CD34 glycoprotein identifies putative stem cells located

in the isthmic region of canine hair follicles

Blackwell Publishing Ltd

Luisa Pascucci*, Francesca Mercati*,

Anna Maria Gargiulo*, Vera Pedini*,
Silvia Sorbolini and Piero Ceccarelli*
*Dipartimento di Scienze Biopatologiche ed Igiene delle Produzioni
Animali ed Alimentari Facolt di Medicina Veterinaria, Universit di
Perugia, Perugia, Italy
Dipartimento di Patologia, Diagnostica e Clinica Veterinaria Facolt
di Medicina Veterinaria, Universit di Perugia, Perugia, Italy
Correspondence: Luisa Pascucci, Dipartimento di Scienze
Biopatologiche ed Igiene delle Produzioni Animali ed Alimentari
Facolt di Medicina Veterinaria, Via S.Costanzo, 4 06126, Perugia,
Italy. Fax: +39-75-5857631; E-mail: luipas@unipg.it

What is known about the topic of your paper

There is general agreement in considering the bulge
region of humans and mice hair follicle as the elective
site of a pool of multipotent stem cells.
CD34 glycoprotein is a defining hallmark of
haematopoietic stem and progenitor cells.
A strong relationship between CD34+ bulge
keratinocytes and multipotent follicular stem cells
has been recently demonstrated in mice.
What your paper adds to the field of veterinary
dermatology
In our work, for the first time, the stem cell marker
CD34 was tested to evaluate the location of hair
follicle stem compartment in dogs.
In this study, we demonstrated that CD34 identifies a
discrete subpopulation of keratinocytes residing in a
bulge-like region of the canine hair follicles.
Our results strongly suggest that CD34+ keratinocytes
in dogs are potentially related to the stem
compartment of this skin appendage.

Abstract
It is widely documented that a pool of multipotent
stem cells located in humans and mice hair follicle
outer root sheath (bulge region) is involved in the
restoration of the whole follicular unit during each
anagen phase. To the authors knowledge, data regarding the location and characterization of hair follicle
stem compartment in dogs have not been reported in
the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor
cell antigen CD34 as a marker of putative stem cells
located in a bulge-like region of canine hair follicles.
The presence of CD34 mRNA and glycoprotein was
assessed on formalin-fixed, paraffin-embedded canine
skin samples by in situ hybridization technique and
by standard immunohistochemistry, respectively. A
strong expression of CD34 mRNA and glycoprotein
was observed in a well-defined area of the hair follicle
244

isthmic region and appeared uniformly concentrated

at the level of the basal layer of the outer root sheath.
These findings provide compelling support to the
hypothesis that in dogs, a subpopulation of basal
keratinocytes located in the hair follicle isthmic region
and characterized by the selective expression of CD34
is potentially associated with the stem cell compartment
of this skin appendage.
Accepted 12 May 2006

Introduction
The hair follicle is a highly attractive and fascinating model
for the study of stem cell biology. Its easy accessibility,
cyclical growth, and the periodical reactivation of its stem
compartment, make the hair follicle an excellent target
for studying the dynamics of somatic stem cells and for
testing their isolation and manipulation techniques.13
In humans and mice, a specialized region of the midhair follicle called bulge is thought to be the main repository of cells with stem properties.48
This area, prominent in embryonic hair follicles, but
relatively inconspicuous in adult hair follicles, has been
described as a swelling of the isthmic outer root sheath
(ORS) extending below the opening of the sebaceous gland
duct to the insertion of the arrector pili muscle. The bulge
marks the lower end of the permanent region of the follicle
that appears to be unaffected by dramatic changes occurring
below this area during the hair growth cycle.4,7,8
Several lines of evidence make bulge cells the ideal follicular
stem cell candidates. They are rarely cycling cells and this
feature has been demonstrated by pulse-chase experiments
with labelled nucleotides showing that slow cycling bulge
cells retain the label (label-retaining cells) in contrast to the
rapidly dividing cells.4 Once cultured in vitro, bulge cells
yield larger colonies than those from any other follicular
site, suggesting that most cells with high proliferative
potential reside in the bulge region.912 They are relatively
undifferentiated showing an elementary ultrastructural
morphology13 and reside in a typical niche within the hair
follicle protecting them not only from normal hair cycling
changes, but also from environmental injuries.14 When taken
outside of their native niche and induced to proliferate
in vitro, bulge cells withstand multiple passages giving
rise to increasing number of holoclones (self-renewal).15
Moreover, when grafted in vivo, donor bulge cells and
the progeny derived from a single bulge cell amplification
(holoclone) are able to repopulate the entire new hair follicle
and the sebaceous gland in normal homeostasis, and
the epidermis after injury (multipotency).10,12,15,16 Finally, it

