**6. Stem‐cell therapy in alopecia**

There are various studies showing the positive effects of laser/light treatments in AGA. In a previous study, the effects of laser on cancer were investigated in mice. The dorsal hair of mice was shaved and the low‐powered ruby laser (694 nm) therapy was given toward this area. They did not find any evidence of cancer but observed accelerated hair growth in laser‐ treated sides [100]. In a clinical study, seven patients with a diagnosis of AGA were exposed to LLLT twice weekly for 20 min for 3–6 months. An increase in the number of terminal hair, a decrease in the number of vellus hair, and an increase in shaft diameter were observed in this

To assess the effect of a 1550 nm fractional erbium‐glass laser in a female pattern hair loss, 28 patients received 10 treatments at 2‐week interval. At the end of the study, a marked increase in hair density and hair shaft thickness and significant improvement at the frontal hair recess were seen in patients. It was revealed that 1550 nm fractional erbium‐glass laser may be a safe and effective treatment option for female pattern hair loss (FPHL) [101]. In a clinical study, the effects of a 1550 nm fractional erbium‐glass laser on the hair cycle in an alopecia mouse model and on the treatment of male pattern hair loss were investigated. In the human pilot study, an increase in hair density and an improvement of growth rate were observed. In the animal study, the effect on hair stimulation was dependent upon the energy levels, densities, and irradiation intervals. Fractional laser irradiation can promote anagen hair growth and induce transition from the telogen phase to the anagen phase. It was shown that Wnt 5‐α and β‐catenin expressions play a role in hair growth were induced by laser irradiation [102]. In a study of 32 patients with male and female androgenetic alopecia, the efficacy and safety of LLLT were evaluated. A Laser comb (655 nm) was used as monotherapy or as a concomitant therapy with minoxidil and finasteride. Eight patients showed significant improvement, 20 patients showed moderate improvement while no improvement was observed in four patients. Improvement was observed in both monotherapy and the dual therapy group [103]. Previously, a Laser comb has been tested in 110 patients with AGA in a double‐blind, sham device‐controlled, multicenter, and 26‐week trial. Significant increase in mean terminal hair density was observed in patients in the LLLT group when compared to patients in the sham device group [104]. Jimenez et al. reported a statistically significant increase in terminal hair density after 26 weeks of low‐level laser comb device treatment compared with sham treat-

study but these changes were not statistically significant [89].

ment in patients with FPHL and male pattern hair loss (MPHL) [105].

As there is no cure for alopecia areata which is an autoimmune disease and may improve spontaneously in 34–50% of patients, clinicians search for new treatment modalities such as

There are limited studies about laser irradiation for alopecia areata. In a study, clinicians used 308 nm xenon chloride excimer laser (XeCl) for two patients with alopecia areata for 11–12 sessions within a 9–11 weeks period. They observed homogeneous and thick hair growth. The exact mechanism was not clear, but immunosuppressive effects of laser irradiation by inducing T‐cell apoptosis and interrupting autoaggressive immune cascade were

**5.2. Alopecia areata**

330 Hair and Scalp Disorders

laser/light sources [86, 106, 107].

### **6.1. Hair follicle, stem cells, and dermal papilla**

Hair follicle (HF) is a complex structure that contains important units in the development of hair shaft including dermal papilla, matrix, and bulge region [3].

The HF undergoes cycles of growth and degeneration that a new hair shaft is formed in each cycle [115]. The signaling in this cycling is not completely understood. Fundamentally, there is a bidirectional communication between the mesenchymal and stem cells within the hair follicle that controls the formation, growth, and cycling of hair follicle [3, 116, 117].

Dermal papilla (DP) is located at the bottom of hair follicle (hair bulb) and consists of specialized mesenchymal cells which produce signals regulating the hair cycling of follicular epithelium and also driving the formation of hair follicle [116, 117]. Bulge region of hair follicle houses epithelial stem cells that become progenitor cells forming the hair follicle. Upon the stimulatory signals from DP cells, progenitor cells move down to the deep dermis where they turn into matrix cells which differentiate to form different parts of hair follicle [3, 117, 118]. It can be understood from these information that although the immediate formation of hair shaft and follicle is achieved by the matrix cells in the DP, reservoir stem cells reside in the upward bulge region that maintain the follicle regeneration [115].

