Alopecia Areata is an autoimmune disorder characterized by patches of non-scarring alopecia affecting scalp and body hair; it is clinically heterogeneous, and its natural history is unpredictable. There is no preventative therapy or cure at this moment. Hair transplantation – which takes plugs of natural hair from hair-rich occipital areas and transplants them to the hair-poor areas- may no longer be a viable option for treatment, as there might not be sufficient donor hair follicles (available from the scalp or other areas) that can be transplanted without causing collateral damage elsewhere.
Female pattern hair loss (FPHL) is the most common type of baldness in women, showing a prevalence of approximately 6% in women aged under 50 years and 38% in women aged 70 years and over (Birch et al., 2001). Currently, there are few therapeutic options for women with FPHL: anti-androgen drugs and topical Estrogen are used, but they do not always achieve successful results and are not safe in reproductive women. In order to overcome these problems, cell therapy to restore bald scalp areas has become an emerging focus in addressing this medical condition.

SCIENTIFIC EVIDENCE
Adipose derived cells (ADC) are one of the latest break-troughs in hair regeneration, since these cells and their conditioned media (ADC-CM) have been reported to promote hair growth in vitro and in some clinical reports of treatments for androgenic alopecia or female pattern hair loss (FPHL).
First proven in animal models (Won et al., 2010), in vitro expanded ADC have been demonstrated to produce molecules (secretome) that induce hair regeneration and stimulate hair regrowth also in humans affected by androgenic alopecia or FPHL (Fukuoka et al., 2015; Shin et al., 2015).
Moreover, ADC-CM increases the proliferation of human follicle dermal papilla cells (DPC) and human epithelial keratinocytes, two cell types present in hair follicles, inducing anagen phase and stimulating hair regeneration. (Won et al., 2010)

ADC: WHY THE FAT?
Fat tissue is an accessible and rich source of mesenchymal cells (ADC), adult non-hematopoietic stromal cells able to differentiate into mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and fat.
ADC can be easily isolated from adipose tissue and can be rapidly expanded in culture.
Recent studies have shown that subcutaneous adipose tissue provides a clear advantage over other mesenchymal stromal cells sources due to the ease with which adipose tissue can be accessed (under local anesthesia and with minimum of patient discomfort) as well as to the ease of isolating cells from the harvested tissue.
Moreover, ADC frequency is significantly higher in adipose tissue than in bone marrow and the maintenance of the proliferating ability in culture seems to be superior.

THE TREATMENT
The treatment is based on the administration of ADC-CM alone or in association to autologous, in vitro expanded ADC.
ADCs are isolated starting from a fat tissue sample which can be collected during a surgical liposuction or in one outpatient session. 
The cells are expanded under sterile conditions and culture media is collected for future use. 
About two weeks after the small FAT collection about ten millions of cells are ready for an immediate treatment and the remaining cells are cryopreserved for future treatments at the Bioscience Clinic facility.  

ADVANTAGES
ADC-CM contains various regenerative factors secreted by ADC that can limit hair loss by recruiting endogenous cells to hair follicles sites and triggering regeneration. Moreover, ADC-CM increases the poor engraftment of the ADC transplanted alone and the clinical outcomes in hair regeneration.
The sample can be taken using a simple micro-liposuction procedure with no need for surgery, the collection does not require dedicated equipment and/or kits, simply a standard syringe.
The possibility of freezing part of the material offers the advantage of repeating the treatment several times over the years without the need to undergo again to fat tissue collection.

GENERAL INTRODUCTION
Adipose Derived Stem Cells (ADSC) are mesenchymal stem cells (MSC): non-hematopoietic stromal cells able to differentiate into mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and fat. The International Society for Cellular Therapy has established minimal criteria for defining MSC. These basal attributes include the abilities to adhere to plastic under normal cell culture conditions, to express a set of cell surface antigens (CD105, CD73, and CD90) while not expressing antigens indicative of other cell lineages, and to differentiate into adipocytes, osteoblasts, and chondroblasts under specific conditions.
ADSC can be easily isolated from adipose tissue and can be rapidly expanded in culture. Recent studies have shown that subcutaneous adipose tissue provides a clear advantage over other MSC sources due to the ease with which adipose tissue can be accessed (under local anesthesia and with minimum of patient discomfort) as well as to the ease of isolating stem cells from the harvested tissue. Moreover, stem cell frequency is significantly higher in adipose tissue than in bone marrow and the maintenance of the proliferating ability in culture seems to be superior.
ADSC have been shown to have immuno-suppressive and tissue regenerative capacities, to improve angiogenesis and prevent fibrosis. Moreover, ADSC can potently modulate immune responses, showing antiproliferative and anti-inflammatory capacities. The mechanisms underlying tissue regeneration and immune modulation by therapeutic doses of ADSC require further elucidation, particularly the extent to which the two processes intersect.
The effects of ADSC can be through differentiation toward a target cell lineage but is more likely to involve trophic modulation by paracrine and autocrine

