‫الرحيم‬ ‫الرح ن‬ ‫ال‬ ‫بسم‬‫الرحيم‬ ‫الرح ن‬ ‫ال‬ ‫بسم‬
””‫إل‬ِ ‫نا‬َ ‫ل‬َ ‫م‬َ ‫ل‬ْ ‫ع‬ِ ‫ل‬ ‫سبحانك‬ ‫قالوا‬‫إل‬ِ ‫نا‬َ...
BY
KHALID M. GHARIB
MD
LECTURER OF
DERMATOLOGY AND
VENEREOLOGY
ZAGAZIG UNIVERISTY
INTRODUCTIOINTRODUCTIO
NN
ANDROGENETIC ALOPECIA
Androgenetic alopecia (AGA) also referred to as
male-pattern hair loss, common baldness or
female-p...
 The pathogenesis of AGA is poorly understood. It is
assumed that the genetically predisposed hair
follicles are the targ...
Hair follicle is the main source of multipoint stem
cells in the skin. These stem cells reside in a
quiescent niche in bu...
STEM CELL MARKERS
Recently, due to discovery of cell surface markers, it
has become possible to characterize the hair fol...
Several studies, reported loss of CK15+ cells in some
scarring alopecia, implying invovement of bulge
region in its patho...
Recently, few studies have been investigating the status
of the hair follicle stem cell compartment in AGA.
Some studies...
AIM OF THEAIM OF THE
WORKWORK
The aim of the present study wasThe aim of the present study was
to investigate the possible role ofto investigate the po...
PATIENTS&METHOPATIENTS&METHO
DSDS
This study has been conducted on 20 patients with
AGA selected from the outpatient clinics of the
Dermatology Department ...
INCLUSION CRITERIAINCLUSION CRITERIA::
 Patients more than 20 years old, both sex.
 Patients diagnosed as male AGA, type...
Norwood -Hamilton scaleNorwood -Hamilton scale Ludwig scaleLudwig scale
I II III Stage
EXCLUSION CRITERIAEXCLUSION CRITERIA:
Pregnancy
Malignancy or other systemic disease that might
interfere with diagnosis...
IMMUNOHISTOCHEMICAL STUDY
Four mm punch biopsy specimens were
obtained from each patient from both frontal
affected area ...
Serial 4 µm sections from paraffin blocks were stained with:
 Hx.& E. stain for histopathological diagnosis.
 Immunohist...
EVALUATION OF STAINING RESULTSEVALUATION OF STAINING RESULTS:
CK15 positive staining was noted by ascertaining
CK15 expre...
CRITERIA FOR GRADING STAINED SECTIONSCRITERIA FOR GRADING STAINED SECTIONS
 Negative (-): if <5 % positive cells.
Weakly...
STAISTICAL ANALYSIS
 The data were tabulated and statistically analyzed
using the SPSS for Windows 10.0 statistical packa...
RESULTRESULT
SS
This study was conducted on 20 AGA patients.
Seventeen patients (80%) were males and three patients
(15%) were females. T...
Using Ludwig's classification in females, 1 patient (5%)
was of type II and 2 patients (10%) were of type III AGA.
 The ...
Table (1): Demographic data and clinical characteristics
Immunohistochemical examination of scalp biopsies
using CK15 antibody showed mild positive expression of
CK15 in both fro...
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
o...
1
Fig (2): CK15 positivity within occipital hair
follicle (X200).
Fig(3): CK 15 positivity of frontal hair follicle
of sam...
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 fron...
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...
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 p...
Figure (5): CD34 positive expression
in control occipital scalp (X200).
Figure (6): CD34 negative expression in
frontal sc...
Figure (7): CD 34 positive expression in
control occipital scalp (X200).
Figure (8): CD34 negative expression in
frontal s...
CD34 immunoreactivity was not detected in
catagen or telogen hair follicles. Only anagen
hair follicles showed CD34 immun...
Figure (9): CD34 negative expression in telogen hair (X200).
P63 ANTIBODY
Immunohistochemical exam. using P63 antibody
showed positive expression in 85% (17 patients)
of frontal scal...
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 pos...
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...
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
&&
RECOMMENDATIORECOMMENDATIO
NSNS
In conclusion, although the pathogenesis ofIn conclusion, although the pathogenesis of
AGA is still not understood, this ...
The loss of CD34+ cells in AGA provides insightThe loss of CD34+ cells in AGA provides insight
into the possible mechanis...
Future studies are recommendedFuture studies are recommended
using other stem cell markers tousing other stem cell marker...
Role of stem cells in ANDROGENETIC ALOPECIA
Role of stem cells in ANDROGENETIC ALOPECIA
Role of stem cells in ANDROGENETIC ALOPECIA
Role of stem cells in ANDROGENETIC ALOPECIA
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Role of stem cells in ANDROGENETIC ALOPECIA

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Role of stem cells in androgenetic alopecia using immunohistochemistry markers as CK15, CD34 AND P63.

