithelial tumor progression. How
important is the difference in the origin of stroma between the oral mucosa
(predominantly neural crest) and skin (predominantly mesoderm) in the process of wound
healing and tumor progression is not clear. Wounds in human oral mucosa heal mainly by
regeneration. The rate of healing is faster than that in the skin or other connective tissues
and seems to be affected negligibly by age and gender (Szpaderska et al., 2003). Human
oral mucosa-derived fibroblasts behave in some respects similarly to fetal-derived
fibroblasts (Schor et al., 1996). Knowing that stroma in the oral cavity is neural crest
derived further characterization of the stromal component of the oral mucosa may help to
understand how this specific environment affects wound healing and tumor progression.
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CHAPTER 4: CONCLUDING REMARKS
Understanding the role of stem cells in the regulation and homeostasis is extremely
important as it provides the answers to better characterize the mechanisms underlying the
process of repair and onset of disease.
In this dissertation, I have demonstrated three vital points; understanding the role of adult
stem cells in the tongue and the salivary gland, defining their potential as tumor initiating
cells and to characterize the underlying stroma in the tongue.
The salivary gland is required for everyday normal functioning such as mastication and
maintenance of oral hygiene. In this study, I have shown that there is a specialized slow
cycling stem cell population that resides in the keratin 5 positive basal layer. In vivo
wound healing assays have demonstrated that these cells can be redirected into an
activated state where they are proliferative and participate in wound closure. These cells
are not only able to self-renew and maintain themselves but are also bipotent where they
can give rise to both the keratin 5 basal cells and the keratin 8 luminal cells. This is an
important observation as it dictates the idea of how adult stem cells have the ability to be
multipotential when provided with the right cues for differentiation. For the first time we
have also molecularly characterized these cells using a microarray analysis.
Transcriptional analysis have further identified that salivary gland slow cycling cells
require TGFβ1 target gene activation while the progenitor cells require BMP signaling. In
an in vivo tumor initiating cell assay, we have also defined the role of these slow cycling
population to adapt a highly proliferative state to give rise to adenocarcinoma phenotype
tumors in mice. Employing serial transplantation assays post purification via FACS sort,
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we have also demonstrated that these cells continue to maintain their tumor initiating
capacity. These findings have not only helped us understand the normal homeostasis of
these cells but it has also shed light on treatment options. Many patients lose the ability
maintain normal salivary gland function and could develop conditions such as xerostomia.
In patients that undergo chemotherapy and radiation, these cells can potentially be used to
regenerate lost function. The molecular targets identified in these cells can be potentially
used as drug targets for creating a more specialized treatment where the source of “cancer
stem cells” can be depleted. K5tetoffTREH2BGFP model is well defined and can be used
to further characterize the mechanisms of tumor initiation, progression and metastasis.
Epithelial mesenchymal interactions play a vital role during development. Epithelial cells
receive instructive signals from the underlying mesenchyme to direct their cell fate and
migration path. These processes are replicated during wound healing where the
underlying mesenchyme via cytokine signaling recruits cells for repair at the wound site.
Recent advances have also revealed that the stroma in the heterogeneous tumor is in an
active state where it provides the necessary signals for cancer progression. Using lineage-
tracing analysis, we have identified a novel neural crest derived stromal stem cell
population in the tongue that persists through adult hood and maintains the homeostasis
in the stroma of the tongue. We have also identified that this stromal stem cell population
first appears at E9.5 as marked by Lgr5 in the migrating neural crest population. These
embryonic cells persist as an undifferentiated stem cell population in the tongue
throughout adulthood. In vivo wound healing assays have revealed that these cells are
capable of not only participating and infiltrating the wound bed but also preferentially
localizes in the stromal layer post transplantation. RNA Seq analysis revealed that these
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cells share homology with neural crest cells and have the potential to differentiate into
several neural crest lineages under appropriate differentiation conditions in vitro.
Comparative gene analysis has suggested that these cells share similarities with the
embryonic neural crest population thus suggesting their origin to be neural crest. These
findings have not only suggested that the stroma of the tongue is quite dynamic and play
an active role in everyday maintenance but that they could potentially be the activated
stromal population that could provide the necessary cues for metastasis and progression.
Given that the stroma of the tongue is quite different from that of the skin, it would be an
interesting direction to study the role of these stromal stem cells in cancer progression.
