Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice.
ABSTRACT In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin alpha6, and thus represent a putative anal stem cell population.
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ABSTRACT: The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3 distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.Cell cycle (Georgetown, Tex.) 09/2013; 12(17):2888-98. · 5.24 Impact Factor
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ABSTRACT: Anorectal malformations are congenital anomalies that form a spectrum from the most benign type with excellent functional prognosis, to very complex, such as cloaca in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs early during embryonic development of the organs derived from the cloaca. Due to the inability to directly investigate human cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca may underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective Shh signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of Keratins in the cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild to severe forms of anorectal malformations including cloaca. We found striking parallels with the Shh mouse model including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early cloacal epithelial differentiation defects may be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged hedgehog and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.Disease Models and Mechanisms 02/2014; · 4.96 Impact Factor
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ABSTRACT: The anorectal and urogenital systems arise from a common embryonic structure termed cloaca. Subsequent development leads to the division/septation of the cloaca into the urethra, urinary bladder, vagina, anal canal, and rectum. Defective cloacal development and the resulting anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. In the most severe form in females, the rectum, vagina, and urethra fail to develop separately and drain via a single common channel known as a cloaca into the perineum. In this review, we summarize our current knowledge of embryonic cloaca development and malformation, and compare them to what has already been described in the literature. We describe the use of mouse models of cloaca malformation to understand which signaling pathways and cellular mechanisms are involved in the process of normal cloaca development. We also discuss the embryological correlation of the epithelial and stromal histology found in step sections of the common channel in 14 human cloaca malformations. Finally, we highlight the significance of these findings, compare them to prior studies, and discuss their implications for the pediatric surgeons. Understanding and identifying the molecular basis for cloaca malformation could provide foundation for tissue engineering efforts that in the future would reflect better surgical reconstruction and improved quality of life for patients.Pediatric Surgery International 09/2014; · 1.06 Impact Factor
www.landesbioscience.com Cell Cycle 3039
Cell Cycle 9:15, 3039-3045; August 1, 2010; © 2010 Landes Bioscience
Identification of epithelial label-retaining cells
at the transition between the anal canal
and the rectum in mice
*Correspondence to: Géraldine Guasch: email@example.com
Submitted: 04/14/10; Accepted: 05/20/10
Previously published online: www.landesbioscience.com/journals/cc/article/12437
Tissue stem cells are thought to proliferate, self-renew and
differentiate throughout the entire life of an animal. Their cell
progeny participate in tissue homeostasis and repair.1,2 Being
long-term residents in an epithelium, stem cells are uniquely sus-
ceptible to the accumulation of multiple oncogenic changes by
repeated divisions giving rise to tumors. One explanation of this
apparent paradox is that tissue stem cells are relatively quiescent
cells, which divide infrequently and give rise to another stem
cell daughter and to a rapidly proliferating, transient amplifying
(TA) daughter cell.3,4 TA cells, unlike stem cells, have a limited
growth potential. Yet, for a short period of time they divide
repeatedly to make a large number of progeny, thereby fulfilling
the need to maintain adequate number of cells of the tissue of
origin. In the absence of specific molecular markers, the slow-
cycling property of stem cells has been used to identify stem
cells in their tissues of origin.5-9 Slowly cycling cells are identi-
fied by a repeated “pulse” with bromodeoxyuridine (BrdU) to
label all the proliferating cells in a tissue, followed by a “chase”
In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types
of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction,
although the reason for this is not known. one possibility is that the abrupt transition of the simple columnar epithelium
of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem
cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent
epithelium. We utilized a tetracycline-regulatable histone H2B-GFp transgenic mice model, previously used to identify
hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination
with a panel of putative stem cell markers. We identified a population of long-term GFp label-retaining cells concentrated
at the junction between the anal canal and the rectum. these cells are BrdU-retaining cells and expressed the stem cell
marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only
gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal
junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell
marker CD34 and integrin α6, and thus represent a putative anal stem cell population.
