Journal of Dermatological Treatment

ISSN: 0954-6634 (Print) 1471-1753 (Online) Journal homepage: http://www.tandfonline.com/loi/ijdt20

Stem cell therapy as a novel therapeutic

intervention for resistant cases of alopecia areata
and androgenetic alopecia

Zeinab Abel Samad Ibrahim, Iman Hamed Elmaadawi, Basma Mourad

Mohamed, Said Mohamed Abdou MD, Yasmina Ahmed El Attar, Amira
Youssef MD, Maha Mostafa Shamloula MD, Atef Taha MD, Hala Gabr
Metwally MD, Mohamed M El Afandy MD & Mohamed Labib Salem MD

To cite this article: Zeinab Abel Samad Ibrahim, Iman Hamed Elmaadawi, Basma Mourad
Mohamed, Said Mohamed Abdou MD, Yasmina Ahmed El Attar, Amira Youssef MD, Maha
Mostafa Shamloula MD, Atef Taha MD, Hala Gabr Metwally MD, Mohamed M El Afandy MD
& Mohamed Labib Salem MD (2016): Stem cell therapy as a novel therapeutic intervention
for resistant cases of alopecia areata and androgenetic alopecia, Journal of Dermatological
Treatment, DOI: 10.1080/09546634.2016.1227419

To link to this article: http://dx.doi.org/10.1080/09546634.2016.1227419

Accepted author version posted online: 24

Aug 2016.
Published online: 24 Aug 2016.

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Download by: [Cornell University Library] Date: 25 August 2016, At: 01:05
 Stem cell therapy as a novel therapeutic intervention for resistant

cases of alopecia areata and androgenetic alopecia

The authors:

Prof dr/ Zeinab Abel Samad Ibrahim

Professor of Dematology and venereology department ,
Faculty of Medicine Tanta University, Tanta, Egypt
ibrahimzeinab85@yahoo.com

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01001709414

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Prof dr/ Iman Hamed Elmaadawi

Professor of Dematology and venereology department ,

Faculty of Medicine Tanta University, Tanta, Egypt
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dr_maadawy@hotmail.com
01227440715
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Prof dr/ Basma Mourad Mohamed

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Professor of Dematology and venereology department ,

Faculty of Medicine Tanta University, Tanta, Egypt
basmamourad@gmail.com
01007961931
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Prof dr/ Said Mohamed Abdou

MD
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Professor of Clinical Pathology Department,

Faculty of Medicine Tanta University, Tanta, Egypt
Saidmohamed_2000@yahoo.com
01222818651

Dr/ Yasmina Ahmed El Attar

Master of Dermatology and Venereoloy/

A student of doctorate degree

Assistant lecturer of Dematology and venereology department ,

 Faculty of Medicine Tanta University, Tanta, Egypt
yasminaelattar@yahoo.com
01225649650

Dr/ Amira Youssef

MD
lecturer of Clinical Pathology Department,
Faculty of Medicine Tanta University, Tanta, Egypt
damirayoussef@yahoo.com
01149712900

Prof dr/ Maha Mostafa Shamloula

MD
Professor of Pathology Department,
Faculty of Medicine Tanta University, Tanta, Egypt.
mshamloula@gmail.com

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01000571583

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Prof dr/ Atef Taha
MD
Professor of Internal Medicine Department,
Faculty of Medicine, Tanta University, Tanta, Egypt.
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Ateftaha1966@yahoo.com
01003070077

Prof dr/ Hala Gabr Metwally

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MD
Professor of Clinical Pathology Department,
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Faculty of Medicine. Cairo University , Cairo, Egypt.

halagabr@yahoo.com
01001500871
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Dr/ Mohamed M El Afandy

MD
Lecturer of Anathesia and intensive care Department,
Faculty of Medicine, Tanta University, Tanta, Egypt.
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Mohamedafandy811@yahoo.com
01225131526

Prof dr/ Mohamed Labib Salem

MD
Professor of Immunology and Biotechnology Unit, Zoology Department, Faculty of Science, Center of
Excellence in Cancer Research, Tanta University, Tanta, Egypt
Mohamed.labib@science.tanta.edu.eg;
CECR_mohamed.labib@unv.tanta.edu.eg
01274272624
Corresponding author is
Dr/ Yasmina Ahmed El Attar
Master of Dermatology and Venereoloy/

A student of doctorate degree

Assistant lecturer of Dematology and venereology department ,

Faculty of Medicine Tanta University, Tanta, Egypt
yasminaelattar@yahoo.com
01225649650
1- Moawya street -el taameen building- fifth floor –( IN FRONT OF DAR EL SHEFAA HOSPITAL)
Tanta- Eygpt (home address).
2- El geigh street- tanta university hospital- dermatology and venereology department Tanta- Eygpt (work address)

_____________________________________________________________________

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Abstract

Background: Management of alopecia areata and androgenetic alopecia is often challenging as patients may be
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resistant to currently available modalities of treatment. The use of stem cells may be a novel option for resistant

cases.
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Objective: To evaluate the safety and efficacy of the use of autologous bone marrow derived mononuclear cells
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(including stem cells ) as compared to follicular stems cells for the management of resistant cases of alopecia

areata and androgenetic alopecia.