In these past 5 years, I have observed that the perspective of cancer treatment has
changed to traditional single drug treatment approach to using multiple combination
therapies. These combination therapies can range from providing a drug cocktail to the
patients to treating multiple cell types at once to trying to deplete the root cause of cancer
stem cells within the tumor. FDA has currently approved cisplatin and radiation or
cisplatin with 5-fluorouracil for both primary and recurrent metastatic head and neck
cancer while a more vigorous combination such as cisplatin with cetuximab and 5-
fluorouracil for metastatic recurrent cancer cases. All these therapies have been proven
successful in many patients but the key question of whether the root causal cancer stem
cell population is being affected by this is still debatable. The treatments now are
focusing on how these cancer stem cells could be targeted more specifically as they
escape all the traditional treatment options. Using the findings in this study, I believe the
future of head and neck cancer treatment lies in combination therapy wherein not only is
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the mass of the tumor targeted for treatment, but also the underlying stroma, the causal
cancer stem cell population are also targeted.
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CHAPTER 5: MATERIALS AND METHODS
5.1 Mice
Two transgenic mouse lines were used: keratin 5-driven tetracycline repressor mice (K5-
tTA) (Diamond et al., 2000) and tetracycline response element-driven histone fusion,
H2BGFP transgenic mice (pTRE-H2BGFP) (Tumbar et al., 2004). In this double
transgenic line, H2BGFP expression is turned on (“pulse”, no doxycycline treatment)
from early embryogenesis. By feeding the animals doxycycline (Dox diet pellets 1mg/g
from BioServ for 4 weeks starting at P21-28), H2BGFP expression is turned off for the
duration of the treatment (“chase”). In addition two reporter mice line from Jackson
Laboratory were used: Tg (TCF/Lef1-HIST1H2BB/EGFP) 61Hadj/J 013752 and Gli1tm3
(cre/ESR1) Alj/J007913). For CreER induction, tamoxifen (Sigma) was dissolved in corn
oil (20 mg/ml) and injected intraperitoneally (i.p. - 10 mg daily for 3 days). For tumor
induction, 4NQO obtained from Sigma-Aldrich was dissolved in propylene glycol
(Sigma-Aldrich) as stock solution (4 mg/mL) (1h warm water bath to dissolve), stored at
4°C, and diluted in the drinking water to a final concentration of 50µg/mL. Water was
changed every 3 days. Lgr5-EGFP-ires-CreERT2 and Rosa26-Tomato mice were
ordered from Jackson Laboratories, Wnt1Cre mice were kindly provided by Dr. Yang
Chai. For CreER induction, Lgr5-EGFP-ires-CreERT2 x Rosa Tomato mice were treated
with 2 consecutive doses of 200 uL or 1000ug/dose of stock 5mg/mL tamoxifen in
ethanol. Tamoxifen in ethanol was applied to the shaved back skin of mice. All mice
work was conducted according to the Institutional Animal Care and Use Committee
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(IACUC) at the University of Southern California. The protocol No. 11172 was approved
by the IACUC Committee.
All mice work was conducted according to the Institutional Animal Care and Use
Committee (IACUC) at the University of Southern California. The protocol No. 11172
was approved by the IACUC Committee. All surgeries were performed under either
isoflurane or ketamine anesthesia and all efforts were made to minimize the suffering
with analgesics (Buprenex prior and post-surgery was administrated).
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5.2 Genotyping
All mice were weaned at 3 weeks of age and clipped to collect tail sample for DNA
isolation. The tails were digested overnight in 200uL tail lysis buffer (50mM Tris pH 8.0,
20mM NaCl, 1mM EDTA, 1% SDS) with 5 uL of Proteinase K solution in a 55°C water
bath overnight. The next day, 60uL of NaCl was added to the solution and mixed
thoroughly for about 45 seconds. The sample was then centrifuged at full speed for 5
minutes at RT. The clear supernatant was transferred to a new tube. Equal volume of 200
proof 100% ethanol was added. The tube was inverted to precipitate the DNA about 5
times. The samples were centrifuged at RT for 5 minutes at full speed to pellet the DNA.
The precipitated DNA is washed with 70% ethanol to remove any salt residues, air dried
and dissolved in 100uL of milliQ water. This was used to genotype the mice.