activity; secretion of angiogenic, chemoattractant, and antiapoptotic factors, and specific anti-inflammatory effects through reduced T-cell activity and MHC suppression (Filomeno et al., 2012).
Finally, thanks to their extensive proliferative capacity, it is possible to produce in vitro relatively large numbers of ADSC for potential clinical applications.
Given these properties, it is unsurprising that ADSC are continuously entering clinical trials for numerous applications.
Indeed, the broad range of clinical applications for ADSC largely depends on their potential for differentiation and on their ability to migrate and to recruit endogenous stem cells from the niches. Now it is largely recognized that, far from building new tissues at site of administration, ADSC exert immune-modulatory functions, secreting several bioactive molecules that inhibit apoptosis and scarring at site of injury and stimulate angiogenesis and mitosis of tissue specific progenitors (Caplan et al., 2010; Park et al., 2010).
Question remain, however, regarding the safety of ADSC clinical application, and their long-term fate. Tumorigenic potential is especially worrisome. To date, any studies suggesting that ADSC have this ability are either inconclusive or have been retracted (Rubio et al., 2005; De la Fuente et al., 2010). In a study culture-expanded human-derived ADSC were applied to immunosuppressed mice. At one year, animals were no different in weight nor life span from controls and showed no signs of tumorigenesis (MacIsaac et al., 2011). Moreover, there are over 100 clinical trials with ADSC registered on clinicaltrials.gov. While a majority of these trials are not completed, the available data suggest safety of this cell population.
In the light of the above, the stage seems to be set for clinicians to translate ADSC from the bench to the bedside: ADSC clinical potential represents an opportunity to coordinate basic research and translational efforts using the principles of evidenced-based medicine.

Adipose Derived Stem Cells’ Conditioned Medium
As stated above, an essential function of ADSC, probably responsible for their regenerative therapeutic effects, is the production and secretion of growth factors and cytokines that activate neighboring cells. ADSC have paracrine effects on their surrounding environment through the secretion of bioactive concentrations of growth factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), transforming growth factor (TGF)-β1, insulin-like growth factor-binding protein (IGF-BP) precursors, fibronectin, and superoxide dismutase (SOD) (Park et al., 2008). Through their secretomes, ADSC mediate diverse skin regenerative effects such as wound healing, antioxidant protection, anti wrinkling and whitening effects (Park et al., 2008).

ADIPOSE DERIVED STEM CELLS AND HAIR LOSS
Adipose derived stem cells (ADSC) are one of the latest break-troughs in hair regeneration, since these cells and their conditioned media (ADSC-CM) have been reported to promote hair growth in vitro and in some clinical reports of treatments for androgenic alopecia or female pattern hair loss (FPHL).
FPHL is a very common type of alopecia seen in women. Its prevalence is approximately 6% in women aged under 50 years and 38% in women aged 70 years and over (Birch et al., 2001). Although FPHL rarely progresses to the total hair loss seen in male androgenic alopecia, hair loss is more distressing to women than men because women with FPHL have a more negative body image in comparison with balding men (Cash et al., 1993). Nevertheless, FPHL patients have fewer therapeutic options than male androgenetic alopecia patients. Lower percentage topical minoxidil is the only medication approved by the US Food and Drug Administration for FPHL. Anti-androgen drugs and topical estrogen are used for the treatment of FPHL. However, they do not always achieve successful results and are not safe in reproductive women. Moreover, topical estrogen still lacks convincing clinical trials (Blume Peytavi et al., 2007).
Alopecia areata is an autoimmune disorder characterized by patches of non-scarring alopecia affecting scalp and body hair that can be psychologically devastating. It is clinically heterogeneous, and its natural history is unpredictable. The disease may be limited to one or more discrete, well-circumscribed round or oval patches of hair loss on the scalp or body, or it may affect the entire scalp (alopecia totalis) or the entire body (alopecia universalis). Also the course of disease is unpredictable, with spontaneous regrowth of hair occurring in 80% of patients within the first year, and sudden relapse at any given time. There is no preventative therapy or cure.