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Methods: This study included forty patients (twenty alopecia areata patients and twenty androgenetic alopecia

patients), all patients were treated with a single session of intradermal injection of autologous SCs therapy.
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They were divided into four groups according to the applied modality [either autologous bone marrow derived

mononuclear cells (BMMCs or autologous follicular stem cells (FSC)].

Results: Six months after stem cell therapy injection, there was a significant improvement, confirmed by

immunostaining and digital dermoscopy. The mean improvement in all groups was “very good”. There was

no significant difference between both methods in either type of alopecia. No serious adverse events were

reported.
Conclusion: Autologous BMMCs and FSC seem to be a safe tolerable and effective treatment for the

management of both resistant alopecia areata and androgenetic alopecia.

Keywords: Bone marrow mononuclear cells _ Follicular stem cells_ Alopecia areata _ Androgenetic alopecia

_ Digital dermoscopy- CK 15- CD34- CD200.

Introduction

Alopecia areata (AA) is a variant of alopecia; it is an autoimmune disease, with a projected lifetime risk of

1.7%. (1) Androgenetic alopecia (AGA) is another type of alopecia, it is androgen dependent, and commonly

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known as male pattern hair loss (MPHL) or female pattern hair loss (FPHL). It affects up to 50% of men

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worldwide. (2) Conventional treatments of both types of alopecia may fail to treat the patients completely; they

include drugs or phototherapy for AA and medical treatments or surgical hair transplantation for AGA. (3, 4, 5)
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The alternative solution of particular interest is so called "Regenerative medicine" based on the therapeutic
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potential of stem cells (SCs). (6,7) Stem cells can be isolated from BM, hair follicles and other tissues such as
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adipose tissue, umbilical cord and skin tissue. Both bone marrow mononuclear cells (BMMCs) (including

SCs) and FSCs may contribute to tissue repair or regeneration of many tissues including myocardium, blood

vessels, damaged bone, tendon, cartilage, skin and hair. Different SC types were tried in alopecia with variable
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results. (8, 9, 10) Moreover, the occipital hairs maintain their resistance to AGA when transplanted to the
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vertex that makes them a good source for stem cell therapy with excellent expectations success. (11)

There are several FSC markers; their expression reflects the SC presence and action. Cytokeratin-15 (CK15)

CD34 and CD200 are markers for bulge stem cells. (12,13) CK15 immunoreactivity is decreased in affected

bulge of AA lesions. (14) It is found in FSCs, in frontal and occipital areas of AGA patients suggesting that

FSC is still present in AGA. (15,16)

 The hair follicle appears to be the only human structure in which expression of CD34 is noted in

epithelial cells. (12,13,17) It was found that CD34-positive hair follicle SCs are deficient in AGA in the frontal

scalp, while it is preserved within hair follicles of the occipital scalp. (18,19)

CD200 is a hair germ marker, its immunoreactivity is decreased in affected bulge of AA lesions. (14)

Insufficient CD200 expression may be involved in the collapse of immune privilege in the epithelial FSC niche

and may contribute to the pathogenesis of AA. On the other hand, CD200-rich positive hair follicle stem cells

are deficient in AGA. (20,21,22)

The purpose of this study was to evaluate the safety and efficacy of the use of autologous bone marrow derived

mononuclear cells (including stem cells) as compared to follicular stems cells for the management of resistant

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cases of alopecia areata and androgenetic alopecia.

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Patients and methods

The study protocol and the consent were approved by the local research ethical committee (REC) NO
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877/12/11 (as extension of M.D. thesis protocol) it is also a scientific project Code:tu-07-13-06
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Patients
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This study was carried out on 40 patients, 20 patients with resistant AA (seven males and 13 females) and

20 patients with AGA (eight males and 12 females ). The ages of the patients ranged from 10-50 years

(mean 26 + 8 ). They did not receive any treatment for alopecia for six months before enrollment in the
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study. All patients were resistant to conventional treatments. Exclusion criteria were as follows: pregnancy,

breastfeeding, systemic disease or immunosuppression.