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology. 17; 244251

CD34 expression in canine hair follicles

has been recently demonstrated that follicular bulge cells

are pluripotent and can trans-differentiate into Schwann
cells.17
By the activation and proliferation of quiescent bulge
keratinocytes in early anagen, a pool of rapidly dividing
progenitor cells (transit amplifying) is derived that populate
the follicular matrix and give rise to the new hair follicle
(bulge activation hypothesis).18 According to the predetermination hypothesis, the bulge region is the source of a
group of cells that migrate downward along the ORS
during the mid-anagen and locate at the base of the hair
bulb as a distinct apoptosis-resistant population known
as the lateral disc. Under the influence of follicular papilla
growth factors, they transform into the hair germ and give
rise to the ascending part of the hair follicle (hair shaft and
inner root sheath), whereas bulge-located stem cells and
their progeny produce the descending ORS.3
Several of the potential candidate molecules proposed
as putative markers of hair follicle stem cells are 1 integrin,19
cytokeratin 15,5,20,21 cytokeratin 19,22,23 6-integrin,24 CD71,25
p63,26 S100A,27 Caveolin-1,28 CD200,29 PHLDA1,29 follistatin,29
frizzled homolog 1,29 nestin,30 and CD34 glycoprotein.7,31
CD34 glycoprotein is a glycosilated transmembrane protein considered as a defining hallmark of haematopoietic
stem and progenitor cells. It is believed to act as a stagespecific antigen as its expression is progressively lost
during cell maturation.3133
In addition to being expressed on stem and progenitor
cells during haemopoiesis, CD34 glycoprotein has been
demonstrated in several non haematopoietic tissues, including satellite cells of skeletal muscles,34,35 interstitial cells
of Cajal in the gastrointestinal tract,36 vascular endothelium,37
neuronal bodies of the brain,38 dermal dendritic cells39 and
numerous neoplasias such as angiosarcoma, solitary fibrous
tumours, epithelioid sarcomas, spindle cell lipomas, myofibroblastomas, dermatofibrosarcoma protuberans and stromal
spindle-shaped cells in Kaposis sarcoma.40 43 Recently,
studies have shown the expression of CD34 glycoprotein
in murine bulge keratinocytes, demonstrating the value
of this molecule as a marker of the stem cell-containing
follicular compartment.7 Therefore, the aims of this study
were to investigate the expression and distribution of CD34
glycoprotein in canine hair follicles and to evaluate the
existence of a bulge-like area in this species. The presence
of CD34 mRNA and glycoprotein was assessed by in situ
hybridization technique and by standard immunohistochemistry, respectively.

Materials and methods

Tissues collection and preparation
Three male and seven female healthy dogs were selected
for sample collection. Their ages ranged from 3 to 9 years
and their breeds included miniature pinscher (2), doberman pinscher (1), boxer (1), beagle (1), Labrador retriever
(1) and mixed breed (4). Fresh normal skin samples were
collected from the dorsal neck, cheek and abdominal region
of each dog by excisional biopsy. Immediately after collection, samples were fixed in 10% neutral-buffered formalin,
dehydrated and embedded in paraffin wax for light microscopic procedures. Fivemicrometre-thick sections were
mounted onto poly-l-lysine-coated glass slides and air dried