Moreover, often the degree of degeneration in patients with alopecia is so severe that the permanent portion of the follicle is lost, the adult human body being unable to regenerate the hair follicle and restore homoeostasis. In these cases, hair transplantation –which takes plugs of natural hair from hair-rich occipital areas and transplants them to the hair-poor areas- may no longer be a viable option for treatment, as there might not be sufficient donor hair follicles (available from the scalp or other areas) that can be transplanted without causing collateral damage elsewhere. In addition, type and quality of follicle from other body parts than the scalp do not give rise to the same type of hair, with long shafts as usually grow on the scalp.
In order to overcome these problems, stem-cell therapy to restore bald scalp areas has become an emerging focus in addressing this medical condition.
First proven in animal models (Won et al., 2010), in vitro expanded ADSC have been demonstrated to produce molecules that induce hair regeneration and stimulate hair regrowth also in humans affected by androgenic alopecia or FPHL (Fukuoka et al, 2012; 2015; Shin et al., 2015).

SCIENTIFIC BACKGROUND of ADSC and their CM for Hair Regeneration
Hair growth is stimulated both ex vivo and in vivo by growth factors secreted by growing ADSC: in a VEGF-transgenic mouse model, VEGF controlled hair growth and follicle size by modulation of angiogenesis (Yano et al., 2001). PDGF isoforms induce and maintain the anagen phase in murine hair follicles (Tomita et al., 2006). HGF and IGF also up-regulate hair follicle growth in various systems (Jindo et al., 1998; Lindner et al., 2000; Su et al., 1999; Weger et al., 2005).
These observations suggest that ADSC secretome may have a therapeutic effect on hair loss.
When cultured under particular growing conditions (i.e., hypoxia), ADSC secrete regenerative factors that can positively affect hair growth, as demonstrated by the subcutaneous administration of ADSC growth medium to mice (Park et al., 2010).
Moreover, ADSC-CM increased the proliferation of human follicle dermal papilla cells (DPC) and human epithelial keratinocytes, two cell types present in hair follicles, inducing anagen phase and stimulating hair regeneration. Won et al. showed that ADSC-CM treatment enhanced the proliferation of cultured human DPC by up to 130% in 48 h, while CM from adipocytes inhibited their proliferation. This result was explained by the activation by ADSC-CM of both Erk and Akt signalling pathways (Won et al., 2010). In addition, ADSC-CM modulates the cell cycle of DPC. Cyclin D1 and CDK2, key cell cycle related molecules, are also upregulated by ADSC-CM (Won et al., 2010).
These results are meaningful with regard to hair growth because dermal papilla size is known to correlate with the hair growth cycle, and the number of DPC increases in the anagen phase (Elliot et al., 1999). In the same paper, the back skin of 7-week-old male C3H/HeN nude mice was shaved and their hair follicles were synchronized in telogen stage. ADSC (5 x 105 cells/50 ml PBS) or PBS alone were subcutaneously injected into dorsal skin every 3rd day for 9 days. Concurrently, ADSC-CM (1 ml) or control medium was topically applied on the back of the other C3H mice. After intradermal injection of ADSC on the back of C3H/HeN mice at the age of 7 weeks, the conversion of telogen to anagen was induced earlier than in controls. Accelerated hair growths was also found after topical application of ADSC-CM. Histologically, the back skin of ADSC-CM treated mice showed increased number of hair follicles (Won et al., 2010). These findings indicate that locally injected ADSC and ADSC-CM might stimulate hair growth in vivo and paved the way for analysis in patients.