Table 1: PCR primer list
Gene Annealing
Temperature
Primer Sequences (5’-3’)
Cre Recombinase 62°C TGCTGTTTCATGGTTATGCGG
TTGCCCCTGTTTCACTATCCA
OCT 4 58°C CAAGGCAAGGGAGGTAGACA
TGCCAGACAATGGCTATGAG
CCAAAAGACGGCAATATGGT
Rosa Tomato 60°C AAGGGAGCTGCAGTGGAGTA
CCGAAAATCTGTGGGAAGTC
GGCATTAAAAGCAGCGATGG
Rosa26 YFP 62°C GGAGCGGGAGAAATGGATATG
AAAGTCGCTCTGAGTTGTTAT
AAGACCGCGAAGAGTTTGTC
K5tTA 54°C CTGCCCAGAAGCTGGTGT
CCATGCGATGACTTAGTA
KRAS 62°C AGCTAGCCACCATGGCTTGAGTAAGTCTG
TTTACAAGCGCACGCAGACTGTAGA
GFP 58°C ATCCTGGTCGAGCTGGACGGCGACG
TCAGGTAGTGGTTGTAGGGCAGCAG
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5.3 Immunohistochemistry and Immunofluorescence Staining
All frozen sections were fixed in 4% paraformaldehyde before embedding in OCT.
Tissue sections were stained with hematoxylin and eosin for H&E visualization. For
immunofluorescence staining, sections were permeabilized with 0.1% Triton X-100 for
10min and blocked in 0.1% Triton-PBS, 0.25% goat normal serum, 0.25% donkey
normal serum and 0.1% BSA for 1h at room temperature. Primary antibodies were
incubated in 0.1% BSA overnight at 4°C and washed thrice with PBS the following day.
Secondary antibodies were incubated in 0.1% BSA for 1h at room temperature. The
sections were counterstained with DAPI (4’-6 Diaminidino -2-phenylindole) for nuclear
visualization. Images were obtained using AxioImager Zeiss Inverted Fluorescence
Microscope.
Table 2: Immunohistochemistry antibody list
Antibody Dilution Company
Keratin 5 1:200 Gift from Colin Jamora (UCSD)
Keratin 8 1:100 DSHB #TROMA-I
CD 104 1:300 BD Pharmingen #553745
E Cadherin 1:200 Zymed Laboratories #13-1900
Vimentin 1:300 Novus Biologicals #NB300-223
Nestin 1:400 DSHB #Rat-401
Smooth Muscle Actin 1:400 Sigma-Aldrich #A5228
CD 44 1:400 BD Pharmingen #550538
Cdh 2 1:300 GeneTex # ZF127530
Tenasin C 1:200 AbCam #ab88728
Tspan 6 1:300 Sigma-Aldrich #HPA004109
Green Florescent protein 1:4000 AbCam #ab13970
Phospho Smad 2 1:50 Cell signaling Ab# 8828
Phospho Smad 1, 5, 8 1:50 Cell signaling Ab # 9516
Sox 9 1:200 SIGMA HPA 001758
B catenin 1:400 Sigma-Aldrich #C7207
Glial Fibrilary Acidic Protein 1:200 Abcam #ab4674
96
5.4 Isolation of minor SG LRCs
K5TetOff/TreH2BGFP animals were fed 1mg/g doxycycline food for 4 weeks starting
around P21-28. H2BGFP+ upper palate were dissected out of 20-30 mice and treated
with 1000U/ml Collagenase type I for 1h at 37°C with shaking. SGs were washed with
DPBS and digested in 0.25% Trypsin-EDTA for 20min at 37°C with shaking. Trypsin
was neutralized and the solution was filtered through both 70um followed by 40um cell
strainers to obtain a single cell suspension. For colony formation equal numbers of sorted
cells were plated onto mitomycin-treated 3T3 fibroblasts in E-media (Rheinwald and
Green, 1977) supplemented with 10% serum and 0.3mM calcium. Number and the
colony size were visualized using a fluorescent microscope as the cells expressed
H2BGFP.
97
5.5 Fluorescence Activated Cell Sorting (FACS)
For FACS, upper palates were dissected 4 weeks post chase from K5tetH2BGFP mice.
Lgr5-EGFP-ires-CreERT2 x Rosa26-Tomato mice were divided into treated and
untreated groups (n=3). One-week post induction with tamoxifen, the mice was sacrificed
and the tongues were dissected out. The tissues were treated with 1000U/ml Collagenase
type I for 1h at 37°C with shaking. SGs were washed with DPBS and digested in 0.25%
Trypsin-EDTA for 20min at 37°C with shaking. Trypsin was neutralized and the solution
was filtered through both 70um followed by 40um cell strainers to obtain a single cell
suspension. The single cells were then labeled with PE conjugated anti-α6 integrin
(CD49f) (1:200; BD Pharmingen) and APC conjugated CD44 (1:200); BD Pharmingen)
for 30min and sorted using the FACS Aria II cell sorter (BD, Bioscience) for
H2BGFP+/CD44+/α6+ and H2BGFP-/CD44+/α6+ populations. Cells were collected in
RNAprotect Cell Reagent (Qiagen) for RNA isolation or in media for expansion in vitro.
