Alopecia areata (AA) is a nonscarring type of hair loss. It may be patchy or may extend to involve the entire scalp (alopecia totalis), or the whole body (alopecia universalis). It is a relatively common condition affecting 0.15% of the population. Although in many cases it can be a self-limiting condition, nevertheless hair loss can often have a severe social and emotional impact .
The origin of AA is not fully understood. However, T cell-mediated autoimmune process, genetical, immunological, and psychological factors are all implicated in the pathogenesis of AA .
Till date, there is no best treatment and no Food and Drug Administration (FDA) approved drug for AA. Conventional treatments include topical corticosteroids, tacrolimus, topical immunotherapy, psoralen followed by ultraviolet A exposure, or intravenous pulse corticosteroids .
The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has not only encouraged researchers to use the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, but has also drawn attention toward the use of hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. Thus, the hair follicle provides an ideal model system not only for the study of fundamental issues in SC biology such as plasticity and SC niches, but also for the identification of reliable, specific SC markers .
On the basis of these data, it seemed prudent to start a research study to evaluate the follicular SCs as a hopeful therapeutic option for this emotionally devastating disorder.
Patients and methods
The study was carried out at the Departments of Dermatology, Clinical Pathology, and Histology, Kasr El Aini Faculty of Medicine, Cairo University, Egypt.
A pilot prospective study was carried out.
Eight patients with AA of the scalp were included in this study. The patients were diagnosed clinically and by scalp biopsy of the affected area. Any medications given earlier were stopped at least 6 months before the treatment. The study has been approved by the Research Ethics Committee of the Dermatology Department, Cairo University. The Declaration of Helsinki protocols were followed. A full-detailed written informed consent for treatment participation, biopsy, and photography was signed by each patient or the patient's parents before starting the treatment.
No age or sex was specified. The surface area of AA ranged between 30 and 80% surface area.
Pregnancy, alopecia totalis, systemic diseases, and immunosuppression.
All participants were subjected to the following
- (1) Complete history taking (age, sex, family history, onset, course, duration, and any associations of the disease);
- (2) Thorough clinical examination and hair counts in the affected area;
- (3) Photography before and after treatment;
- (4) Three scalp biopsies were performed.
Two scalp biopsies (4 mm skin punch) were performed before treatment. One biopsy was from the affected (nonhairy) site, which was fixed in 10% formol saline (10% solution of formalin in 0.9% aqueous NaCl), processed into paraffin sections, and stained with hematoxylin and eosin (H&E) to document the diagnosis, and the other one was from a nonaffected (hairy) area. The latter was subdivided into two parts: the first part was fixed in 10% formol saline and processed into paraffin blocks to be subjected to staining with H&E, Prussian blue (counter sainted with neutral red), and with S100 A4 immunohistochemistry as a marker for follicular SCs ; while the second part was put in Hank's balanced salt solution (Invitrogen, Carlsbad, California, USA) for subsequent cell culture.
A third scalp biopsy was performed after 3 months of treatment, from the injected area, and was stained with H&E to assess the morphology and with Prussian blue for the tracking of iron oxide labeled cells as discussed later. Plucked hairs from each patient were also taken for coculture.
Paraffin sections (5 μm thickness) from nonaffected hairy scalp region were stained with S100 A4 (Ab-8 Rabbit Polyclonal Antibody Cat. ♯RB-1804-R7), supplied as prediluted antibody by NeoMarkers (NeoMarkers, Fremont, California, USA). Staining of formalin-fixed tissues required boiling of tissue sections in 10 mmol/l citrate buffer, pH 6.0, (NeoMarkers, Cat. ♯AP-9003), for 10–20 min followed by cooling at room temperature for 20 min. The Super SensitiveTM Link-Label Detection Systems Concentrated Format (BioGenex, San Ramon, California, USA), avidin–biotin complex technique and 3,3′-diaminobenzidine tetrahydrochloride (DAB chromogen, BioGenex) were used as a revelation system. Nuclei were counter stained with Mayer's hematoxylin.
Hair follicle isolation
Under complete aseptic conditions in laminar flow cabinet, scalp tissues received were rinsed, trimmed to remove excess adipose tissues, cut into small pieces, and subjected to enzymatic dissociation in 12.5 mg/ml dispase (Invitrogen) in Dulbecco's modified Eagle's medium for 24 h at 4°C.
