role of stem cells in androgenetic alopecia
DESCRIPTION
Role of stem cells in androgenetic alopecia using immunohistochemistry markers as CK15, CD34 AND P63.TRANSCRIPT
الرحمن الله الرحمن بسم الله بسمالرحيمالرحيم
ل�م� ل�ن�ا ”” ل�م� ل�ن�ا قالوا سبحانك ال ع قالوا سبحانك ال ع�ن�ت� ن�ك� أ ت�ن�ا إ ا ع�ل�م� �ن�ت� إال م� ن�ك� أ ت�ن�ا إ ا ع�ل�م� إال م�
�كيم كيم�ال�ع�ليم� ال�ح� ““ال�ع�ليم� ال�ح�
العظيم الله العظيم صدق الله صدق((3232)البقرة:)البقرة:
الرحمن الله الرحمن بسم الله بسمالرحيمالرحيم
ل�م� ل�ن�ا ”” ل�م� ل�ن�ا قالوا سبحانك ال ع قالوا سبحانك ال ع�ن�ت� ن�ك� أ ت�ن�ا إ ا ع�ل�م� �ن�ت� إال م� ن�ك� أ ت�ن�ا إ ا ع�ل�م� إال م�
�كيم كيم�ال�ع�ليم� ال�ح� ““ال�ع�ليم� ال�ح�
العظيم الله العظيم صدق الله صدق((3232)البقرة:)البقرة:
BYKHALID M. GHARIB MDLECTURER OF DERMATOLOGY AND VENEREOLOGY ZAGAZIG UNIVERISTY
INTRODUCTIONINTRODUCTION
ANDROGENETIC ALOPECIAAndrogenetic alopecia (AGA) also referred to as
male-pattern hair loss, common baldness or female-pattern hair loss, is a slowly progressive form of alopecia that begins after puberty.
It affects at least 50% of men by the age of 50 years, and up to 70% of all males in later life.
AGA is characterized by androgen dependent, progressive, patterned hair loss from the scalp (Zhao and Hantash, 2011).
The pathogenesis of AGA is poorly understood. It is
assumed that the genetically predisposed hair
follicles are the target for androgen-stimulated hair
follicle miniaturization, leading to gradual
replacement of terminal hairs by barely visible,
depigmented vellus hairs.
The miniaturization process occurs over many
years during repeated hair cycles with shortened
anagen phase and hair follicle reduction in size and
depth (Trüeb , 2002).
Hair follicle is the main source of multipoint stem
cells in the skin. These stem cells reside in a
quiescent niche in bulge region near the insertion of
arector pill muscle. Bulge cells generate all the
epithelium lineages within the follicle and their
selective destruction leads to loss of hair follicle.
Also, bulge cells give rise to progenitor population
called secondary germ cells that reside adjacent to
bulge during telogen and produce the new hair shaft
at anagen onset (Zhang et al., 2009).
STEM CELL MARKERS
Recently, due to discovery of cell surface markers, it has become possible to characterize the hair follicle stem cells.
Cytokeratin15 is the best marker for bulge epithelial stem cells in human hair.
Other markers as Nestin, p63 and CD34 allow assessment of stem and progenitor cell populations in human scalp.
Several studies, reported loss of CK15+ cells in some
scarring alopecia, implying invovement of bulge
region in its pathogenesis (Tsuboi et al., 2012).
AGA represents the most common type of non
scarring alopecia; however permanent alopecia may
occur in long standing cases. This irreversibility of
the ability to grow hair again may give rise to the
possibility of stem cell invovement in this type of
alopecia (Al-Refu , 2012).
Recently, few studies have been investigating the status
of the hair follicle stem cell compartment in AGA.
Some studies suggested that compromising integrity of
follicular bulge area or sebaceous gland may play a role
(Lee and Lee, 2012).
While others suggested diminished conversion of hair
follicle stem cells to progenitor cells. whether these data
will assess the role of hair follicle stem cells in AGA is still
unknown.
AIM OF THE AIM OF THE WORKWORK
The aim of the present study was The aim of the present study was
to investigate the possible role of to investigate the possible role of
hair follicle stem and progenitor hair follicle stem and progenitor
cells in the pathogenesis of cells in the pathogenesis of
androgenetic alopeciaandrogenetic alopecia.
PATIENTS&METHODSPATIENTS&METHODS
This study has been conducted on 20 patients with AGA selected from the outpatient clinics of the Dermatology Department of Zagazig University Hospitals during the period from January 2011 to December 2012.
They were 17 males and 3 females with their ages ranging from 20 to 29 years with a mean of 24.05±1.6.
Diagnosis of AGA was clinically established in males by the characteristic distribution of frontal and vertex hair and in females by the Christmas tree pattern of diffuse hair loss at middle hairline.