Laura A. Runck,1 Megan Kramer,1 Georgianne Ciraolo,2 Alfor G. Lewis1,3 and Géraldine Guasch1,*,
1Division of Developmental Biology; 2Department of pathology; Cincinnati Children’s Hospital Medical Center; Cincinnati, oH USA; 3Division of endocrinology;
University of Cincinnati; Cincinnati, oH USA
Key words: stem cells, transitional epithelium, keratinocyte, slow-cycling, label retaining cell
abbreviations: BrdU, bromodeoxyuridine; TA, transient amplifying; H2B-GFP, histone 2B-green fluorescent protein; LRCs, label-
retaining cells; doxy, doxycycline; TZ, transition zone; K5, keratin 5
period. The rapidly dividing TA cells divide and dilute the
label, while infrequently dividing cells retain the label (label-
retaining cells or LRCs). Therefore, label retention reflects the
growth history of the cells. Pulse-chase labeling schemes are
highly variable and depend on the growth kinetics of the tissue
LRCs can be found in many tissues including the oral mucosa
and the skin epidermis,10 the hair follicle,11 the eccrine glands,12
the bone marrow,13-15 the mammary gland,16 the kidney,17 the
liver,18 the trachea,19 the esophagus,20 the tibia,21 the pancreas,8
the heart,22 the bladder,23 the limbus of the eye,5,24,25 and the
Stem cells are critical for wound healing, due to their potent
ability to regenerate their own tissue. However, their extensive
capacity for proliferation also implicates them in tumorigen-
esis.27 Transitional epithelia are defined by the abrupt change
from one type of epithelium to another and have been shown to
exist in the human eye, esophagus, stomach, bladder, cervix and
anus. Interestingly, these transition zones have been reported to
be susceptible to tumor formation, which raises the possibility
3040 Cell Cycle Volume 9 Issue 15
of GFP label. Compared to BrdU label retention,
the histone H2B-GFP pulse-chase system is more
sensitive, ensures initial uniform labeling of cells
within tissues, and affords enhanced sensitivity
in monitoring the infrequent division behavior of
stem cells.38 We have detected a minor population
of LRCs in the basal layer of the anal epithelium
at the transition zone that meet the simple epithe-
lium of the rectum. The stem cell surface marker
CD34, also expressed in hair follicles39 and esoph-
ageal stem cells,20 has been found to colocalize
within the anal LRCs.
mouse anorectal transition zone and anal dif-
ferentiation. We began by analyzing how cells
connect at the dentate line where squamous
anal cells abut columnar cells from the rectum
(fig. 1a). We analyzed four adult CD-1 mice
from four to six weeks-old. Ultrastructural analy-
ses revealed that cells at the anorectal transition
zone show fewer desmosomes than anal cells that
connect between each other in the basal layer
(figs. 1b–b’ and s1). Hemidesmosomes can be
detected between the basement membrane and
the anal keratinocytes (fig. 1b”). We next exam-
ined how cells differentiate in the anal epithelium.
As the electron microscopy images indicated, the
mouse anal epithelium is composed of a basal
layer of cells that differentiate into a spinous,
granular and terminally differentiated stratum
corneum layer (fig. s1a). The markers of anal
differentiation that we examined were Keratin
6, Keratin 10, Loricrin and Filagrin. Keratin 6,
a keratin naturally expressed in hair follicles, but
aberrantly induced in epidermis under hyperproliferative condi-
tions, is expressed in the suprabasal layers of the anal epithelium
(fig. 2a). Similarly, Keratin 10 (a marker of the spinous layer
of the epidermis) is highly expressed in the suprabasal layers of
the anal epithelium (fig. 2b). These results were not surprising
as we have previously shown that the anal epithelium is natu-
rally more proliferative than the epidermis of the skin.35 As in
the epidermis, Loricrin marks the granular layer (fig. 2c) and
Filagrin marks the terminally differentiated stratum corneum
layer (fig. 2d). The anal epithelium expresses typical markers
for stratified squamous epithelium and therefore may provide
a niche for a population of stem cells similar to the stratified
squamous epithelium of the skin and the esophagus.
slow-cycling h2b-GfP lrcs are detected in the basal
layer of the squamous anorectal transition zone. To determine
whether slow-cycling cells were present in the anal epithelium,
we used the in vivo pulse-chase experiments previously employed
for labeling adult hair follicle bulge cells with histone H2B-
GFP.37 In this system, double transgenic tetracycline-induc-
ible mice express H2B-GFP driven by the keratin 5 promoter
that a stem cell niche exists within the transition zone.28-32 For
instance, in the cervix, cancers arise exclusively in the vaginal-
cervical squamocolumnar junction.33 In anal cancers, tumors
can develop in the perianal skin, anal margin and anal canal.