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Subjects were selected from Outpatient Clinic of Dermatology and Venereology Department within Tanta

University Hospital in Tanta, Egypt. This study was done in cooperation of Dermatology, Clinical

Pathology, Internal Medicine, and Anesthesia and Intensive Care departments of (Tanta university), together

with Clinical Pathology department Cairo university.

Study objectives: To evaluate the clinical efficacy and safety of autologous bone marrow mononuclear

(including stem cells) cells versus follicular stem cells in resistant cases of alopecia areata and androgenetic

alopecia.

Study design: The study design is double randomized clinical disciplined attempt. The patients were

divided into four groups as following: Group 1 (10 resistant alopecia areata patients) and group 3 (10

androgenetic alopecia patients) received single session of intradermal injections of autologous bone marrow

derived mononuclear cells (BMMCs). Group 2 (10 resistant alopecia areata patients) and group 4 (10

androgenetic alopecia patients) received single session of intradermal injections of autologous follicular

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stem cells (FSC). The clinical types of AA subjects in this study were patchy, ophiasis and alopecia totalis

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(Table 1). After taking an informed consent from the patients, they were subjected to routine laboratory

investigations including bleeding, prothrombin and coagulation time tests, to exclude any hematologic
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diseases. Chest X-ray and abdominal ultrasound were done to exclude any systemic illness. Dermoscope

and digital dermoscope , histological examination of the lesion were done for all patients before treatment ,
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while after therapy histological examination was repeated at three months period and digital dermoscope
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was repeated at six months.

The procedures:
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I- Bone marrow aspiration Assay procedure (Groups 1 and 3): (23)

Each patient received granulocyte colony-stimulating factor (G-CSF) for three successive days before BM
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collection, by a dose of 5 mg/kg/day by subcutaneous route till white blood cells reach 17 000/ml (or more).

Then, BM samples were collected under complete aseptic conditions. Subjects were taken to the operating

room and general anesthesia was administered. Fifty milliliters (mL) of bone marrow(BM) were harvested

from the upper iliac crest and placed in a conical tube with four mL of heparin. Mononuclear cells were

isolated with density gradient centrifugation. The aspirated BM was diluted at a ratio of 7:1 with buffer.

Cells were passed through a 100-mm filter to remove bone fragments and cell clumps. Thirty milliliters of
diluted cell suspension was carefully layered over 15ml of ficoll-paque in a 50-ml conical tube.

Centrifugation at 2000 rpm for 20 minutes at 20 _C in a swinging bucket rotor without brake was done.

The BMMCs (mixture of progenerating cells, hematopoietic cells, a variety of inflammatory cell types, and

mesenchymal stem cells) were carefully transferred at the interphase to a new 50-ml conical tube. Cells

were washed twice by adding up to 40 ml of buffer, mixed gently and centrifuged at 1500 rpm for 10– 15

minutes at 20 _C. The supernatent was carefully removed. For removal of platelets, the cell pellet was

resuspended in 50 ml of buffer and centrifugation at 1500 rpm for 10 minutes at 20 _C “was performed.

(final volume of 300 µl of clinical buffer for up to 108 total cells).

II- Follicular stem cell culture ( Groups 2 and 4):

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For subjects receiving follicular stem cells, a four mm skin punch biopsy was performed at the clinically

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unaffected areas. There samples were place in Hank's balanced salt solution (Invitrogen, Carlsbad,

California, USA) for subsequent cell culture. Plucked hairs from each patient were also taken for coculture.
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10 mmol/l citrate buffer, pH 6.0, (NeoMarkers, Cat. #AP-9003), for 10–20 min followed by cooling at room

temperature for 20min. The Super Sensitive [TM Link- Label Detection Systems Concentrated Format]
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(BioGenex), avidin–biotin complex technique and 3, 30-diaminobenzidine tetrahydrochloride (DAB

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chromogen, BioGenex) were used as a revelation system. Nuclei were counter stained with Mayer’s

hematoxylin.
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Cell culture ; Hair follicle isolation was done according to the method described by Yu et al, with

additional androgen blocking factor( SIGMA) to (group 4) samples from day 10Within a laminar flow
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cabinet, scalp tissue was rinsed, trimmed of adipose, dissected to expose the bulge region, and subjected to

enzymatic dissociation in 12.5mg/ml collagenase (SIGMA) for 1 h at 37 C. After treatment, the epidermis

was peeled off from the dermis, and hair follicles were plucked from the dermis. To isolate hair follicle