The cells for that had endogenous expression of GFP or tomato were sorted based on
their inherent expression.
98
5.6 Glandular Injections
The sorted cell populations (H2BGFP+/CD44+/α6+) and (H2BGFP-/CD44+/α6+) were
plated in 4-well plates with 3T3 feeder cells. After 14 days in culture, the sorted cells
were collected and mixed with matrigel (BD) (1:1). The unsorted dissected clumps were
micro dissected directly from 4 weeks chased minor SG tissue. The sorted cells
suspension and dissociated unsorted minor SG cell clumps were injected into
submandibular gland of immunocompromised NOD.Cg mice. 8 weeks after injection,
mice were sacrificed, and transplants were dissected out and analyzed.
99
5.7 RNA Isolation, Microarray, RT-PCR, and qPCR
Total RNAs were purified from FACS-sorted SG LRCs, SG non-LRCs, using Qiagen’s
RNeasy Micro kit according to the manufacturer’s instructions. Equal amounts of RNA
were reverse transcribed using the Superscript III First-Strand Synthesis System
(Invitrogen) according to the manufacturer’s instructions. cDNAs were amplified by PCR
and used in triplicates for each qPCR sample primer set with all primer sets designed to
work under the same conditions. Real-time PCR amplification of the particular genes of
interest was performed using an Applied Biosystems 7900HT Fast Real-Time PCR
System and the fold difference between samples and controls were calculated based on
the 2
-ΔΔCT
method, normalized to β-actin levels. Total RNAs from FACS of minor SG
LRCs (H2BGFP+/CD44+/ α6+) and minor SG non-LRCs (H2BGFP-/CD44+/ α6+) were
purified using RNeasy Micro Kit (Qiagen), and quantified using Nanodrop. RNA 6000
Pico Assay (Agilent Technologies) was used for RNA quality check. Microarray analysis
was performed by the University of Southern California Microarray Core Facility using
Affymetrix GeneChip® Mouse 430Plus 2.0 Arrays.
100
5.8 In vitro expansion, growth curve analysis and colony formation assay
Post FACS sorting, the cells were expanded in vitro in specific neural crest media. This is
a conditioned media collected after overnight exposure to SNL feeder cells and
supplemented with bFGF (25ng/mL) and LIF (10^3 U/mL) prior to use. (DMEM with
10% FBS, 4.5 g/L D-Glucose, 1mM Sodium Pyruvate, 1X MEM Non-Essential Amino
Acids, 1X Mercaptoethanol, 100U/mL PenStrep, 1X Glutamine). Equal number of cells
(100 cells/well) were plated in triplicates and counted using the hemocytometer at day 2,
4, 6, 10 and 12. Average values were calculated and plotted to obtain a growth curve.
Equal number of cells (50 cells/well) was plated in triplicates for assessing the ability for
colony formation. The colonies were visualized after 10 days using crystal violet dye.
101
5.9 In vitro Differentiation Assay
Osteogenic Differentiation
Lgr5+/Tom+ cells and Lgr5+ cells were treated with osteogenic specific (MEM with
10% FBS, 10mM B glycerophosphate, 0.2mM Ascorbic Acid, 100 ng/mL BMP-4 media)
for 21 days and stained with Alizarin red for visualization.
Smooth Muscle Differentiation
Lgr5+/Tom+ and Lgr5+ cells were treated with Smooth muscle specific media (DMEM
with 10% FBS) for 7 days and stained with SMA (Sigma- Aldrich #A5225) for
visualization.
Neural Differentiation
Lgr5+/Tom+ and Lgr5+ cells were treated with neural specific media (DMEM/ F12 with
1X B-27, 20 ng/mL FGF2, 10 ng/mL) for 7 days and stained with Tuj1/ P75 for
visualization.
102
5.10 Chamber graft transplantation and wound healing assays
A wound of 2cm diameter was created by removal of skin on the back of a nude mouse.
Chamber (company name) was placed to prevent closing of the wound. 1 X 10^6
Lgr5+/Tom+ cells were mixed with mouse epithelial cells in DMEM and coated on the
wound area. The chamber was removed after 7 days post implantation. The mouse was
sacrificed after complete healing of the wound. The tissue was prefixed as previously
described for sectioning. Lgr5-EGFP-ires-CreERT2 x Rosa26-Tomato mice were
induced with 2 doses of tamoxifen at P40. Post 3 days of tamoxifen treatment, using a
biopsy punch, a wound was created mid line in the tongue damaging both the epithelium
and the stroma. The mice were then allowed to heal and sacrificed 10 days post injury.