Stem cells are characterized by the capacity of self‐renewal and ability to differentiate into various cell lineages. Hair follicle stem cells (HFSCs) which are found in hair bulge are quiescence cells that divide infrequently [3]. HFSCs are multipotent that they can give rise to all cells of a hair follicle, sebaceous gland, and interfollicular epidermis [3, 115, 118]. In addition, hair follicle bears other types of stem cells including interfollicular epidermal stem cells, sebaceous gland stem cells, follicle nestin + pluripotent stem cells, etc. [3, 115].

The induction of hair cycling and hair follicle regeneration from the HFSCs is a complex process which starts with the signals from DP cells. This interaction involves several signaling pathways, growth factors, specific protein ligand‐receptor binding, upregulation of various hair‐related genes and activation of different transcription factors [3, 116, 118].

#### **6.2. The rationale behind the stem‐cell therapy in the treatment of alopecia**

As the current treatment options for most types of alopecia including AGA and AA are not satisfactory, new therapies are still being under investigation for various types of alopecia. Development of bioengineering technologies has provided the use of HFSCs as a promising treatment in the management of alopecia. Since the conventional drugs for alopecia are unable to target all the pathophysiologic factors, stem‐cell therapy is considered as a potential solution to correct the main pathology in various types of alopecias [3, 115, 117, 118].

It has been suggested that the distinct pathophysiologic pathways may be targeted by stem cell therapies in different diseases. An important point is that in order to specifically manage the alopecia, it is important to clarify the exact etiologic mechanism underlying various types of alopecias [3, 118]. For example, in AGA, the main etiology is that the HF is miniaturized by the effect of 5‐DHT and the signaling that drives the HF regeneration is impaired. Although the stem cells in bulge region are undamaged, the production of new hair formation is interrupted in AGA [3, 116]. Another example for the impaired induction of hair formation by the destruction of DP region is the chemotherapy induced alopecia. Induction of hair generation by DP cells has been suggested to be achieved by stem‐cell therapy in this type of alopecia [116]. In AA, DP (bulbar region of HF) is attacked by the immune cells [3, 115]. Stem‐cell therapy has been suggested to be effective in the suppression of autoimmune destruction and recovery of immune balance in AA patients [119]. In cicatricial alopecia, inflammation leads to the destruction of the bulge region where the normal immune privilege has been lost by pathologic triggers and stem cells are destroyed [115]. Producing a new hair follicle unit via transplantation of stem cells has been suggested as a major innovation for the treatment of most forms of alopecias including scarring alopecia [3, 115, 117, 118].

#### **6.3. Preliminary hair follicle generation studies and obstacles in stem cell therapies**

The HF undergoes cycles of growth and degeneration that a new hair shaft is formed in each cycle [115]. The signaling in this cycling is not completely understood. Fundamentally, there is a bidirectional communication between the mesenchymal and stem cells within the hair

Dermal papilla (DP) is located at the bottom of hair follicle (hair bulb) and consists of specialized mesenchymal cells which produce signals regulating the hair cycling of follicular epithelium and also driving the formation of hair follicle [116, 117]. Bulge region of hair follicle houses epithelial stem cells that become progenitor cells forming the hair follicle. Upon the stimulatory signals from DP cells, progenitor cells move down to the deep dermis where they turn into matrix cells which differentiate to form different parts of hair follicle [3, 117, 118]. It can be understood from these information that although the immediate formation of hair shaft and follicle is achieved by the matrix cells in the DP, reservoir stem cells reside in the

Stem cells are characterized by the capacity of self‐renewal and ability to differentiate into various cell lineages. Hair follicle stem cells (HFSCs) which are found in hair bulge are quiescence cells that divide infrequently [3]. HFSCs are multipotent that they can give rise to all cells of a hair follicle, sebaceous gland, and interfollicular epidermis [3, 115, 118]. In addition, hair follicle bears other types of stem cells including interfollicular epidermal stem cells, seba-