for 2 h at 37 C. The slides were stored at 4 C in a dry

atmosphere until further processing.
In situ hybridization
CD34 riboprobes synthesis
Canine CD34 cDNA cloned in PCDM8 vector (Invitrogen,
Carlsbad, CA, USA) was kindly provided by Dr Richard Nash
(Fred Hutchinson Cancer Research Center, Seattle, WA,
USA). The canine CD34 sequence was published by Mc
Sweeney et al. in 1996 (NCBI, Accession no. U49457).44
To allow the synthesis of sense and antisense riboprobes,
the region spanning two internal primers 5-TGACACCCCAAGTACCATCA-3 (dog CD34 2a) and 3-TGCGGGAATAGCTCTGGTG-5 (dog CD34 2c) was amplified by polymerase
chain reaction (PCR). The selected region, corresponding to
nucleotides 513877 of CD34 coding sequence, codifies
for a tract of the amino acid sequence located in the extracellular domain of the protein.44 The PCR fragment was
subcloned into pCR4-TOPO (TOPO TA Cloning Invitrogen)
and then sequenced to verify the orientation of the insert.
The region extending from T3 primer and dog CD34 2a
(antisense filament) and the region extending from T7 primer
and dog CD34 2c primer (sense filament) were amplified by
PCR. The purified 417 pb products were used to synthesize
antisense and sense digoxigenin (DIG)-labelled riboprobes.
The transcription was performed with a DIG RNA labelling
kit (Roche Molecular Biochemicals, Mannheim, Germany)
according to the manufacturers protocol. To assess the
efficiency of DIG-labelled nucleotide incorporation, the
probes were spotted on a nylon membrane, detected with
sheep anti-DIG-alkaline phosphatase-conjugated antibody
(Roche Molecular Biochemicals) and revealed using 5-bromo4-chloro-3-indolyl-phosphate (BCIP)/nitro blue tetrazolium
(NBT) as chromogen substrates for alkaline phosphatase.
Hybridization
CD34 mRNA distribution was assessed as described by
Braissant and Wahli with minor modifications.45 Deparaffinated skin sections were permeated using 1 g mL1
proteinase K (Sigma-Aldrich Co., St. Louis, MO, USA) in 0.05
mol L1 Tris-HCI pH 7.5 for 20 min at 37 C and acetylated
in a solution of 0.25% acetic anhydride in 50 TAE (Tris
acetateEDTA) buffer at room temperature for 10 min. After
rinsing in distilled water, the sections were dehydrated
through 70, 96 and 99% ethanol steps. Thereafter, sections
were air dried for 1 h at room temperature and incubated
with a 150250-L hybridization mixture prepared with
500 ng mL1 CD34 DIG-labelled sense or antisense probe
in hybridization buffer (50% formamide, 300 mmol L1 NaCl,
10 mmol L1 Tris pH 7.5, 1 mmol L1 EDTA, 1 Denhardts
solution, 10% dextran sulphate, 70 mmol L1 DTT, tRNA
denaturated at 85 C, 500 g mL1 Poly A denaturated
at 85 C) and were denatured at 70 C for 1 min. Slides
were covered with coverslips and incubated overnight
at 45 C in a moist chamber containing 50% formamide
and 2 sodium chloride-sodium acetate buffer (SSC). After
hybridization, the coverslips were carefully removed in 2
SSC. The slides were then subjected to three high stringency
washes in 0.02 SSC for 25 min at 55 C and treated with
20 g mL1 RNAseA (Roche Molecular Biochemicals) in
NTE (NaCl, Tris HCl, EDTA) buffer for 30 min at 37 C.

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Pascucci et al.