SCs, the lower bulge areas were treated twice with 0.25% trypsin/EDTA (Invitrogen) for 30 min at 37 0C,

5% CO2, according to the method described by Yu et al. ,where enzymatic digestion is used then 1ml of

sterile serum is added to stop the action of trypsin. (10, 24)

The cell suspension was filtered through a 40-mm cell strainer (BD Falcon, Bedford, Massachusetts, USA),

and cell numbers were counted. Single cells were cultured in noncoated flasks containing 5ml of Dulbecco’s

modified Eagle’s medium (DMEM) /F-12 medium (Invitrogen),antibiotics, 200 mmol/l L-glutamine

(Invitrogen), 0.1mmol/l b-mercaptoethanol (Sigma, St Louis, Missouri, USA), 1% nonessential amino acids

(Invitrogen) 20 ng/ml stem cell factor, and 20 ng/ml basic fibroblast growth factor (Research Diagnostics,

Concord, Massachusetts, USA). With additional androgen blocking factor (SIGMA) to (group 4) samples

from day 10. Media were changed every 72 h for two weeks. Non dissociated parts were left in trypsin

and the single cells were separated after 24 and 48 h. Plucked hairs from the same individual were treated

with trypsin as described above and co cultured with hair follicle epithelial SCs. Cell harvest ; The cells

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were washed, counted and subjected to the following prior to injection. Viability assessment: cell

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suspension in a density of 100 000 cell/ml are added to 100 ml trypan blue and incubated for five min at 37

0C. Cells were evaluated under high power microscope. Cells stained with the dye are counted as dead and
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the percentage of viable cells was calculated. (10,13,24)

Method of applications
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Under aseptic technique, SC-containing solution was injected intradermally, using a 26-gauge needle. One
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millilter (in a density of 100 000 cell/ml) was injected at per centimeter square of the treated site.

Assessments
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I- Clinical evaluation of the patients

The patients were photographed by using a Sony camera 18 mega pixels. All patients were assessed
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clinically at the time of enrolment and at the end of the study by the Severity of Alopecia Tool (SALT )

score in AA (groups 1 and 2) and quantitative grading in AGA (groups 3 and 4). (26)

Clinical improvement was assessed at three and six months.

The National Alopecia Areata Foundation working committee has devised

“Severity of Alopecia Tool score” (SALT score). Scalp is divided into 4

areas namely, Vertex - 40% (0.4) of scalp surface area; right profile of
scalp - 18% (0.18) of scalp surface area; left profile of scalp -18%

(0.18) of scalp surface area; Posterior aspect of scalp - 24% (0.24) of

scalp surface area. Percentage of hair loss in any of these areas is

percentage hair loss multiplied by percent surface area of the scalp in

that area. SALT score is the sum of percentage of hair loss in all above

mentioned areas.

For example, the percentage of improvement at the third month=

% of baseline extent_ % of affection at 3rd:month

% of baseline extent

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Improvement was graded as follows: excellent response if > 75% improvement, very good response if

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>50-75% improvement, good response if >25-50% improvement, fair response if < 25% improvement

II- Histopathologial examination:

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Two scalp biopsies were taken from the affected area of alopecia, one before therapy and another after three
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months of therapy for assessment of improvement using CK 15, CD34 and CD 200 for detection of FSC
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before and after stem cell therapy (SCT).

Immunohistochemical staining procedure: (the improved streptavidin-biotin amplified system. (16,27)

Primary antibodies used were: Cytokeratin-15 [is a rabbit polyclonal antibody. (US Biological). IgG
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concentration 1.0mg\ml], CD 34 [(clone QBEnd/10) , Mouse Monoclonal Antibody (ScyTek). IgG1, kappa

.Concentrate] and CD 200 [ is a rabbit polyclonal antibody. (Gene tex). IgG concentration 1 mg\ml]
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Semiquantitative evaluation of all three markers were applied by detection of positive cells over entire scalp

specimen , based on the proportion (positive cell number) and intensity Criteria for grading stained sections

: Percentage of positive cells were subdivided into four score categories; 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. (16)

Statistical analysis
The analysis was done using IBM SPSS software package version 20.0. Data were expressed as using range

(minimum and maximum), mean, standard deviation and median). Parametric tests such as t-test were

applied for data that followed a normal distribution. Nonparametric tests such as Mann–Whitney U test,

Wilcoxonsigned ranks test and Chi-squared test were applied for data that did not follow a normal

distribution. Correction for chi-square was conducted using Fisher’s Exact test or Monte Carlo correction.

