Adipose derived stem cell based therapies or male/female pattern hair loss

In the United States, Alopecia Androgenetica, more commonly known as male or female pattern hair loss, affects approximately 50% of the male population and 45% of the female population [1,2]. Alopecia Androgenetica can be characterized by the gradual thinning of hair emphasized at the crown and frontal scalp, attributed to interactions among various genetic and endocrine factors [3,4]. Individuals impacted by Alopecia Androgenetica are subject to general psychological trauma, as many report a decreased quality of life, lack of self-confidence, and limited social contacts [4-6].


Introduction
In the United States, Alopecia Androgenetica, more commonly known as male or female pattern hair loss, affects approximately 50% of the male population and 45% of the female population [1,2]. Alopecia Androgenetica can be characterized by the gradual thinning of hair emphasized at the crown and frontal scalp, attributed to interactions among various genetic and endocrine factors [3,4]. Individuals impacted by Alopecia Androgenetica are subject to general psychological trauma, as many report a decreased quality of life, lack of self-confidence, and limited social contacts [4][5][6].
The hair growth cycle in the scalp is separated into 3 phases: anagen (growth), catagen (regression), and telogen (resting). The 26 year long anagen phase is classified as the growing period, whereas the catagen phase of regression lasts 23 weeks and is followed by a 12 week telogen resting phase [4]. Typical anagen to telogen ratio in an individual experiencing regular hair growth is established at 9:1 4. Individuals with Alopecia Androgenetica, however, experience a decrease in the duration of anagen phase as the hair cycle proceeds [4]. As the anagen phase decreases, the proportion of hair follicles in the telogen phase increases, inducing hair growth that is more fine [4]. Additionally, an increase in the turnover time between hair shedding and the anagen (growth) phase diminishes hair density at the scalp [4]. The degree to which individuals are affected by Alopecia Androgenetica is classified using the Norwood-Hamilton scale for men ( Figure 1) [7], and the Savin scale for women ( Figure 2) [8]. An alternative scale for females is the Ludwig scale, which is nearly identical to the Savin scale. These scales are based on standardized visual parameters and are often used to evaluate drugs in clinical hair growth studies [4]. Grade II on the Norwood-Hamilton scale is the most prevalent kind of Alopecia Androgenetica, affecting approximately 27.2% of patients [9].

Current treatments
There has been a relative lack of new therapeutic options for the treatment of hair loss in the last 30 years. While a significant amount of research and capital have been invested in the sector, investigated treatments have not proven effective enough to replace current treatment modalities (Table 1). Prevalent remedies for Alopecia Androgenetica include two leading pharmaceutical treatments, topical Minoxidil and oral Finasteride. However, both treatments are notorious for producing a myriad of undesirable side effects. While the mechanism of topical Minoxidil, a piperidinopyrimidine derivative, is not fully understood, it is known to cause contact dermatitis and facial hypertrichosis, mainly in women [4]. Topical Minoxidil  requires application to treatment areas twice a day, and must be used consistently before results are seen within the first 46 months. Maximum remedial effects are established at 12 years, at which point hair counts plateau, and hair density quickly returns to baseline values in the event that treatment is discontinued [4]. Oral Finasteride, a synthetic azosteroid, acts as a 5alphareductase inhibitor that prevents conversion of testosterone to dihydrotestosterone (DHT) [4]. Oral Finasteride must be taken consistently and indefinitely as progressive balding will be induced once treatment comes to a halt. Negative side effects of oral Finasteride include decreased Prostate Specific Antigen (PSA), libido and ejaculate volume, as well as erectile dysfunction [4]. When ingested for longer than 2 years, oral Finasteride is not shown to facilitate continued hair growth, but results were maintained at 5 years (longest reported follow up) [4]. Additionally, Finasteride is only prescribed for male patients as it has shown limited efficacy in women and may cause birth defects in pregnant women. Dutasteride, commercially available as Avodart, is a 5alphareductase inhibitor used to treat enlarged prostates, similar to Finasteride which is also used by some for treatment of male pattern baldness, but has similar efficacy to Finasteride and is again not recommended for use by women. While finasteride and dutasteride are not indicated for use in women, come offlabel studies have shown efficacy at higher doses [10][11][12][13][14]. Efficacy results for finasteride and dutasteride are conflicting across different studies, with some showing efficacy and others not. Apart from topical Minoxidil, oral Dutasteride and oral Finasteride, the current state of the art non pharmaceutical method is the Follicular Unit Extraction (FUE), more commonly known as a hair transplant, which can be costly and result in noticeable donor sites. Additionally, there is a risk that the transplanted hairs will not take to the donor site and will die off shortly after transplant. New therapies that are more effective, have fewer side effects and are applicable to the female patient population are needed.