Immunological detection
After rinsing in distilled water, the sections were treated with
buffer 1 (100 mmol L1 Tris pH 7.5, 150 mmol L1 NaCl, 1%
blocking reagent) for 1 h and with buffer 2 (100 mmol L1
Tris pH 7.5, 150 mmol L1 NaCl, 0.5% fetal bovine
serum, 0.3% Triton 100) for 30 min at room temperature.
For immunological detection, the sections were incubated
with sheep anti-DIG-alkaline phosphatase-conjugated
antibody (Roche Molecular Biochemicals) diluted to 1 : 1000
in buffer 2 for 2 h at room temperature in a moist dark
chamber. The slides were then washed in NT (100 mmol L1
Tris pH 7.5, 150 mmol L1 NaCl) and incubated in buffer 3
(100 mmol L1 Tris pH 9.5, 100 mmol L1 NaCl, 50 mmol L1
MgCl2) for 10 min. The reaction was developed using BCIP
and NBT as chromogen substrate for alkaline phosphatase.
The alkaline-phosphatase reaction was stopped by 10 mmol
L1 TrisHCl pH 8.0 and 1 mmol L1 EDTA.
Immunohistochemistry
The immunohistochemical demonstration of CD34 antigen
was performed using standard techniques. After deparaffinization with xylene and hydration, endogenous peroxidase
activity was quenched with 3% hydrogen peroxide in water
for 10 min. The slides were then treated with normal goat
serum (Dako Corporation, Carpinteria, CA, USA) 1 : 10 and
incubated overnight at room temperature with a mouse
anticanine CD34 biotinylated monoclonal antibody (BD
Biosciences-San Jos, CA, USA) at a dilution of 1 : 50. Bound
antibodies were sequentially reacted with an avidinbiotin
peroxidase complex (Vector Laboratories, Peterborough,
UK). The reaction was finally visualized by exposure to fresh
diaminobenzidine chromogen substrate solution (Dako
Corporation, Carpinteria, CA, USA) according to the
manufacturers instructions. Negative controls without
the primary antibody were run in parallel.

Results
In a large number of hair follicles from each examined skin
sample, a slight swelling of the ORS variably extending
throughout the isthmic tract of the hair follicle was evidenced
(Fig. 1a). It consisted essentially of cubic-shaped basal
keratinocytes characterized by a large nucleus and a subtle
cytoplasm. This structure, topographically resembling the
bulge region previously described in humans and mice,
appeared to be present only in primary follicular units.
In situ hybridization
In situ hybridization of skin samples from healthy adult dogs
with CD34 antisense riboprobe, revealed the presence
of abundant CD34 transcripts in the ORS keratinocytes of
the isthmic region (Fig. 1a). CD34 mRNA was observed in
a wide area of the hair follicle, extending below the region
of the sebaceous gland opening to the site of insertion of
the arrector pili muscle (Figs 2 and 3). CD34-positive cells
were clearly detectable in the basal cell layer of the ORS
(Fig. 4) both in the growing and in the regressive to resting
phases of hair follicle growth cycle. In the latter one (telogen
hair follicle) the reaction clearly involved the residual lower
portion of the hair follicle surrounding the club hair. Dermal
papilla cells were always negative (Fig. 5a). CD34 mRNA was
always detected in the subnuclear cytoplasm and was mostly
246

Figure 1. (a) Low power magnification of a longitudinal-cut anagen

hair follicle. Arrows point to bilateral swelling of the outer root sheath
at the level of the isthmic region. Reactivity of basal keratinocytes to
CD34 riboprobe appears as a dark purple outline profiling the follicular
wall. Note the lack of labelling in the bulb area. The epithelial structure
on the right showing a strong signal represents the wall of an irregularly
cut hair follicle. In situ hybridization with CD34-specific riboprobe.
Bar = 300 m. (b) Square bracket encloses the mid-tract of a
longitudinal-cut anagen hair follicle where CD34+ cells are located
(brown). Note the lack of labelling in the bulb area. Immunostaining
with anti-CD34 antibody. Bar = 300 m.

Figure 2. Anagen hair follicle, longitudinal section.The CD34+

keratinocytes (purple) are located in a bilateral swelling of the outer
root sheath just below the sebaceous gland. In situ hybridization with
CD34-specific riboprobe. HC, hair canal; ORS, outer root sheath;
SG, sebaceous gland. Bar = 40 m.

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.