Results

I-Assessment of clinical efficacy:

Regarding the clinical improvement, in AA subjects receiving autologous BMMC, five patients (50%)

showed very good. In AA subjects receiving autologous FSC, three patients (30%) showed excellent

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response and two patients (20%) showed very good response (Figure 1). Regarding in AGA subjects

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receiving autologous BMMC, there was one patient (10%) with excellent response and five patients (50%)
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with very good response (Figure 2). In AGA subjects receiving autologous FSC, there was five patients

(50%) with very good response (Table 2). The mean percentage of improvement in AA patients was 45± 22
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in AA subjects receiving autologous BMMC and 58 ± 34 in AA subjects receiving autologous FSC with
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non-statistically significant difference. The mean percentage of improvement in AGA patients was 52– 28

in AGA subjects receiving autologous BMMC and 42± 27 in AGA subjects receiving autologous FSC with

non-statistically significant difference (Table 3).

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After BMMC the mean improvement was higher in the ophiasis type compared to other types of AA, but the

difference was statistically non-significant. While, after FSC the mean improvement was higher in the
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patchy type as compared to other types of AA. ( p= 1 and 0.134 respectively ) (Gragh 1). There was more

improvement in females compared to males after both BMMC and FSC therapies (P= 0.016 and 0.008

respectively), but no significant difference was found between both types of therapy (Gragh 2).

There was significant negative correlation between the SALT score of AA and the therapeutic efficacy after

six months in group 2 (treated with FSC) and total AA patients. There was significant negative correlation

between the duration of AA and therapeutic efficacy after six months in group 2 (treated with FSC).
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 Safety and tolerability

Generally, subjects tolerated study procedures well, however some patients suffered from "Granulocyte

colony stimulating factor induced fatigue and chills relieved with analgesics. Eighty percent of subjects

experienced bone pain and hematoma also managed with analgesics. Scalp dermatitis in 20% of the patients

that was treated with emollient creams was the only noticed complication in FSC therapy. 45% of AA

patients suffered recurrence of disease activity after one year of follow up mostly due to their stressful life

events.

II-Results of dermoscopic examination and Digital dermoscopy examination:

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Regarding the dermoscopic examination of AA,; before therapy, there were tapered hair in seven patients (35%)

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, yellow dots in nineteen patients (95%), black dots in seven patients (35%) , short vellus hair in seventeen

patients (85%) , honey comb appearance and white dots in two patients (10%). While six months of therapy,
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there were tapered hair in two patients (10 %), yellow dots in fourteen patients (70%), black dots in one patient
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(5%), short vellus hair in fifteen patients (75%), broken dystrophic hair in one patient (10%), honey comb

appearance in two patients (20%) and white dots in one patient (5%). Regrowing hair was found in nineteen
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patients (95%).

Regarding dermoscopic examination of AGA before therapy , there were hair diameter diversity in
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twenty patients (100%) , peri pilar sign in nineteen patients (95%) , yellow dots in twelve patients (60%) , and

single hair pilo-sebaceous units hair in twenty patients (100%). While six months of therapy, there were hair
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diameter diversity in eighteen patients (90%), peri pilar sign in nineteen patients (95%), yellow dots in nine

patients (45%) and single hair pilo-sebaceous units hair in nineteen patients (95%). Regrowing hair was found

in twenty patients (100%).

There was no significant difference in dermoscopic findings of AA and AGA lesions before and after therapy,

however, it showed some signs of improvement after SCT in regrown hairs, and other few changes (figure 3).
Both AA and AGA showed significant improvement with digital dermoscopy examination. It showed increase

in hair width as well as hair density after six months of single SC injection therapy in all groups of patients

(Figure 4) (Table 4).

III- Histopathological results

Interpretation of CK15, CD 34 and CD200: (16)

Immunoreactivity was regarded as positive when brown staining was localized , positive staining was in

cytoplasm but the distribution differed according to type of immune staining as following; CK15, CD34 and

CD200 were noted in the peripheral layer of the outer root sheath of the hair follicles. CD200, was additionally

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noted in hair germ cells.

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Immunohistochemichal examination, showed that the immunoreactivities of CK15 , CD200 and CD 34 ( that

were decreased in most of the AA and AGA lesions (CK15 ranging from weak to moderate positive, while
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CD200 and CD34 ranging from negative to weak positive in the ORS in both AA and AGA) increased in

expression after stem cell therapy except in the non-responding alopecia totalis (AT) cases and some MPHL.
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In the majority of cases CK15 ranging from moderate to strong positive, while CD200 and CD34 ranged from
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weak to moderate positive in the ORS in both AA and AGA, On the other hand, CD 200 immunoreaction

was noted in hair germ cells with strong positivity (+3) in scalp samples after therapy (Figure 5).
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Discussion

This study is comparing BMMC and FSC intradermal injection in the management of AA and AGA. SCs
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possess multipotency and self-renewal, enabling them with the ability to differentiate into the multiple cell

types.