After treatment, the epidermis was peeled off from the dermis, and hair follicles were plucked from the dermis. Hair follicles were rinsed thoroughly with phosphate-buffered saline, to prevent the contamination of the epidermal or dermal cells, and examined under an inverted microscope.
To isolate hair follicle SCs, the lower bulge areas were treated twice with 0.25% trypsin/EDTA (Invitrogen) for 30 min at 37°C, according to the method described by Yu et al. , where enzymatic digestion is used. Nondissociated parts were left in trypsin and the single cells were separated after 24 and 48 h.
The cell suspension was filtered through a 40-μm cell strainer (BD Falcon, Bedford, Massachusetts, USA), and cell numbers were counted. Single cells were cultured in noncoated flasks containing Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen), 200 mmol/l L-glutamine (Invitrogen), 0.1 mmol/l β-mercaptoethanol (Sigma, St Louis, Missouri, USA), 1% nonessential amino acids (Invitrogen), and 4 ng/ml basic fibroblast growth factor (Research Diagnostics, Concord, Massachusetts, USA). Media were changed every 72 h for 2 weeks. Plucked hairs from the same individual were treated with trypsin as described above and cocultured with hair follicle eSCs.
The cells were washed, counted, and subjected to the following before injection
- (1) Viability assessment: cell suspension in a density of 100 000 cell/ml are added to 100 μl trypan blue and incubated for 5 min at 37°C. Cells were evaluated under high power microscope. Cells stained with the dye are counted as dead and the percentage of viable cells is calculated;
- (2) Cell tagging was done using iron oxide particles [Feridex I.V. (ferumoxides) 11.2 mg/ml, Bayer Healthcare Pharmaceuticals, Wayne, New Jersey, USA] by adding the cell suspension in a density of 100 000 cells/ml to 20 μl iron oxide and incubated for 4 h at 37°C. Cells were washed with phosphate-buffered saline to remove excess iron particles. The cell suspension was washed and the pellet was suspended in 5 ml sterile saline for injection.
Method of applications
Every patient was instructed to apply local anesthetic cream on the affected areas half an hour before injection. The injection was performed intradermally with a 23 gauge needle. Each patient was injected once. One millilter (in a density of 100 000 cell/ml) was injected at per centimeter square of the treated site. After injection, a local antiseptic was prescribed for 2 days later.
Assessment of engraftment
Another scalp biopsy was performed after 3 months of injection, from treated areas. Histological sections were prepared, stained with H&E to assess the morphology and with Prussian blue to detect the iron oxide tagged cells.
Clinical assessment of the patients
The clinical improvement was assessed by calculating the percentage of the difference in the AA at the end of 3 months and 6 months in relation to the baseline extent. For example, the percentage of improvement at the third month=
Grading of the response
Degree of response was graded as follows: excellent response if there was an improvement of 50% or more, good if the improvement was less than 50–10%, and poor if the improvement was less than 10%.
Data were statistically described in terms of range, mean±standard deviation, frequencies (number of cases), and relative frequencies (percentages) when appropriate. Comparison was made using Mann–Whitney U-test for independent samples. Correlation between various variables was done using Pearson's correlation equation for nonnormal variables. A probability value (P value) of less than 0.05 was considered statistically significant. All statistical calculations were done using computer programs such as Microsoft Excel (Microsoft Corporation, New York, USA) and SPSS version 10 for Microsoft Windows (Statistical Package for the Social Science; SPSS Inc., Chicago, Illinois, USA).
Eight patients with AA were included in this study (five girls and three boys). Family history was positive in three cases. All cases had gradual onset and progressive course. Their age ranged from 6 to 17 years. The mean age of the patients was 10.12±3.68 years. There was a negative correlation between the age of the patients and the grade of response they achieved, that is, the younger the patient, the better the response, but this correlation was statistically insignificant (P=0.647). The duration of disease ranged from 1 to 4 years (mean: 2.31±0.96 years). The extent of affection ranged from 25 to 70% (mean: 48±15%). The duration and extent of the disease were both correlated with the grade of response and showed negative correlations, but without statistical significance (P=0.993 and P=0.646, respectively) (Table 1).