CD34 expression in canine hair follicles

Figure 3. Longitudinal-cut hair follicle. The arrector pili muscle

insertion (arrow) is clearly detectable just beneath the lower limit of
CD34+ tract (arrowheads). In situ hybridization with CD34-specific
riboprobe. APM, arrector pili muscle; HC, hair canal; ORS, outer root
sheath; asterisks (*), inner root sheath. Bar = 150 m.

concentrated inside cellular basal expansions (Fig. 6). Primary

hair follicles showed repeated reactivity, while secondary
ones were negative (Fig. 7). The expression of CD34 mRNA
appeared unaffected by body region. No or little variability
in the intensity of labelling was observed among the
tested animals. The sense probe produced no signal.
Immunohistochemistry
The monoclonal antibody anti-CD34 reacted consistently
with the ORS cells of canine hair follicles. Staining was limited
to the follicular isthmic region (Fig. 1b) and involved the
basal layer of the ORS (Fig. 8). The suprabasal layers were
essentially negative although, occasionally, the layer
immediately above basal cells was discontinuously reactive
(Fig. 9).The CD34-positive cells showed remarkably sharp

Figure 4. Longitudinal-sectioned hair follicle. Labelled riboprobe

localizes at the subnuclear cytoplasm of basal keratinocytes in the
outer root sheath. In situ hybridization with CD34-specific riboprobe.
HC, hair canal; ORS, outer root sheath; BL, basal layer; FS, fibrous
sheath. Bar = 12 m.

staining, demarcating the lateral and apical cytoplasmic

membrane. The reaction was never detected at the
level of the basal cellular membrane (Fig. 9). Expression of
CD34 glycoprotein appeared prominent during the whole
hair growth cycle. Telogen hair follicles, in particular, showed
that CD34 expression was restricted to the permanent
isthmic tract of the hair follicle that is not affected by
catagen-associated apoptotic changes (Fig. 5b).

Discussion
In this study, the authors examined adult canine hair follicles
and determined that a discrete subpopulation of basal
keratinocytes, located in the follicular isthmic region,
specifically expressed CD34 glycoprotein. The techniques
used to evaluate CD34 mRNA and glycoprotein expression

Figure 5. Low power magnification of a longitudinal-cut telogen hair follicle showing CD34+ cells in the basal layer of the outer root sheath
surrounding the club hair (arrowheads). (a) In situ hybridization with CD34-specific riboprobe. (b) Immunostaining with anti-CD34 antibody.
APM, arrector pili muscle; H, club hair; DP, dermal papilla; P, pigment granules. Bar = 100 mm.

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.

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Pascucci et al.

Figure 6. Longitudinal-sectioned hair follicle. High power magnification

of outer root sheath basal keratinocytes. The signal is concentrated
inside basal cytoplasmic protrusions. In situ hybridization with
CD34-specific riboprobe. N, nucleus; BP, basal protrusions;
FS, fibrous sheath. Bar = 5 m.

Figure 8. Longitudinal-sectioned anagen hair follicle. At the centre of

the field, a large pigmented hair can be observed. At the sides of the
hair shaft, note the symmetric reactivity of the outer root sheath basal
layer to anti-CD34 antibody. Immunoistochemistry. H, hair shaft; HC,
hair canal; ORS, outer root sheath. Bar = 45 m.

Figure 9. High power magnification of longitudinal-sectioned follicular

wall. Positive keratinocytes of the basal layer show an intense and
clear-cut membrane reactivity limited to the lateral and apical boundaries
of the cells. Note the presence of several reactive cells in the suprabasal
layer of the outer root sheath. Immunostaining with anti-CD34 antibody.
N, nucleus; BL, basal layer; FS, fibrous sheath; H, hair shaft. Bar = 10 m.

Figure 7. This figure demonstrates the negative reactivity of

secondary hair follicles and the positive bilateral staining (arrows) in
the outer root sheath of a primary hair follicle. SHFs, secondary hair
follicles; PHF, primary hair follicle; H, hair shaft. In situ hybridization
with CD34-specific riboprobe. Bar = 250 m.