The induction of hair cycling and hair follicle regeneration from the HFSCs is a complex process which starts with the signals from DP cells. This interaction involves several signaling pathways, growth factors, specific protein ligand‐receptor binding, upregulation of various

As the current treatment options for most types of alopecia including AGA and AA are not satisfactory, new therapies are still being under investigation for various types of alopecia. Development of bioengineering technologies has provided the use of HFSCs as a promising treatment in the management of alopecia. Since the conventional drugs for alopecia are unable to target all the pathophysiologic factors, stem‐cell therapy is considered as a potential

It has been suggested that the distinct pathophysiologic pathways may be targeted by stem cell therapies in different diseases. An important point is that in order to specifically manage the alopecia, it is important to clarify the exact etiologic mechanism underlying various types of alopecias [3, 118]. For example, in AGA, the main etiology is that the HF is miniaturized by the effect of 5‐DHT and the signaling that drives the HF regeneration is impaired. Although the stem cells in bulge region are undamaged, the production of new hair formation is interrupted in AGA [3, 116]. Another example for the impaired induction of hair formation by the destruction of DP region is the chemotherapy induced alopecia. Induction of hair generation by DP cells has been suggested to be achieved by stem‐cell therapy in this type of alopecia [116]. In AA, DP (bulbar region of HF) is attacked by the immune cells [3, 115]. Stem‐cell

solution to correct the main pathology in various types of alopecias [3, 115, 117, 118].

follicle that controls the formation, growth, and cycling of hair follicle [3, 116, 117].

upward bulge region that maintain the follicle regeneration [115].

332 Hair and Scalp Disorders

ceous gland stem cells, follicle nestin + pluripotent stem cells, etc. [3, 115].

hair‐related genes and activation of different transcription factors [3, 116, 118].

**6.2. The rationale behind the stem‐cell therapy in the treatment of alopecia**

As the epithelial‐mesenchymal interaction is crucial in the development of HF, it is essential to coculture DP cells with stem cells in order to generate a complete HF in laboratory condition. However, it has to be in mind that it is not easy to obtain and grow stem cells in laboratory experiments and their turnover is low [115, 117].

Marazzi et al. have isolated human follicle DP and bulge cells and cultured them in human skin sample (organotypical culture). After injection of the cultured bulge and DP cells into deep dermis, epidermis forming ability of the cells was assessed. The authors suggested their methodology as a relevant source of bioengineered hair follicles for hair transplantation therapies in alopecia [120].

In a previous report, mouse embryonic skin‐derived stem cells were used to form a hair germ and the resultant bioengineered follicle germ was intracutaneously transplanted to create a structurally correct hair follicle. On the back skin of a nude mouse, the transplanted follicle germ was able to form hair shaft, construct appropriate connection with surrounding tissue, and undergo cycling [121]. As the transplantation of a mature bioengineered hair follicle rather than follicle germ is considered to be more favorable in hair regeneration, Asakawa et al. in their animal study, have shown that ectopic transplantation of bioengineered hair follicles (created by follicle germ cells from embryonic pelage skin and regenerated *in vitro* culture) could develop a fully functional hair follicle in host. Authors reported that the results of the study have indicated transplantation of the bioengineered hair follicles could replace the conventional FUT therapy in alopecia treatment [122].

An important problem in the hair follicle regeneration studies is that cultured DP cells lose their inductive capacity after a few passages. Attempts including co‐culturing with keratinocytes and adding growth factors to the medium have been done to effectively expand DP cells *in vitro* culture [117]. As the laboratory conditions and *in vitro* assays are far from the *in vivo* ambience of DP cells, to better simulate the real hair follicle, three‐dimensional (3D) dermal spheric cultures have been generated [123]. To further increase the inductive capacity of DP cells and to enhance the reproducibility of assays, novel membranes for spheric culturing have been used [124]. One of the most important obstacles in hair regeneration studies is the results of animal or *in vitro* studies differ from those on human. Despite an intact HF can be formed in murine and embryonic cell experiments, incomplete HF are formed with human DPCs. Subcutaneous implantation of isolated human HFSCs and human scalp DPCs resulted in the formation of hair follicle‐like structures in nude mice [125]. To overcome this problem strategies such as culturing DPCs with keratinocytes have been formulated [126]. Recently an acellular dermal matrix has been used to grow human epithelial and dermal cells from scalp tissue with promising results [127]. Recently, human DP cells from scalp tissue have been embedded into dermal–epidermal composites (DECs) and formation of complete HF has been observed [128].