In this study, gradual improvement started by the end of the first month and reached its maximum improvement

after six months. Interestingly, BMMCs and FSCs gave significant improvement in AGA and AA with no

statistically significant difference between both methods. (p = 0.426) In AA patients, the patchy type and the

ophiasis type showed the highest response to SC therapy compared to AT types. There was significant
improvement in females compared to males after both BMMC and FSC therapies, but with non significant

difference between both types of therapy.

Increased immunereactivities with CK 15, CD34 and CD200 in all treated groups confirmed the effectiveness

of SCT in AA and AGA treatment. Moreover, CD200 expression in hair germs that were in scalp samples of

both AA and AGA after SCT suggests that the stem cells were able to progress to hair germs and subsequent

hair follicle organogenesis.

The effect of BMMC intradermal injection in the treatment of both types of alopecia may be the result of its

content of a mixture of progenerating cells, hematopoietic stem cells, a variety of inflammatory cell types, and

mesenchymal stem cells (MSCs). (28) Based on BMMCs properties they can stimulate hair growth in AA

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(group 1) and AGA (group 3) due to: (1) the ability to home to sites of inflammation following tissue injury

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when injected intravenously, (2) the ability to differentiate into various cell types, (3) the ability to secrete

multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation, (4)
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the lack of immunogenicity and the ability to perform immunomodulatory functions. 5) They have anti-

apoptotic and regeneration-stimulating effects. 6) Hematopoietic stem cells also have an indirect role through
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secretion of factors such as vascular endothelial growth factor (VEGF) (which controls hair growth and follicle
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size through angiogenesis). These effects can be either direct or indirect. (8, 23, 29)

Following FSC, in group 2 (AA) and group 4 (AGA) the multipotent epithelial stem cells give rise to cycling
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hair follicles. Besides, the signalling between epithelial keratinocytes and underlying specialized mesenchymal

dermal papilla cells induces stem cell proliferation and initiates the cascade of cell differentiation into the HF
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cell lineages. (30) Consequently, this therapy may activate already existing stem cells on the scalp or their

progenitor cells. This therapy may be able to simply signal follicle stem cells to give off chemical signals to

nearby follicle cells which have shrunk due to androgenetic alopecia, in addition to the immune modulatory

character of SCs may help in AA recovery, and once again making healthy hair.

A prior study have demonstrated feasiblity, effiacy and short-term safety of local FSC therapy for AA." (10)
BMMCs was also effective and safe in treatment of acne scars and leg ischemia. (23,31) It was reported that,

long-lasting alopecia universalis, coincidentally and completely recovered after allogeneic hematopoietic SCT

that was given to treat chronic myeloid leukemia. (32) The Stem Cell Educator therapy was tried for the

treatment of AT. (30) Both of these studies provided intense immunosuppression combined with replacement

of the immune system by donor cells can induce regrowth of scalp and body hair, but in our study AT showed

poor response because the SCT effect on immunosuppression was mild, local and temporary.

The significant improvement in females coincides with the previous finding that adipose derived SCT was

effective on females, but not in males with AGA. This was explained as hairs are regulated by various factors,

including androgen and cytokines, therefore, the efficacy of combination therapy should be elucidated in a

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future study. (33)

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It was reported that both CK15 and CD200 immunoreactivities were decreased in affected bulge of AA lesions,
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but the immunereactivities after therapy of AA was not evaluated. (14) CK15 was found in follicular stem cells

in the bulge region, in frontal and occipital areas of AGA patients. (16) CD200-rich and CD34-positive hair
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follicle stem cells may be deficient in AGA. (18, 22) These studies coincide with our study as we reported that
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CK 15 ,CD200 and CD34 showed weak immune reactivities in AA and AGA lesions.

Using skin reconstitution assays, they confirmed that analogous CD200 rich cells from murine hair follicles

were multipotent and able to generate new hair follicles. (34) Moreover, CD34 acts as a specific marker for
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the epithelial keratinocytes present in the hair follicle bulge and these cells with characteristic progenitor marker

could be used for tissue engineering. (35) Therefore the increased immune reactivities of CD200 and CD34
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in AA and AGA after SCT confirm its effectiveness.