Interestingly, tumors of the anal canal develop at the transition
zone between the stratified squamous epithelium of the anal
canal and the columnar epithelium of the rectum. These tumors
are more frequent than those at the anal margin and the peri-
anal skin and their prognosis is less favorable.34 In the absence of
TGFβRII, mouse anal transitional epithelia spontaneously gener-
ate squamous cell carcinomas.35 Similarly, mice with a targeted
disruption of BMPR1A develop polyps in the intestinal epithe-
lium, but carcinomas result in the gastrointestinal transitional
zone.36 Transitional epithelia are poorly characterized and the
presence of putative slow-cycling cells has not previously been
In this study, we utilized a previously developed strategy to
detect cells in anal epithelium based on their proliferation his-
tory.37 Specifically, we used tetracycline-inducible mice driving
histone H2B-GFP to follow cell proliferation through the dilution
Figure 1. Characterization of the adult mouse anal canal and the anorectal junction. (A)
Semi-thin section stained with toluidine blue of the anorectal transitional epithelium
from 4–6 weeks old CD-1 mice. the dashed line indicates the basement membrane. Like
the epidermis, the anal canal is composed of a basal layer and differentiating spinous,
granular and stratum corneum layers (Fig. S1A). (B) Ultrastructural analysis of the ano-
rectal transitional epithelium. the dentate line separates the anal transition zone from
the rectal epithelium (Fig. S1D). the dotted line denotes the presence of the basement
membrane. Boxed areas B’ and B” are shown below at higher magnification. (B”) Intercel-
lular junctions between basal anal keratinocytes are rich in desmosomes (Fig. S1B and
C). the anorectal junction possesses fewer desmosomes. (B”) Basal anal keratinocytes
display numerous hemidesmosomes that connect with the basement membrane. tZ,
transition zone; BL, basal layer; Sp, spinous layer; Gr, granular layer; Sc, stratum corneum;
Dm, Desmosome; M, Mitochondria; Hm, Hemidesmosome.
www.landesbioscience.com Cell Cycle 3041
(K5-TetVP16xTRE-H2B-GFP) specifically in skin.
H2B-GFP expression is activated upon tetR-VP16
protein binding to the tetracycline responsive ele-
ment DNA fragment, and can be turned off by addi-
tion of a tetracycline analogue (doxycyline, doxy)
to the mouse diet. Upon H2B-GFP repression in
the adult and embryo, the brightest label-retaining
cells have been found in the hair follicle bulge.37,40
Here, we used this tet-off system to examine the
presence of H2B-GFP LRCs in the anal epithe-
lium. In unchased mice, all perianal skin and anal
epithelial cells displayed H2B-GFP epifluorescence,
consistent with strong keratin 5 promoter activ-
ity in these tissues (fig. 3a and b). No H2B-GFP
expression was detected in the rectum of H2B-
GFP founder mice or in any double transgenic
mice expressing the K5-driven tetracycline-regu-
latable transactivator mouse strain confirming that
there is no leaky expression of GFP in this organ
(fig. 3b). After three to four weeks of doxycycline
chase, H2B-GFP LRCs clustered prominently
within the anal transition zone at the junction with
the dentate line (fig. 3c and d). This pattern of
expression was consistent in all the mice analyzed (n
= 10). After five weeks of chase almost no LRCs were
detected in the anal transition zone, in contrast to
the hair follicle bulge which can retain label for ten
weeks of chase.37,38
brdu-retaining cells colocalize with h2b-
GfP lrcs in the basal layer of the squamous ano-
rectal transition zone. To verify the presence of the
LRCs in the anal transitional epithelium, we uti-
lized a second pulse-chase technique. Slow-cycling
cells have been detected in the past by a repeated
“pulse” of the nucleotide analog bromodeoxyuri-
dine (BrdU) to label all the proliferating cells in a
tissue, followed by a “chase” period.11,41 We applied
the BrdU pulse and chase strategy to the double
transgenic mice K5-TetVP16xTRE-H2B-GFP to
analyze the presence of H2B-GFP and BrdU LRCs
in the anal epithelium. We labeled the tissue with
BrdU for five days (P22-P26) and chased until 43
or 56 days of age. Simultaneously, we turned off
the H2B-GFP expression by feeding the mice with
doxycycline (fig. 4a). Before the chase, immuno-
fluorescence staining of perianal skin and anorectal
frozen sections indicated that most of the cells were
labeled with BrdU (fig. 4b and c). After three
weeks of chase, BrdU and H2B-GFP LRCs were
found in the bulge area of the perianal hair follicle
(fig. 4d) and in the anorectal transition zone
(fig. 4e). This pattern of expression was con-
sistent in all mice analyzed (n = 6). Due to the
requirement that cells be actively undergoing
DNA synthesis to incorporate BrdU,42 not all of
the H2B-GFP LRCs colocalized with the BrdU
Figure 2. Differentiation markers of the adult mouse anal canal and the anorectal
junction. (A–D) Immunofluorescence analysis for the indicated markers. Differentia-
tion markers of the anal canal include Keratin 6, Keratin 10, Loricrin and Filagrin.