Recently, satisfactory results for hair regeneration have been achieved in 12 women with FPHL and 13 men, 12 with androgenic alopecia and 1 with androgenic alopecia and alopecia areata, as determined by a visual analog scale. All patients were treated with an ADSC-CM which includes various cytokines and growth factors: a commercial advanced adipose-derived stem cell protein extract (AAPE™) was used. This extract contains numerous growth factors and regeneration-promoting proteins. Its main components include PDGF (44.41 ± 2.56 pg/ml), bFGF (131.35) ± 30.31 pg/ml), KGF (86.28 ± 20.33 pg/ml), TGF-β1 (103.33 ± 1.70 pg/ml), HGF (670.94 ± 86.92 pg/ml), VEGF (809.53 ± 95.98 pg/ml), collagen (921.47 ± 49.65 pg/ml), fibronectin (1466.48 ± 460.21 pg/ml), and SOD in 5 ml saline solution, to which, in the present study, buflomedyl, cysteine, coenzyme Q10, and vitamins were added. This solution was applied 4 to 6 times every 3 to 5 weeks by mesotherapy techniques, such as nappage and papule injections with 30g or 32G needle.
Patients received follow-up examinations at 2- to 4-month intervals for at least 1 year after the final treatment session. Patients experienced increased hair growth with good results and without adverse effects including bacterial infections and granuloma. Hair regrowth was maintained for at least 1.5 years, but this was dependent on patient age. Men older than 60 years had the least maintenance of hair regrowth, followed by men 50–60 years old and women older than 60 years. Men younger than 50 years and women younger than 60 years maintained hair regrowth between 1.5 and 2.5 years. (Fukuoka et al., 2012).
In another study by the same group, ADSC-CM (enriched AAPE™, as prepared in the previous study) was intra-dermally injected in 22 patients (11 men and 11 women) with alopecia. Intradermal injections with a 31G needle provided about 0.02 ml/cm2 of solution. Patients received treatment every 3 to 5 weeks for a total of 6 sessions. Some male patients received finasteride, as well. Hair numbers were counted using trichograms before and 1 to 3 months after treatment. A half-side comparison study was also performed in 10 patients (8 men and 2 women). Hair numbers were significantly increased after treatment in both male (including those without finasteride administration, without significant differences in the two groups) and female patients. The mean increase was 29 ± 4.1 in male and 15.6 ± 4.2 in female patients. In the half-side comparison study, the increase in hair numbers was significantly higher on the treatment side than on the placebo side (Fukuoka et al., 2015).
Shin and coworkers performed a retrospective, observational study of outcomes in 27 patients (men patient age 41.9 ± 13.4 years) with female pattern hair loss (FPHL) treated with ADSC-CM. The commercial product AAPE™, produced from human subcutaneous adipose tissue obtained by medical liposuction from healthy persons from which ADSC were isolated and in vitro expanded till passage 4 under hypoxic conditions, was used.
Finally, conditioned media were collected and microfiltered: for fresh use, 4-ml vials containing equal protein concentrations were freeze-dried as a single lot sample preparation of AAPE™. The treatment course consisted of repeated applications of ADSC-CM once per week for 12 consecutive weeks. To evaluate the efficacy of the treatment, patients’medical records and phototrichographic images were analysed. The application of ADSC-CM showed efficacy in treating FPHL after 12 weeks of therapy. Mean hair density increased from 105.4 to 122.7 hairs/cm2 (P < 0.001), representing an increase of 16.4%. Mean hair thickness increased from 57.5 μm to 64.0 μm (P < 0.001), an increase of 11.3%. None of the patients reported severe adverse reactions or irritation or itching, suggesting application of ADSC-CM is a potential treatment option also for FPHL (Shin et al., 2015).

PERSPECTIVES
Given the broad implications of ADSC and of their secreted factors on hair regeneration, further research are ongoing in the field.
In particular, given the background described above, we will investigate two approaches:
administration of ADSC-CM in association to autologous, in vitro expanded ADSC. This approach is based on the idea that ADSC-CM contains various regenerative factors secreted by ADSC and can increase the poor engraftment of the ADSC transplanted alone and the clinical outcomes in hair regeneration.
administration of ADSC-CM alone, taking advantage of the various factors and cytokines secreted by ADSC in culture, which could limit hair loss and recruit endogenous cells to hair follicles sites, triggering regeneration.

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