In this study, the good responder AT patient was a child who received BMMC therapy. The childhood onset of

AA is a poor prognostic sign. (36) This can be explained by the good efficacy of BMMCs in pediatric patients

as the ageing significantly increases the senescence-of the SCs, decreased their proliferative capacity and

production of GFs. However, the poor response of other AT patients can be explained by that the therapy can

induce hair regrowth in AA but do not change the immune dysfunction which is multifactorial and not only
localized in HFs but also outside the HFs, that is why AT needs intense systemic immune suppressive therapy,

which may be provided by systemic (IV or IM) SCT not local as our study. (36,37) This may be also explained

by the important findings in scalp specimens of AT lesions that makes the treatment trial is challengeable , as

they showed, marked hair follicle miniaturization which are situated slightly deeper than normal follicles, and

the nonsclerotic fibrous tracts (streamers) extending along the original site of the previous terminal follicles into

the subcutis. Besides, the small number of AT studied cases may be not sufficient to show the accurate SCT

efficacy.

In this study using human SC in the treatment of alopecia, we tried to avoid any possible risks to the patients.

Although bone marrow harvesting may have potential side effects as bleeding, infection and persistent pain. No

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serious side effects were reported in this study. Careful assessment of the patients and proper investigations

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before the procedure will avoid these risk. Moreover, complete sterilization and strict aseptic techniques are

mandatory. In addition FSC therapy showed only scalp dermatitis in 25% of patients which was resolved by
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emollients.

The forty five percent relapse that occured in AA subjects after one-year follow-up, indicates that these clinical
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procedures just temperately improve the hair growth, without significantly effects on fundamentally
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correcting the autoimmunity of AA that hides several immune-privellage collapse attacks , which are induced

by several environmental factors including the severe stress they live in as we did not referred them to
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psychiatric consultations. To overcome this, we suggest that SCT may be tried in multiple sessions instead of

single session as our study, or use it in combination with immune modulators in addition to psychiatric
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consultations for severely stressed patients.

Conclusions

Autologous BMMCs and autologous FSC seem to be a safe, tolerable and effective treatment option for the

management of resistant AA and androgenetic alopecia as well.

SC therapy seems to be more effective in females than in males AGA patients.

Although SCT is a promise therapy, but it is expensive, time consuming and need well professional physicians

and laboratory of excellences.

Recommendations

Further studies on large number of patients and using different types of SCs are required to assess the stem

cell therapy efficacy and safety. Use of combined SCT in the treatment of various types of alopecia and

systemic SCT may be needed in AT.

Acknowledgment This work was funded by a grant from the Research Development Fund, Tanta University,

Egypt.

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Conflict of interest The authors indicate no potential conflicts of interest.

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 Table (1): Comparison between studied groups according to clinical data

Group 3 Group 4
Clinical data Group 1 Group 2
(n = 10) (n = 10)
(n = 10) (n = 10)
No. % No. %
No. % No. %

Alopecia areata types

Ophiasis 6 60 3 30
Patchy AA 2 20 6 60
Alopecia totalis 2 20 1 10
 (
2 MC
p): 3.248 (0.324)
Androgenetic alopecia types
MPHL 3 30 5 50

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FPHL 7 70 5 50

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2 (MCp): 0.833 (0.650)

Duration (years) 1.1 – 4 1–3

Min. – Max. 0.8 – 4 0.8 – 3 2 ± 0.9 1.9 ± 0.8
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Mean ± SD. 2±1 1.5 ± 0.7
1. 8 1.8
Median 1.8 1.3
Z (p) 0.957 (0.339) 0.114 (0.909)
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Table (2): Evaluation of clinical efficacy after stem cells therapy in all studied groups.

Clinical efficacy Group 1 Group 2 Group 3 Group 4

No. % No. % No. % No. %
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Excellent 0 0 3 30 1 10 0 0
Very Good 5 50 2 20 5 50 5 50
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Good 3 30 3 30 1 10 0 0
Fair 2 20 2 20 3 30 5 50
Table (3): Comparison between the studied groups according to clinical efficacy after six months of end

of treatment

Stem cell therapy type Alopecia areata AGA p1

BMMC Group 1 Group 3
Min. – Max.( of clinical response) 0– 75 10 – 85
Mean ± SD. 45 ± 22 52 ± 28 0.364
Median 50 63
FSC Group 2 Group 4
Min. – Max. (of clinical response) 0– 100 10 – 75
Mean ± SD. 58 ± 34 42 ± 27 0.288
Median 52 40

p2 0.426 0.445

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p1: p value for Mann Whitney test for comparing between Alopecia areata and AGA

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p2: p value for Mann Whitney test for comparing between BMMC and FSC

*: Statistically significant at p ≤ 0.05

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 Table: (4) Comparison between the studied groups of patients by digital dermoscopic measures