Keratin 8 marks the simple epithelium of the rectum. DApI labels all the nuclei in blue.
BL, basal layer; Gr, granular layer, Sc; stratum corneum; K6, Keratin 6; K8, Keratin 8; K10,
Keratin 10; α6, α6 integrin; K5, Keratin 5.
Figure 3. In vivo detection of label-retaining cells in the anorectal junction. (A–D)
Anorectal sections of ptRe-H2B-GFpxK5ttA mice before (26 days old) and after 4 week
chase (56 days old). Shown are epifluorescence of H2B-GFp (green) and 4’,6’-diamidino-2-
-phenylindole (DApI) (blue) and indirect immunofluorescence with indicated antibodies
(Rhodamine Red). (A and B) Before the chase, all Keratin 5 expressing cells and their
progeny show nuclear GFp expression in the perianal skin, hair follicles (A) and anal
epithelium. (B) Note that the rectum is GFp-negative. (C and D) GFp-positive cells are
retained in the anal region adjacent to the dentate line (anal transition zone). Keratin
17,58 is found expressed at the anal transition zone in adult mice. p26, 26 days old; p56,
56 days old; K17, Keratin 17. the asterisk denotes autofluorescence.
3042 Cell Cycle Volume 9 Issue 15
factor involved in embryonic stem cell main-
tenance51,52 and in stratified squamous epi-
thelium such as the esophagus53 is strongly
expressed in the basal layer of the anal epi-
thelium and its expression is decreased upon
differentiation. In contrast to p63, Sox2 is not
expressed in the anal gland (fig. 5d and e).
slow-cycling h2b-GfP anorectal lrcs
give rise to differentiated anal epithelium. To
track the fate of anal LRCs during the differ-
entiation of the anal epithelium, we monitored
GFP fluorescence intensities relative to those of
the differentiation-associated markers Keratin
6 (K6), Filagrin and Keratin 10 (K10) (fig.
6). Over-exposure of the anorectal sections,
from the K5-TetVP16xTRE-H2B-GFP double
transgenic mice chased for four weeks (P56),
verified that suprabasal and differentiated cells
were largely GFP-positive, deriving from anal
LRCs. We conclude that there is a small popu-
lation of anal LRCs at the anorectal transitional
epithelium that may give rise to progeny of the
label-retaining cells at transitional epithe-
lium. The identification and characterization
of anal stem cells would significantly advance
our understanding of the biology of the anal
epithelium in health and disease. However, the
proliferative compartment within the mouse
anal epithelium remains poorly characterized
despite the clinical importance of this tissue.
The mouse anal epithelium can be divided into
two compartments: a superficial stratified layer
consisting of large, differentiated and keratiniz-
ing squamous cells, and a basal layer composed of densely packed
columnar cells. Transitional epithelia are defined by the abrupt
change from one type of epithelium to another. Whether or not
there are stem cells present in the transition zone responsible for
maintaining the anal epithelium has never been investigated.
In other stratified epithelia (such as the epidermis of the skin
and esophagus) stem cells have been shown to reside in the basal
layer.20,54 We have identified a rare population of slow-cycling
cells that retains both H2B-GFP and BrdU for prolonged chase
times (3–4 weeks) and resides in the basal layer of the anal epi-
thelium, at the transition between the stratified anal epithelium
and the simple epithelium of the rectum. The anal epithelium
is naturally more proliferative than the perianal skin; therefore,
it is not surprising that H2B-GFP LRCs may be detected fol-
lowing shorter chase periods than their counterparts in the hair
follicle bulge. No LRCs were found in epithelial cells of the anal
canal close to the perianal skin.
label-retaining cells and stem cell markers. Epithelial
stem cells are frequently described as part of a population of
positive cells, as the K5-TetVP16xTRE-H2B-GFP pulse stage is
cell cycle independent. These results clearly indicate the presence
of a population of LRCs in the mouse anal transition zone.