Adipose tissue, adipose derived stem cells and hair growth
There is growing evidence supporting a relationship between dermal adipose deposits and proper follicle function. A variety of studies have shown in mice that a reduction of intradermal adipose tissue has been associated with abnormalities in skin structure including hair loss, epidermal hyperplasia and abnormal sebaceous gland function [15][16][17]. A 2011 study by Festa et al. examined the role of intradermal adipocytes on follicular stem cell activity [18]. They observed a dynamic process of adipogenesis that parallels the activation of hair follicle stem cells. Additional functional analysis revealed that immature adipocyte cells are necessary and sufficient to drive follicular stem cell activation and they attributed this relationship to the production of plateletderived growth factor (PDGF) by immature adipocyte lineage cells. They ultimately established that adipose tissue plays a crucial role in the normal hair cycle and even went as far as to suggest that telogen may be the result of a localized absence of adipose tissue.

Adipose derived stem cells: A promising future for hair loss
Adipose derived stem cells (ASCs) are of increasing interest in the treatment of Alopecia Androgenetica. Adipose derived stem cells are being explored for a variety of regenerative applications based on their ability to differentiate down a variety of cellular pathways as well as the regenerative benefits afforded by the growth factors produced [20][21][22]. A variety of studies have been published examining the effects of adipose derived stem cells and therapies based on adipose derived stem cells, such as cell assisted lipotransfer (CAL) or adipose derived stem cell conditioned media (ADSC CM), in relation to the reversal of male and female pattern hair loss. The early results published are promising.
ADSC CM is the growth factor rich media produced when adiposederived stem cells are cultured. During culturing, the ASCs constantly release growth factors into the surrounding culture media. The ASCs are subsequently removed, leaving a cell free, growth factor rich solution which has been shown to exhibit regenerative potential similar to treatment using adipose derived stem cells. The culture conditions alter the growth factor content, with lower oxygen being associated with enhanced regenerative potential [23].  [24]. They began by examining the effect of ADSC CM on human dermal papilla cells (hDPCs) and immortalized keratinocyte cells (HaCaT cells) in vitro. ADSC CM was shown to significantly enhance the proliferation of both HaCaT cells and hDPCs. Next, cell cycle analysis was conducted on ADSC CM treated hDPCs. They noted increased proliferation of hDPCs as a result of ADSC CM exposure, which they attributed to enhanced activation of Erk and Akt signaling pathways. The Erk pathway has been associated with mitogenesis and the Akt pathway mediates cell survival signals, and both have been associated with enhanced survival and proliferation of hDPCs [25,26]. With the promising results afforded by the early stages of this study, they then examined the effect of ADSC CM on hair shaft elongation in 5 male volunteers. Intact hair follicles were extracted and cultured with ADSC CM, minoxidil or nothing added (control). The ADSC-CM treated follicles elongated by 40% compared to the control group, similar to those in the minoxidil group. The final facet of this study examined in vivo effects. 48 mice received injections of PBS or 5 × 10 5 ASCs every 3 days for 9 days. Concurrently, ADSC CM or control media was topically applied. They noted that in mice who received ASC injections that there was earlier conversion of telogen to anagen. Additionally, after 4 weeks they noted significantly accelerated hair growth and increased number of hair follicles after topical application of ADSC CM.
Another 2010 study by Park et al. reported on the effects of ADSC-CM [27]. They examined the effects of ADSC CM produced under normoxic conditions (NorCM) and hypoxic conditions (HypoCM). In culture experiments using hDPC and human epidermal keratinocytes (HEKs), they noted that ADSC CM exposure increased proliferation of hDPCs and HEKs in a dose dependent manner. When comparing hypoCM and norCM, no significant difference was observed in hDPF proliferation but hypoCM showed a significant increase in HEK proliferation compared to norCM treatment. This study also examined the effects of norCM and hypoCM in a mouse model (n=21). Mice received 3 subcutaneous injections of 100ul of control media (n=5), norCM (n=5) or hypoCM (n=5) at 3 day intervals. Overall, they noted that mice treated with hypoCM induced the anagen phase and the development of dark spots (hair growth) on treated mice more rapidly than the norCM treated mice.
Another study published in 2013 by Jeong et al. [28] examined differences between various preparations of ASCs and ADSC CM (with and without UVB irradiation preconditioning) and their effect on hair growth in mice. Telogen matched mice were shaved and injected with ASCs or ADSC CM with and without UVB irradiation (four groups total). Mice received injections of 1 × 10 4 cells into the dermis of a shaved area or 50ul of ADSC CM injected into the dermis of the shaved area. Overall, they noted that all groups were able to convert telogen to anagen, but both the ASC and ADSC CM groups which had been preconditioned with UVB irradiation demonstrated accelerated conversion and subsequently more hair growth In 2015, Shin et al. published a retrospective clinical case series on the use of ASCs and ADSC CM for the treatment of female pattern hair loss [29]. Treatment was administered under a 12 week protocol where subjects received ADSC CM applications once per week with a micro needling roller. 27 women with hair loss classified as Ludwig I were treated with ADSC CM and all completed the 12 week treatment course. Hair density and thickness were assessed using phototrichogram captures. They reported an increase in mean hair density from 105.4 hairs/cm 2 to 122.7 hairs/cm 2 , which represented a 16.4% increase. These reports are similar to those reported using topical minoxidil twice daily for 48 weeks, which reported 17.3% and 13.8% increase in hair density using 5% and 2% minoxidil, respectively [30]. Additionally, as a result of 12 week ADSC CM application, mean hair thickness increased from 57.5 um to 64.0 um, an 11.3% increased. In terms of hair thickness, ADSC CM was shown to be superior to reported outcomes of 2% minoxidil (5.0% at 24 weeks) [10]. No serious adverse events were associated with the treatment. The study lacks significant long term follow up data, as a majority of subjects were followed only for the 12 week course of treatment, but clinical benefit was maintained in all patients who were followed up for 6 month (n=9).
In 2012, Fukuoka et al. [31] published their reports using ADSC-CM for hair loss in 24 patients (12 men, 12 female) with androgenetic alopecia. Subjects received ADSC CM injections 4 6 times every 3 5 weeks. The authors reported overwhelmingly favorable results in terms of patient and investigator satisfaction and reported that all treated patients demonstrated increased hair growth. The study lacks quantifiable data in terms of hair density and thickness.
As a follow up to their 2012 study, Fukuoka and Suga once again reported [32] favorable outcomes using ADSC CM in 2015. 22 patients (11 male, 11 female) received intradermal injections of ADSC CM every 3 5 weeks for a total of 6 sessions. Trichogram captures were taken before treatment and 1 3 months after the last treatment session. Overall, they noted a significant increase in the number of hairs in both male and female patients. Male patients showed an average increase of 29 ± 4.1 hairs/cm 2 and female patients showed an average increase of 15.6 ± 4.2 hairs/cm 2 . 6 out of 11 males were concurrently taking Finasteride during the course of treatment. No significant difference was observed in the amount of hair growth observed between men with and without finasteride use. Another group of 10 patients (8 male, 2 female) were treated in a side by side fashion, were the left half of the scalp received ADSC CM and the right side received saline placebo injections. Surprisingly, increases in hair growth were seen in both sides of the scalp as a result of treatment, but the ADSC CM treated side was significantly higher than the saline only side of the scalp. This suggests that mechanical tissue injury could possibly result in marginal hair growth or that the effects of the ADSC CM are not entirely localized to the treatment area, but rather can affect the whole scalp via local circulation.