Tissue samples were collected, prefixed and embedded as previously described.
103
5.11 Immunocytochemistry
Cells were plated on pre-coated fibronectin or collagen glass slides. The cells were fixed
in 4% PFA, blocked in 2.5% NGS, 2.5% NGS, 0.1% BSA in 0.1% PBST for 1 hour at
RT. Primary antibodies were diluted in 0.1% BSA in 0.1% PBST over night at 4°C.
Secondary antibodies were diluted in blocking solution for 1 hour at RT. All slides were
counter stained with DAPI and imaged an inverted fluorescence microscope.
104
5.12 RNA Seq
RNA was prepared from cells using Trizol followed by purification using Qiagen Micro
RNA isolation kit. The paired-end libraries were prepared using the Illumina truseq RNA
sample prep kit V3 and sequenced on Illumina HiSeq2000 at the USC Norris Cancer
Center Next Gen Sequencing Core. The average length of raw reads is 75.5 nt with
average number of raw reads per sample is 59,253,994. RNA-seq data was analyzed
using the RNA-seq workflow in Partek Flow V4 (Partek Inc., St. Louis, MO). Raw reads
were trimmed based on the quality score (Phred QC>=25, min read length=25 nt) and
subsequently mapped to mm10 (Ensembl 72) using Tophat v2.0.8 (Kim et al. 2013) with
mostly default parameter settings. On average, over 90.2% of raw reads were mapped to
the genome. Gencode M3 annotation was used to quantify the aligned reads using Partek
E/M method. Quantified reads were normalized using RPKM method. For each sample,
a list of the genes with RPKM>=10 was generated and functional enrichment analyses
was subsequently carried out using PANTHER.
105
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Abstract (if available)
Abstract
Tongue and salivary glands are the main sites of head and neck squamous cell carcinoma. However, little is known about the origin and progression of such tumors. In this study, we investigated the role of stem cells in tumor initiation, and their cross talk with the surrounding stroma. Using a pulse chase strategy, we identify keratin 5 expressing (K5+) basal cells as slow-cycling bipotent cellular population in the minor salivary gland that self renew and differentiate into keratin 8 expressing luminal cells. Serial cell transplantation assays unraveled K5+ basal cells as tumor-initiating cells in vivo. To gain further insights into the phenomenon of tumor progression and wound healing in the head and neck region, we interrogated the stromal-epithelial interactions in the oral mucosa. After injury, the oral mucosal epithelium rapidly heals with lack of scar formation, and is a site of relatively aggressive epithelial malignancy compared to the salivary gland epithelia. Multipotent stem cell population of putative neural crest origin that persists into adulthood resides in the stroma of the oral cavity. The precise location, and their role in normal tissue homeostasis and tumor progression is not known. Herein, we identify Lgr5 expressing (Lgr5+) cellular population in the cranial neural crest during embryonic development that persists as a distinct stem cell population in the stromal compartment of the adult tongue and oral mucosa. The Lgr5+ oral stromal stem cells (LOSSCs) display properties of neural crest stem cells, including clonal growth, and multipotent differentiation into smooth muscle, neuronal, and osteogenic lineages. RNA-seq analysis of LOSSCs revealed significant enrichment of neural crest markers and lineage-tracing studies demonstrate that these cells give rise to stromal progeny and persist over a year in the tongue and buccal mucosa. In vivo transplantation experiments demonstrated that these cells preferentially reconstitute the stromal layer after injury. Our studies identify for the first time the persistence of Lgr5+ embryonic neural crest cells into adulthood that contributes to the homeostasis of the stromal compartment of the oral mucosa, and participate in wound healing after injury. Taken together, our studies highlight the role of adult stem cell populations in the head and neck region with respect to homeostasis, injury-repair, and tumorigenesis.
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Boddupally, Keerthi
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Core Title
Identification and characterization of adult stem cells in the oral cavity
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Keck School of Medicine
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Doctor of Philosophy
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Genetic, Molecular and Cellular Biology
Publication Date
09/18/2015
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05/07/2015
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epithelial stem cells,head and neck cancer,LGR5,neural crest stem cells,OAI-PMH Harvest,salivary gland,Tongue
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epithelial stem cells
head and neck cancer
LGR5
neural crest stem cells
salivary gland