slow-cycling cells express stem cell markers. To determine
if the anal H2B-GFP LRCs might express stem cell markers, we
conducted immunofluorescence microscopy on anorectal frozen
sections with antibodies directed against a number of adult stem
cell markers. Similarly to the hair follicle stem cell niche,39,43,44 we
found that the surface marker CD34 is expressed in a subset of
H2B-GFP LRCs (fig. 5a). p63, a transcription factor involved
in maintenance of the stratified squamous epithelia and essential
for the proliferative potential of their stem cells,45-47 is expressed
throughout the entire anal epithelium and in the anal glands,
but not in the rectum (fig. 5b–d). In contrast to the hair fol-
licle bulge,48 Keratin 19 marks the simple epithelium of the rec-
tum and is absent in the stratified epithelium of the anal canal
(fig. 5c). Other bulge stem cell markers, such as Keratin 15,
Lhx2 and Sox9,40,49,50 are not detected by immunofluorescence
in the anal epithelium (data not shown). Sox2, a transcription
Figure 4. H2B-GFp retaining cells colocalize with BrdU-retaining cells. (A) Schematic of BrdU
and doxy pulse-chase experiments. Small arrows represent ten intraperitoneal BrdU injec-
tions at the indicated time points. (B–e) Colocalization of BrdU and H2B-GFp before (B and
C) and after the chase (D and e). Before the chase, at 26 days old (p26), all the epithelial cells
expressed H2B-GFp and most of the cells (including the cells in the rectum) are labeled with
an anti-BrdU antibody showing the efficiency of the BrdU pulse (B and C). After the chase,
white arrows indicated label-retaining cells in the bulge of the perianal hair follicle (D) and
in the basal layer of the anal tZ (e). (e’) Higher magnification of the anal tZ, showing colocal-
ization of H2B-GFp and BrdU-retaining cells. SG, sebaceous gland; Bu, Bulge; tZ, transition
zone; the asterisk denotes autofluorescence.
www.landesbioscience.com Cell Cycle 3043
Keratin 6 (a generous gift from Dr. Elaine Fuchs, 1/500),
Keratin 10 (Covance, Emeryville, CA, 1/1,000), Keratin 8
and Keratin 19 (these antibodies, developed by Dr. Brulet and
Dr. Kemler, were obtained from the NICHD Developmental
Studies Hybridoma Bank maintained by the University of Iowa),
Loricrin (Covance, Emeryville, CA, 1/500), Filagrin (Covance,
Emeryville, CA, 1/1,000), CD34 (eBiosciences Inc., San Diego,
CA, 1/50), P-cadherin (R&D Antibodies, North Las Vegas,
NV, 1/100), CD49f (BD Biosciences, San Jose, CA, 1/100), p63
(Santa-Cruz Biotechnology Inc., Santa Cruz, CA, 4A4, 1/50),
Sox2 (a generous gift from Dr. Jeffrey Whittsett, 1/5,000).
4',6-diamidino-2-phenylindole (DAPI) was utilized as a marker
of cell nuclei (Sigma Chemical Co., St. Louis, MO, 1/5,000).
Secondary antibodies conjugated with FITC or Rhodamine
(Jackson ImmunoResearch Laboratories Inc., West Grove, PA)
were used at a dilution of 1/250.
slow-cycling cells or LRCs, based upon the fact that
stem cells divide less frequently than other differentiated
cells.10,24 Therefore, the anatomical finding of LRC dis-
tribution in the anal epithelium is useful in predicting
stem cell distribution because it is likely that many of the
LRCs are in fact stem cells.6 For example, slow-cycling
cells detected in the limbus at the transition zone of the
cornea with the conjunctiva24,25 have been largely dem-
onstrated to be stem cells.55
We found that the anal slow-cycling cell population
expresses the surface marker CD34, which is expressed
by a variety of pluripotent cells and tissue stem cells56
including hair follicle bulge stem cells,39 esophageal stem
cells20 and muscle satellite cells.57 Moreover anal slow-
cycling cells also expressed several other markers con-
sistent with known stem cells.46,51,52,56 As in other stem
cell niches, we found heterogeneity in the LRC and the
presence of stem cell marker such as CD34.7
Further analysis will determine the properties of the
anal LRCs. Mouse anal epithelial cell culture studies
and anorectal injury models will be required to directly
address the stem cell potential of the anal LRCs.