INCLUSION CRITERIAINCLUSION CRITERIA:: Patients more than 20 years old, both sex.
Patients diagnosed as male AGA, type III to VI
using Hamilton –Norwood classification, and as
female pattern hair loss type I-III using Ludwig
classification.
Active hair loss within last 12 months.
Willing to sign informed consent.
Norwood -Hamilton scale Norwood -Hamilton scale Ludwig scaleLudwig scale
I II III Stage
EXCLUSION CRITERIAEXCLUSION CRITERIA:
Pregnancy
Malignancy or other systemic disease that might
interfere with diagnosis as collagen diseases and
chronic thyroiditis.
Unconfirmed diagnosis clinically or pathologically.
Global scalp hair thinning including occipital areas.
IMMUNOHISTOCHEMICAL STUDYFour mm punch biopsy specimens were
obtained from each patient from both frontal
affected area of scalp and the occipital skin that
served as a control.
The specimens were fixed in 10% neutral
buffered formalin and processed for paraffin
embedding.
Serial 4 µm sections from paraffin blocks were stained with:
Hx.& E. stain for histopathological diagnosis.
Immunohistochemical staining using the Streptavidin-Biotin
immunoperoxidase technique for the detection of:
CK15 expression using CK15 Ab-1 mouse monoclonal AB.CK15 expression using CK15 Ab-1 mouse monoclonal AB.
CD34 expression using CD34 mouse monoclonal AB (1:800).CD34 expression using CD34 mouse monoclonal AB (1:800).
P63 expression using P63 Ab-1 mouse monoclonal antibody P63 expression using P63 Ab-1 mouse monoclonal antibody
(Wallace and Smoller, 1997).(Wallace and Smoller, 1997).
EVALUATION OF STAINING RESULTSEVALUATION OF STAINING RESULTS:
CK15 positive staining was noted by ascertaining CK15 expression in cytoplasm. Any nuclear stain was considered background artifact.
P63 positive expression was noted by nuclear localization. CD34 expression was noted when uniform/circumferential staining expression as seen in occipital skin was not observed (Hoang et al., 2009).
Positive slides showed brown colored precipitate. The percentage of positive cells was calculated in 100 cells / 4 HPF.
CRITERIA FOR GRADING STAINED SECTIONSCRITERIA FOR GRADING STAINED SECTIONS
Negative (-): if <5 % positive cells.
Weakly positive (+): if 5% - 25% positive cells.
Moderately positive (++): if >25-50% positive cells.
Strongly positive (+++): if >50% positive cells
(Fiuraskova et al., 2005).
STAISTICAL ANALYSIS
The data were tabulated and statistically analyzed
using the SPSS for Windows 10.0 statistical package
program. Paired data of qualitative variable were
estimated by McNemar,s chi-square test (X2) and
Wilcoxon signed rank test `(Dean et al., 1994)
RESULTSRESULTS
This study was conducted on 20 AGA patients.
Seventeen patients (80%) were males and three patients
(15%) were females. Their ages ranged between 20 and
29 years with a mean of 24.05± 1.6.
Classification of males according to Norwood Hamilton
scale showed that two patients (10%) were of type IV, 3
patients (15%) of type V, 4 patients (20%) of type Va and
8 patients (40%) of type VI AGA.
Using Ludwig's classification in females, 1 patient (5%)
was of type II and 2 patients (10%) were of type III AGA.
The disease duration ranged between 1-6 years with a
mean of 3.175 ± 2.014 years. No family history of a
similar condition was detected.
All patients had received previous therapy, 85% received
topical minoxidil therapy while 15% received intralesional
mesotherapy .
Table (1): Demographic data and clinical characteristics
Immunohistochemical examination of scalp biopsies
using CK15 antibody showed mild positive expression of
CK15 in both frontal and occipital skin in all 20 AGA
patients (100%).
CK15 was expressed in the bulge region and in the
peripheral layer of the outer root sheath, at the level of the
isthmus.
In frontal skin, CK15 was expressed in the bulge region in
95% (19 patients) and in the peripheral layer of ORS
in100% (20 patients) compared to 100% in both the bulge
region and ORS in occipital skin.
Table (2): Summary of CK15 positive expression in frontal versus occipital skin in 20 AGA patients.
Table (3): Staining degree of CK15 in frontal versus occipital skin
Specifically, the CK15
immunostaining extended
from the entrance of the
sebaceous gland duct
down to the insertion site
of the arrector pili muscle.
This CK15 staining was
maintained throughout the
different phases of the
hair follicle cycle.
Figure (1): CK 15 positivity of frontal hair follicle within bulge region and ORS (X400).
1
Fig (2): CK15 positivity within occipital hair follicle (X200).
Fig(3): CK 15 positivity of frontal hair follicle of same patient at the outermost cell layer of
ORS, at the level of isthmus( X400) .