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Role of stem cells in ANDROGENETIC ALOPECIA

  1. 1. ‫الرحيم‬ ‫الرح ن‬ ‫ال‬ ‫بسم‬‫الرحيم‬ ‫الرح ن‬ ‫ال‬ ‫بسم‬ ””‫إل‬ِ ‫نا‬َ ‫ل‬َ ‫م‬َ ‫ل‬ْ ‫ع‬ِ ‫ل‬ ‫سبحانك‬ ‫قالوا‬‫إل‬ِ ‫نا‬َ ‫ل‬َ ‫م‬َ ‫ل‬ْ ‫ع‬ِ ‫ل‬ ‫سبحانك‬ ‫قالوا‬ ‫م‬ُ ‫لي‬ِ ‫ع‬َ ‫ل‬ْ ‫ا‬ ‫ت‬َ ‫ن‬ْ ‫أ‬َ ‫ك‬َ ‫ن‬ّ ‫إ‬ِ ‫نا‬َ ‫ت‬َ ‫م‬ْ ‫ل‬ّ ‫ع‬َ ‫ما‬َ‫م‬ُ ‫لي‬ِ ‫ع‬َ ‫ل‬ْ ‫ا‬ ‫ت‬َ ‫ن‬ْ ‫أ‬َ ‫ك‬َ ‫ن‬ّ ‫إ‬ِ ‫نا‬َ ‫ت‬َ ‫م‬ْ ‫ل‬ّ ‫ع‬َ ‫ما‬َ ‫م‬ُ ‫كي‬ِ ‫ح‬َ ‫ل‬ْ ‫ا‬‫م‬ُ ‫كي‬ِ ‫ح‬َ ‫ل‬ْ ‫ا‬““ ‫لا العظيم‬‫ل‬‫لا ا‬‫ق‬‫صد‬‫لا العظيم‬‫ل‬‫لا ا‬‫ق‬‫صد‬ : )‫البقرة‬: )‫البقرة‬3232 ))
  2. 2. BY KHALID M. GHARIB MD LECTURER OF DERMATOLOGY AND VENEREOLOGY ZAGAZIG UNIVERISTY
  3. 3. INTRODUCTIOINTRODUCTIO NN
  4. 4. ANDROGENETIC ALOPECIA Androgenetic 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).
  5. 5.  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).
  6. 6. 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).
  7. 7. 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.
  8. 8. 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).
  9. 9. 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.
  10. 10. AIM OF THEAIM OF THE WORKWORK
  11. 11. The aim of the present study wasThe aim of the present study was to investigate the possible role ofto investigate the possible role of hair follicle stem and progenitorhair follicle stem and progenitor cells in the pathogenesis ofcells in the pathogenesis of androgenetic alopeciaandrogenetic alopecia.
  12. 12. PATIENTS&METHOPATIENTS&METHO DSDS
  13. 13. 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.
  14. 14. 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.
  15. 15. Norwood -Hamilton scaleNorwood -Hamilton scale Ludwig scaleLudwig scale I II III Stage
  16. 16. 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.
  17. 17. IMMUNOHISTOCHEMICAL STUDY Four 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.
  18. 18. 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 antibodyP63 expression using P63 Ab-1 mouse monoclonal antibody (Wallace and Smoller, 1997).(Wallace and Smoller, 1997).
  19. 19. 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.
  20. 20. 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).
  21. 21. 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)
  22. 22. RESULTRESULT SS
  23. 23. 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.
  24. 24. 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 .
  25. 25. Table (1): Demographic data and clinical characteristics
  26. 26. 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.
  27. 27. Table (2): Summary of CK15 positive expression in frontal versus occipital skin in 20 AGA patients.
  28. 28. Table (3): Staining degree of CK15 in frontal versus occipital skin
  29. 29. 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(.
  30. 30. 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(.
  31. 31. Figure (4): CK15 positivity within frontal hair follicle at level of sebaceous glands, (X200).
  32. 32. 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) .
  33. 33. Table (4): Comparison of CD34 expression in frontal versus occipital skin.
  34. 34. 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 .
  35. 35. Table (5): Staining degree of CD34 in frontal versus occipital skin.
  36. 36. 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.
  37. 37. 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).
  38. 38. Figure (7): CD 34 positive expression in control occipital scalp (X200). Figure (8): CD34 negative expression in frontal scalp of same patient (X400).
  39. 39. 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 .
  40. 40. Figure (9): CD34 negative expression in telogen hair (X200).
  41. 41. P63 ANTIBODY Immunohistochemical 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).
  42. 42. Table (6): Comparison of p 63 expression in frontal versus occipital skin.
  43. 43. 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.
  44. 44. Table (7): Staining degree of P63 in frontal versus occipital skin.
  45. 45. Figure (10): p63 strong positive expression in occipital area (X200). Figure (11): p63 positive expression in frontal area (X200).
  46. 46. Figure (12): p63 strong positivity in occipital area (X400). Figure (13): p63 strong positivity in occipital area (X400).
  47. 47. Figure (14): p63 positivity in telogen germinal center in frontal area (X400).
  48. 48. CONCLUSIONCONCLUSION && RECOMMENDATIORECOMMENDATIO NSNS
  49. 49. In conclusion, although the pathogenesis ofIn conclusion, although the pathogenesis of AGA is still not understood, this study confirmsAGA is still not understood, this study confirms that the follicular stem cells in the bulge regionthat the follicular stem cells in the bulge region are intact and are not the only reservoir of stemare 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 AGAThe preservation of CK15 expression in AGA supports the concept that it is a nonscarringsupports the concept that it is a nonscarring type of alopecia and suggests its potentialtype of alopecia and suggests its potential reversibility.reversibility.
  50. 50. The loss of CD34+ cells in AGA provides insightThe loss of CD34+ cells in AGA provides insight into the possible mechanism of miniaturizationinto the possible mechanism of miniaturization and supports the notion that a defect inand supports the notion that a defect in conversion of bulge stem cells to progenitorconversion 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 thisAlso the decreased expression of P63 in this study suggests a role of this protein in thestudy suggests a role of this protein in the pathogenesis of AGA.pathogenesis of AGA.
  51. 51. Future studies are recommendedFuture studies are recommended using other stem cell markers tousing other stem cell markers to further clarify the role of follicularfurther clarify the role of follicular stem cells in androgenetic alopeciastem cells in androgenetic alopecia and to identify signals responsibleand to identify signals responsible for transformation of these stemfor transformation of these stem cells to progenitor cells.cells to progenitor cells.

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