Materials and Methods
electron microscopy. Anorectal regions from adult CD-1
mice were dissected and fixed in 2% glutaraldehyde, 4%
paraformaldehyde and 2 mM CaCl2 in 0.05 M sodium
cacodylate buffer, pH 7.2 at 4oC for one hour. The sam-
ples were then post fixed in 1% osmium tetroxide in 0.2
M sodium cacodylate buffer, processed through a graded
series of alcohols, infiltrated and embedded in LX-112
resin. After polymerization at 60oC for three days, ultra-
thin sections (100 nm) were cut using a Reichert-Jung
Ultracut E microtome and counterstained in 2% aque-
ous uranyl acetate and Reynolds lead citrate. Images were
taken with a transmission electron microscope (Hitachi
H-6750) equipped with a digital camera (AMT 2k x 2K
histology and immunolabeling. One percent tolu-
idine blue O was prepared in 1% sodium borate solution and used
to stain 1 µm anorectal sections. Cryostat sections (10 µm) of
mouse anorectal regions were fixed for 10 min in freshly prepared
4% paraformaldehyde in 1X phosphate-buffered saline solution
(1X PBS) and washed 3 times for 10 min in 1X PBS at room
temperature. Sections were then permeabilized in 0.1% Triton X
100 for 10 minutes and non-specific staining was eliminated via
the following blocking solution: 2.5% normal goat serum, 2.5%
normal donkey serum, 2% gelatin, 0.1% Triton X 100 and 1%
bovine serum albumin in 1X PBS. Whenever mouse monoclo-
nal antibodies were utilized, the MOM kit (Vector Laboratories,
Burlingame, CA) was used.
Primary antibodies against the following proteins were used
at the dilution indicated: bromodeoxyuridine (Abcam, 1/200),
Keratin 5 (Seven Hills Bioreagents, Cincinnati, OH, 1/2,000),
Keratin 17 (a generous gift from Dr. Pierre Coulombe, 1/5,000),
Figure 5. expression of stem cell markers at the anorectal junction. (A) CD34 is
expressed at a low level in the H2B-GFp retaining cells (LRCs). High expression of
CD34 is detected in the stroma surrounding the anal region. Boxed area is magni-
fied and shown to the right, demonstrating CD34 expression in the GFp-retaining
cells. (B) p63 is expressed through the entire basal layer of the anal epithelium
and the anal glands, colocalizing with CD34 in a small group of cells at the anorec-
tal transitional zone (indicated with white arrows). Boxed area enlarged in inset,
showing the expression of CD34. (C) Keratin 19 is expressed in the simple epitheli-
um of the rectum and not in the anal transitional cells. (D and e) Sox2 is expressed
in the basal cells of the anal epithelium (including the anal transition zone) and its
expression decreases as cells differentiate. Sox2 and p63 are not expressed in the
rectum. K19, Keratin 19; LRCs, H2B-GFp-retaining cells; α6, α6-integrin.
3044 Cell Cycle Volume 9 Issue 15
mice and labeling experiments. All experiments
were approved by the Cincinnati Children’s Hospital
Research Foundation IACUC and carried out using
standard procedures. We generated Tet-off H2B-GFP
mice by crossing heterozygous K5tTA (FVB) mice with
pTREH2B-GFP (CD1) mice37 and identified animals
expressing GFP with a blue led flashlight (Tektite) and
amber eyeglasses (EyeSave Sunglasses and Readers).
Mice were fed with 1 g/Kg of Doxycycline food
(Harlan) to repress H2B-GFP expression. For label
retention studies, 5-bromo-2-deoxyuridine (BrdU,
Sigma-Aldrich, St. Louis, MO) was injected intraperi-
toneally to a final concentration of 50 µg/g of body
weight starting at postnatal days P22-P26 at 12 h
intervals, and was subsequently added to the drinking
water (0.8 mg/ml) during the pulse period. A total of
six mice were analyzed.
We thank Dr. James Lessard for valuable discus-
sions, Drs. Elaine Fuchs, Pierre Coulombe and Jeffrey
Whittsett for providing their antibodies. We thank
Dr. Adrian McNairn for valuable criticism of the
manuscript. We also thank Dr. Amalia Pasolli for her
advice on the electron microscopy procedures. This
work was funded by CCHMC Trustee Grant Award
and part by PHS Grant P30 DK 078392 (G.G.).
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