Figure (4): CK15 positivity within frontal hair follicle at level of sebaceous glands, (X200).
CD34 ANTIBODY`
Immunohistochemical examination using CD34
antibody showed positive expression in 30%
(6 patients) of frontal scalp biopsies, compared to
85% (17 patients) in occipital biopsies with a highly
statistically significant difference, (P<0.001) .
Table (4): Comparison of CD34 expression in frontal versus occipital skin.
A highly statistically significant difference was also
detected in CD34 staining intensity in frontal skin as
compared to occipital (P<0.005).
In frontal skin, weak positivity was detected in 20% (4
patients), moderate positivity in 5% (1 patient) and
strong positivity in 5% (1 patient). While in occipital
skin, 65% (13 patients) showed weak positivity, 5% (1
patient) showed moderate positivity and 15% (3
patients) showed strong positivity .
Table (5): Staining degree of CD34 in frontal versus occipital skin .
The CD34 immunoreactivity was detected only
below the bulge zone, at the most peripheral
layer of ORS, in the transient portion of follicle
below the isthmus and above the matrix cells.
Thus CD34 and CK15 antibodies recognize
different types of cells or cells at different stages
of differentiation.
Figure (5): CD34 positive expression in control occipital scalp (X200).
Figure (6): CD34 negative expression in frontal scalp follicles while positive expression in blood vessels (X200).
Figure (7): CD 34 positive expression in control occipital scalp (X200).
Figure (8): CD34 negative expression in frontal scalp of same patient (X400).
CD34 immunoreactivity was not detected in
catagen or telogen hair follicles. Only anagen
hair follicles showed CD34 immunoreactivity.
Other skin structures that stained with CD34
antibody were the endothelial cells and
perifollicular spindle shaped cells .
Figure (9): CD34 negative expression in telogen hair (X200).
P63 ANTIBODYImmunohistochemical exam. using P63 antibody
showed positive expression in 85% (17 patients) of frontal scalp biopsies, compared to 100% (20 patients) in occipital biopsies with no statistically significant difference, (P>0.08).
The nuclear staining for P63 was detected in all hair follicle epithelial structures. However, comparison of P63 staining intensity in frontal versus occipital skin showed a highly statistically significant difference (P=0.005).
Table (6): Comparison of p 63 expression in frontal versus occipital skin.
In frontal skin, weak positivity was detected in 40% (8 patients), moderate positivity in 30% (6 patients) and strong positivity in 15% (3 patients).
While in occipital skin, 45% (9 patients) showed weak positivity, 25% (5 patients) showed moderate positivity and 30% (6 patients) showed strong positivity.
Thus higher expression of P63 is detected in occipital skin as compared to the affected frontal skin.
Table (7): Staining degree of P63 in frontal versus occipital skin.
Figure (10): p63 strong positive expression in occipital area (X200).
Figure (11): p63 positive expression in frontal area (X200).
Figure (12): p63 strong positivity in occipital area (X400).
Figure (13): p63 strong positivity in occipital area (X400).
Figure (14): p63 positivity in telogen germinal center in frontal area (X400).
CONCLUSIONCONCLUSION
& &
RECOMMENDATIONSRECOMMENDATIONS
In conclusion, although the pathogenesis of In conclusion, although the pathogenesis of
AGA is still not understood, this study confirms AGA is still not understood, this study confirms
that the follicular stem cells in the bulge region that the follicular stem cells in the bulge region
are intact and are not the only reservoir of stem are intact and are not the only reservoir of stem
cells in the hair follicle.cells in the hair follicle.
The preservation of CK15 expression in AGA The preservation of CK15 expression in AGA
supports the concept that it is a nonscarring supports the concept that it is a nonscarring
type of alopecia and suggests its potential type of alopecia and suggests its potential
reversibility.reversibility.
The loss of CD34+ cells in AGA provides insight The loss of CD34+ cells in AGA provides insight
into the possible mechanism of miniaturization into the possible mechanism of miniaturization
and supports the notion that a defect in and supports the notion that a defect in
conversion of bulge stem cells to progenitor conversion of bulge stem cells to progenitor
cells may play a role in disease progression. cells may play a role in disease progression.
Also the decreased expression of P63 in this Also the decreased expression of P63 in this
study suggests a role of this protein in the study suggests a role of this protein in the
pathogenesis of AGA.pathogenesis of AGA.
Future studies are recommended Future studies are recommended using other stem cell markers to using other stem cell markers to further clarify the role of follicular further clarify the role of follicular stem cells in androgenetic alopecia stem cells in androgenetic alopecia and to identify signals responsible and to identify signals responsible for transformation of these stem for transformation of these stem cells to progenitor cells.cells to progenitor cells.