Group Group Group Group

Group Grou Group Group
Hair 1 3 Density/ 1 3
2 p4 2 4
width (cm²)
(AA- (AGA- (AA- (AGA-
(mm) (AA- (AGA (AA- (AGA-
BMMC BMMC Baseline BMMC BMMC
FSC) -FSC) FSC) FSC)
Baseline ) ) ) )

Min. – 0 – 0.01 – 0 – Min. – 0 – 46–

0 – 0.02 0 – 114 62– 218
Max. 0.07 0.08 0.09 Max. 534 136
Mean ± 0.01 ± 0.01 ± 0.02 ± 0.03 ± Mean ± 63±
51 ± 40 119± 47 92± 26
SD. 0.01 0.01 0.01 0.02 SD. 132
p1
p1 0.061 0.547
0.172 0.017*

After 6 months of end of therapy After 6 months of end of therapy

Min. – 0– 0.02 – 0.03 – Min. – 88–
0– 0.04 0– 208 0– 690 83– 231
Max. 0.04 0.04 0.04 Max. 205
Mean ± 0.02 ± 0.03 ± 0.04 ± Mean ± 206± 136±
0.03 ± 0 102± 40 147± 43

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SD. 0.01 0.01 0 SD. 202 27
0.003* 0.550 p1 0.001* 0.352
p1

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<0.001 <0.001 <0.001
P2 <0.001* * <0.001* 0.003* P2 <0.001* * <0.001* *

 : Chi square for Kruskal Wallis test, Sig. bet. grps was done using Mann Whitney test
KW 2
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p1: p value for comparing between group 1 and group 2 also group 3 and group 4

p2: p value for Wilcoxon signed ranks test for comparing between before and after six months
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*: Statistically significant at p ≤ 0.05

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 Legends to the figures

Figure (1) Patients with alopecia areata

A and B : a female patient with AA in group 1 (ophiasis) on the left side of the scalp.

A; before therapy, B; six months after treatment with BMMC showing excellent response ,

C and D: a male patient with AA in group 2 (patchy type) on the back of the scalp.

C; before therapy, D; six months after treatment with FSC showing excellent response.

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Figure (2): patients with androgenetic alopecia
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A and B : A female patient with AGA in group 3.
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A; before therapy (grade III), B; six months after treatment with BMMC showing excellent
response ( changed grade I).
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C and D : A female patient with AGA in group 4 .

; before therapy (grade III), B; six months after treatment with FSC showing very good
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response ( changed to grade II).

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Figure (3) dermoscopic pictures of alopecia areata patients

showing (A) ; Active AA lesion before therapy with tapering hairs (yellow arrows), and black dots (red

arrows), ( B); the same lesion after SC therapy showing normal hair (complete cure) (C); long standing

alopecia totalis lesion with honey comb appearance and white dots of empty follicles,(D) ; the same lesion

after SC therapy showing regrowing vellus hair, but honey comb appearancestill present in the background
 (E);ophiasis lesion with telangiectasia (white arrows), of previous intralesional steroid , (F); the same lesion

after normal hair (cure)

Figure (4) digital dermoscopy of androgenetic alopecia

( A): Hair density before the therapy ,the red dots (thin hairs) are more than green dots (terminal hairs ) (

more hair thinning). (B): Hair density after stem cell therapy the green dots (terminal hairs ) are more than

red dots ( more terminal hairs). (C):Hair width measurements before the therapy , ( more hair thinning).

(D): Hair width measurements after stem cell therapy the hair becomes more thick ( more terminal hairs).

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Figure (5) Horizontal sectionsof immunohistochemichal stainning in hair follicles
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(ORS) of scalp samples of alopecia patients treated with stem cell therapy showing:

A: strong positive (+3) CK 15 immuno stainning in alopecia areata hair follicle (x 400)
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B: strong positive (+3) CK 15 immuno stainning in androgenetic alopecia scalp section (x 400)
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C: strong positive (+3) CD200 immuno stainning in alopecia areata (germenitive epithelium of hair follicle ) (x 100)

D: strong positive (+3) CD200 immuno stainning in androgenetic alopecia (germenitive epithelium of hair follicle ) (x
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100)

E: weak positive (+1) CD200 immuno stainning in alopecia areata hair follicle (x 400)
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F: moderate positive (+2) CD34 immuno stainning in androgenetic alopecia hair follicle (x 100)
Graph (1): Relation between Alopecia areata type and clinical efficacy of stem cell therapy after six

months of treatment.

Graph (2): Relation between androgenetic alopecia type and clinical efficacy after six months of

treatment.

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