248

provided overlapping results concerning the area of reactivity

that was characteristically associated with an enlargement
of the mid-tract of the follicular wall. These findings substantiate results reported by Trempus et al.,7 who described
the expression of the haematopoietic stem and progenitor
cell marker CD34 in mouse bulge keratinocytes.
The reaction pattern of individual keratinocytes differed
in accordance with the technique used. The pattern of
riboprobe-labelled skin sections was cytoplasmic with a
clear localization of the signal at the basal pole of keratinocytes. This distinct subnuclear localization of CD34 mRNA
might be associated with a particular compartmentalization of the intracellular protein synthetic apparatus. The
reactivity of anti-CD34 antibody that sharply demarcated the
latero-apical cytoplasmic membrane of positive keratinocytes

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.

CD34 expression in canine hair follicles

could be explained by the function of CD34 glycoprotein

as an adhesion molecule.46 This particular location of the
signal could suggest that CD34 plays a role in the adhesion
between undifferentiated and early differentiated cells as
its expression is completely lost beyond the layer above
basal cells. The absence of reactivity of the basal cellular
membrane seems to exclude any involvement of CD34
in the physical maintenance of stem cells inside the niche
via a keratinocyte to basement membrane attachment. It
should be emphasized that CD34 expression was restricted
to the permanent portion of the ORS that is not affected
by apoptosis-driven catagen involution.
In this study, CD34 expression was limited to primary
hair follicles. As demonstrated in several recent studies,
multipotent stem cells, located in the permanent region of
vibrissal and pelage hair follicles, are able to regenerate all
epithelial lineages of hairy skin suggesting the possible
existence of a pilosebaceous epithelial unit (PSU).10,12,1416
Based on these observations we hypothesize that in canine
compound hair follicles, a unique stem compartment located
in primary hair follicles could be the source of multipotent stem
cells even for secondary follicular units within the same group.
A recent report carried out in mice emphasized the
strong relationship between CD34+ keratinocytes and
follicular stem cells. This study showed that in addition to
their label-retaining properties, bulge cells expressing high
levels of CD34 glycoprotein are able to self-renew in vitro
and, when grafted, they are responsible for regenerating
pilosebaceous units and epidermis (multipotency). The
attributes of self-renewal and the multipotency of CD34+
bulge cells have led numerous investigators to consider
this glycoprotein as a reliable marker of follicular stem
cells.15 In contrast, Ohyama et al. recently demonstrated
that CD34 expression is low or absent in human bulge
ORS keratinocytes, suggesting that this molecule is not
a marker of the human bulge area where follicular stem
cells are thought to reside. Divergent CD34 expression
between humans and mice suggests that substantial
molecular and biological differences exist in bulge keratinocytes among mammalian species.29
In summary, the expression of CD34 mRNA and glycoprotein in canine hair follicles is limited to a group of basal
keratinocytes of the ORS that are localized in a restricted,
slightly enlarged area of the follicle between the sebaceous
gland opening and the arrector pili muscle insertion. These
findings strongly support the hypothesis that in canine hair
follicles a bulge-like region does exist where a population
of biochemically distinct follicular keratinocytes resides.
The authors believe that these cells, distinguishable on
the basis of CD34 expression, could be considered the
putative stem cells of canine hair follicle. Moreover, the
results of this study add new insight into the investigation
of CD34 stem cell-related molecule in hair follicles, providing
some evidence that, as previously demonstrated in mice,
CD34 may be considered a reliable marker of follicular
stem compartment even in dogs.

Acknowledgement
The authors would like to acknowledge Dr Richard Nash of
the Fred Hutchinson Cancer Research Center (Seattle, USA)
who kindly provided the canine CD34 cDNA.