Follow-up was carried out at the first, third and sixth months after injection. Patients were reviewed for any side effects, locally examined for new erupted hairs by visual method, and percentage of improvement was registered. At the end of the first month, none of the patients showed any response. Along the whole period of follow-up, one patient showed no response and was excluded from the analysis. Patients showed variable degrees of response, 20–80% (mean: 45±22%) from baseline at the end of the third month and 30–100% (mean: 69±27%) from baseline at the end of the sixth month. Two patients who developed pain at the site of injection, which lasted for 12 h, were relieved by rest and not by analgesia. One patient who showed pustular eruption, which lasted for 2 days, was treated with topical antibiotic cream, and one patient developed a new lesion at a distant site away from injection site at the end of 6-months follow-up period. Excellent response was achieved in five patients (62.5%), good response was achieved in two patients (25%), whereas one patient (12.5%) showed poor response at the end of 6-months evaluation (Table 2, Fig. 1a and b).
Histological, histochemical, and immunohistochemical results
Examination of H&E stained scalp biopsies from affected (nonhairy) areas revealed the typical diagnosis of AA where there was perifollicular infiltration by mononuclear cells with remnants of fibrous tracts around miniaturized follicles (Fig. 2).
S100 A4 immunohistochemistry of biopsies obtained from nonaffected hairy scalp region before injection showed positively-stained cells in the outer root sheath of the hair follicle and in the dermal fibroblasts (Fig. 3).
H&E stained sections, prepared from scalp biopsies of treated areas 3 months after injection, showed a normal morphology of hair follicles (Fig. 4).
Sections of scalp biopsies obtained from nonaffected hairy area before treatment stained with Prussian blue showed negative reaction (Fig. 5), whereas Prussian blue stained sections of scalp biopsies obtained from treated areas 3 months after injection showed positively-stained iron particles in the regenerating hair (Fig. 6a and b).
AA is a relatively common condition causing nonscarring hair loss, yet its pathogenesis is unclear . Treatment of AA is a difficult challenge. All tissues in the mammalian adults are ultimately derived from SCs . SCs possess the salient features of multipotency and self-renewal, enabling them with the ability to differentiate into the multiple cell types required to generate new tissue throughout the development and the lifetime of the organism .
In this study, eight patients of AA were included. For demonstration of follicular SCs, scalp biopsies from nonaffected areas were taken and S100 A4 immunohistochemistry was used as a marker for follicular SCs . S100 A4 is a member of S100 calcium-binding proteins implicated in a variety of cellular events, including growth, signaling, differentiation, and motility . In concordance with Ito and Kizawa , we detected positively- stained cells in the outer epithelial layer of the hair follicle and in the dermal fibroblasts.
There are many methods to isolate hair follicle SCs such as a microdissection method described by Roh et al.  and Lako et al. . However, in this study, we followed the method of Yu et al.  where enzymatic digestion was used.
To elucidate the exact fate and mechanism of action of the injected cells, in-vivo cell tracking is important. One of the FDA-approved methods of in-vivo cell tracking in humans is the superparamagnetic iron oxide (SPIO) nanoparticles. These particles do not affect cell viability, function, or immune profiles. They can be detected radiologically by magnetic resonance imaging or in tissue sections by iron-staining dyes . We postulate that the presence of iron-staining cells in the bulge area denotes migration of follicular SCs to their anatomical niche while the presence of iron-staining mature cells or iron-staining hair denotes proliferation and differentiation of follicular SCs. In our study, histological sections from scalp biopsies obtained from treated areas 3 months after injection, stained with Prussian blue to detect iron-oxide tagged cells showed iron particles in the regenerating hair. This particular finding can exclude the possibility that hair regrowth occurred as a response to scalp injection per se.
The mean age of the patients was 10.12±3.68 years, there was a negative correlation between the age of the patients and the grade of the response they achieved; this may be explained by the increased activity of telomerase in children . However, this correlation was statistically insignificant, probably because of small number of patients enrolled in this study.
Although many treatments have been shown to stimulate hair growth in AA, there are limited data on their long-term efficacy and impact on quality of life. In our study, approximately 60% of the patients (the excellent responders) reported improved quality of life as regards, regular school attendance, social interactions, and peer relations. This study faced many limitations such as small sample size and awaiting long-term follow-up.
Due to the lack of availability of SC therapeutic trials, it is too early to judge upon, whereas Nevskaya et al.  showed the efficiency of local CD34(+) cell therapy for systemic sclerosis ischemic complications.
Placebo-controlled study on a larger sample size and with longer follow-up period has to be conducted to confirm our preliminary findings.
This is the first open-label pilot study that shows the feasibility, efficacy, and short-term safety of local follicular SC therapy for AA.
There is no conflict of interest.
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Keywords:© 2011 Egyptian Women's Dermatologic Society
alopecia areata; follicular stem cells; S100 A4