By ongoing studies, it was realized that not only the close environment of HF but also the macro environment of HF is important in the growth induction of HF. As the adipocyte stem cells (ASCs) secrete growth factors and stimulate hair growth pathways and the activation of hair follicle stem cell by adipocyte lineage cells has been shown, ASCs and ASC‐conditioned medium (ASC‐CM) have been investigated in hair regeneration studies. DPCs which are cultured in ASC‐CM showed increased proliferation. These studies suggested a role for ASCs in alopecia treatment [117, 129].

Bone marrow mesenchymal stem cells (BM‐MSCs) have also been used to induce hair induction *in vitro* assays and tested for HF formation capacity in mouse models [116].

#### **6.4. Studies on the stem‐cell therapy in alopecia**

In a randomized placebo‐controlled trial, topical application of a commercially available solution containing HFSCs in male patients with AGA was found to be effective in the induction of hair growth and reduction of hair loss [130]. Supernatant of BM‐MSC culture overexpressing Wnt1a has been shown to increase hair producing ability of DP cells. Additionally, intradermal injection of concentrated solution of the above mentioned supernatant enhanced the transition from telogen to anagen in mouse. Also, negative effect of a 5‐DHT on hair related genes was restored with the addition of Wnt‐CM. Study indicated a role for Wnt1a from MSCs in hair regeneration therapies for alopecia [116].

The effect of intradermal injection of commercially available ASC‐CM product (containing hepatocyte growth factor, fibroblast growth factor‐1, granulocyte colony‐stimulating factor, granulocyte macrophage‐colony‐stimulating factor, interleukin‐6, vascular endothelial growth factor, and transforming growth factor β‐3) to 22 AGA patients (11 males, 11 females) has been studied. Patients were treated in six sessions at 3–5‐ week interval. Six male patients were also on finasteride treatment. Half‐side comparison study has been undertaken in 10 patients. Hair counts were increased in all patients according to trichogram assays. In comparison study, hair count was increased in both side of the scalp, however, the increase was higher in the treatment side compared to the placebo side. The response in the placebo side is suggested to be related to the effect of injection itself or the diffusion of the solution to the other side [131].

In another study with the same product, 27 patients with FPHL were treated with the solution (ASC‐CM) weekly with concurrent use of microneedling roller. Retrospective assessment of the results revealed significant increment in the hair density and thickness after 12 sessions [129].

An evidence to the alternative mechanisms of stem‐cell therapy is the "stem cell educator therapy" which has been used for its immune modulation effect in nine AA patients. Cord blood stem cells (CB‐SCs) have been used to be introduced to patient's blood in a closed loop system. Patient's lymphocytes are separated and cocultured with CB‐SCs *in vitro* and returned to patient's circulation after "education." A significant suppression of CD8Tcell attacking and upregulation of the co inhibitory molecules resulted in the diminishment of autoimmune destruction and reversal of immune balance by shifting the immune response toward Th2. As only a small portion of lymphocytes encounter with CB‐SCs, the educated immune response has been suggested to be expanded systemically leading a generalized outcome [119].

In a recent review, an unpublished study (NCT01286649) has been reported investigating the efficacy of injecting human autologous HF dermal sheath cup cells which have been taken by punch biopsy from the scalp of patients with AGA. The results of the study await publication [118].

#### **6.5. Contraindications and side effects of stem‐cell therapy in alopecia treatment**

The presence of skin disease, inflammation or infection, having an allergic, autoimmune disease or cancer, pregnancy, and the usage of anticoagulant therapy are reported as contraindications of stem‐cell therapy [131]. Most of the studies on stem‐cell therapy in alopecia treatment reported no severe adverse effects [119, 129, 132]. Patients can feel pain when injection technique is used which can be overcome by nerve blockages, local anesthesia, cooling, or prescription of nonsteroidal antiinflammatory drugs [131].