Clinical trials currently underway
The potential for applications of adipose and adipose derived stem cell based therapies looks promising. While ADSC CM and ASCbased therapies are commercially available in some countries, larger, controlled clinical trials are needed to formally establish safety and efficacy of these therapies. There are currently 4 studies listed on Clinicaltrials.gov for the treatment os Alopecia Androgenetica using an ASC based therapy. A phase 3 study (Identifier: NCT02594046) beginning in October 2015 is examining the effects of ADSC CM and is listed under the name "The Effect of Allogeneic Human Adipose Derived Stem Cell Component Extract on Androgenic Alopecia" [33]. CAL is currently under investigation in the STYLE Trial [34] sponsored by Kerastem Technologies, LLC. This Phase II study which began in November 2015 (Identifier: NCT02503852) is investigating the uses of a combination of fat grafting prepared using the Puregraft system (Puregraft, LLC) and adipose derived regenerative cells (ADRCs) isolated using the Cytori Celution 800/CRS system. Another Phase I clinical trial using the GID SVF 2 device to isolate stromal vascular fraction cells is currently recruiting (identifier: NCT02626780) for a trial titled "Adipose derived SVF for Treatment of Alopecia" and is currently recruiting as of December 2015 [35]. The trial does not involve fat grafting, only injections of freshly harvested SVF cells. A second Phase 0 study with similar methodology (Identifier: NCT02729415) listed under the name "Point of Care Adipose derived Cells for Hair Growth (ASVF 2016)" is set to begin recruiting in July 2016 [36].

Conclusion
The body of evidence in favor of the use of adipose derived stem cells and ASC based therapies for male/female pattern hair loss is steadily growing. These therapies offer the benefit of reduced side effects compared to the current treatment modalities and appear to be effective in both males and females. With relatively no new formally approved treatments for hair loss in the last 20+ years, the future of hair restoration may lie in adipose derived stem cell based therapies.