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Rsum Un pool de cellules souches multipotentes existe dans la rgion du bulbe de la gaine folliculaire
externe chez lhomme et chez la souris, qui correspond la zone de restauration de lunit folliculaire
pendant chaque phase anagne. A la connaissance des auteurs, lidentification dune telle rgion na pas
t rapporte chez le chien dans la littrature rcente. Dans cette tude, nous avons utilis lantigne CD34
comme un marqueur possible des cellules souches localises dans la rgion du bulbe des follicules pileux
canins. La prsence dARNm de CD34 et de glycoprotines a t recherche sur des biopsies paraffines,
fixes dans le formol, par tude dhybridation in situ et par immunohistochimie. Une forte expression
dARNm CD34 et de glycoprotines a t observe dans une zone bien dfinie de la rgion de listhme
folliculaire, localise trs uniformment au niveau de la couche basale de la gaine folliculaire externe. Ces
rsultats soutiennent lhypothse que chez le chien, une sous-population de kratinocytes basaux localiss
dans la rgion de listhme du follicule pileux et caractriss par une expression slective du CD34, est
potentiellement associe au compartiment souche de cette annexe cutane.
Resumen Ha sido ampliamente documentada la existencia de un grupo de clulas madre multipotentes
en humanos y ratones, localizadas en la vaina externa de la raiz del pelo (zona del bulbo). Estas clulas juegan
un papel relevante en la restauracin completa de la unidad folicular durante la fase angena. Sin embargo,
basado en nuestros conocimientos, no existe ningn dato en la literatura reciente con respecto a la localizacin
y caracterizacin del compartimento de clulas madre en el perro. En este estudio hemos investigado el
antgeno de clulas madre hematopoiticas y clulas progenitoras CD34 como un marcador de posibles
clulas madre localizadas en la regin equivalente a la regin del bulbo en los foliculos pilosos del perro.
La presencia de ARN mensajero para CD34 y de la glicoprotena fue analizada en piel de perros fijada
con formalina y embebida en parafina. El anlisis se realiz utilizando las tcnicas de hibridacin in situ
e inmunohistoqumica, respectivamente. Una expresin elevada de ARNm para CD34 as como de la
glicoprotena se observ en una zona bien definida en el istmo del folculo piloso y pareca uniformemente
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CD34 expression in canine hair follicles

concentrada al nivel del estrato basal en la vaina radicular externa. Estos hallazgos contribuyen a reforzar la
hiptesis de que en perros, una subpoblacin de queratinocitos basales localizados en la regin del istmo
y caracterizados por la expresin de CD34, est potencialmente asociada con el compartimento de clulas
madre del folculo piloso.
Zusammenfassung Es ist weitlufig dokumentiert, dass ein Pool von multipotenten Stammzellen, die in
der ueren Wurzelscheide (Bulge Region) des menschlichen und des Muse Haarfollikels lokalisiert sind,
beim Wiederaufbau der gesamten Haarfollikeleinheit whrend einer jeden anagenen Phase beteiligt sind.
Nach dem Wissen der Autoren sind in der relevanten jngeren Literatur keine Ergebnisse bzgl. Lokalisation
und Charakterisierung der Haarfollikelstammzellen publiziert. In dieser Studie haben wir das hmatopoetische
Stamm- und Vorluferzellen Antigen CD34 als Marker der mutmalichen Stammzellen in einer Bulge-hnlichen
Region des caninen Haarfollikels untersucht. Das Vorhandensein von CD34 mRNA und Glykoprotein wurde
in Formalin fixierten und in Paraffin eingebetteten caninen Hautproben mittels in-situ Hybridisierungstechnik
bzw. mittels Standard-Immunhistochemie beurteilt. Eine starke Expression von CD34 mRNA und Glykoprotein
wurde in einem gut abgegrenzten Gebiet der Isthmusregion des Haarfollikels beobachtet und erschien
einheitlich konzentriert auf der Hhe der Basalmembran der ueren Wurzelscheide. Diese Ergebnisse
liefern zwingende Beweise fr die Hypothese, dass bei Hunden eine Subpopulation an basalen Keratinozyten,
die in der Isthmusregion des Haarfollikels lokalisiert und durch selektive Expression von CD34 charakterisiert
sind, mglicherweise mit den Stammzellen dieses Hautanhangs assoziiert sind.

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