key: cord-0879734-6a69raj3
authors: Martinez-Lopez, Antonio; Montero-Vilchez, Trinidad; Sierra-Sánchez, Álvaro; Molina-Leyva, Alejandro; Arias-Santiago, Salvador
title: Advanced Medical Therapies in the Management of Non-Scarring Alopecia: Areata and Androgenic Alopecia
date: 2020-11-09
journal: Int J Mol Sci
DOI: 10.3390/ijms21218390
sha: 29b5e461d6872e2c64f2416e37fd161a4ee8a2eb
doc_id: 879734
cord_uid: 6a69raj3

Alopecia is a challenging condition for both physicians and patients. Several topical, intralesional, oral, and surgical treatments have been developed in recent decades, but some of those therapies only provide partial improvement. Advanced medical therapies are medical products based on genes, cells, and/or tissue engineering products that have properties in regenerating, repairing, or replacing human tissue. In recent years, numerous applications have been described for advanced medical therapies. With this background, those therapies may have a role in the treatment of various types of alopecia such as alopecia areata and androgenic alopecia. The aim of this review is to provide dermatologists an overview of the different advanced medical therapies that have been applied in the treatment of alopecia, by reviewing clinical and basic research studies as well as ongoing clinical trials.

Alopecia is one of the most common consultation requests in dermatological daily practice [1] . This condition is usually associated with some psychological disturbances such as anxiety, depression, and distress [2] . In recent decades, many topical, intralesional, oral, or surgical treatments have been employed in order to delay and stop the hair loss as well as to restore the presence of hair in alopecic areas. However, some of the therapeutic drugs employed in some conditions such as minoxidil, finasteride, and dutasteride for androgenic alopecia (AHA) only provide partial and temporary involvement. Moreover, treatments for severe alopecia areata (AA) and many forms of scarring alopecia are usually ineffective and sometimes are associated with serious adverse effects [3, 4] . With this background, some alternative therapeutic strategies are needed for this disease.

The use of genes, cells, and tissues as a new therapeutic resource is one of the characteristics of contemporary medicine. Advances in regenerative medicine has increased interest in applying stem cells to engineered tissue scaffolds to reconstitute damaged tissue and develop regenerative therapies for the skin [5, 6] . This new therapeutic approach is based on gene therapy, cell therapy, tissue engineering, or the combination of any type of drug with medical devices ( Figure 1 ). As defined by the European Commission, Advanced Therapy Medicinal Products (ATMPs) are new medical products based on genes (i.e., recombinant nucleic acids, gene therapy), cells (cell therapy), and/or tissue engineered products that contain or consist of engineered cells or tissues and are presented as having properties for, or are used in or administered to, human beings with a view of regenerating, repairing, or replacing human tissue [7, 8] . According to the Annex I to Directive 2001/83/EC, a somatic cell therapy medicinal product means a biological medicinal product that presents two main characteristics: (a) Contains or consists of cells or tissues that have been subject to substantial manipulation so that biological characteristics, physiological functions, or structural properties relevant for the intended clinical use have been altered, or of cells or tissues that are not intended to be used for the same essential functions in the recipient and the donor; (b) Is presented as having properties for, or is used in or administered to, human beings with a view of treating, preventing, or diagnosing a disease through the pharmacological, immunological, or metabolic action of its cells or tissues.

the European Commission, Advanced Therapy Medicinal Products (ATMPs) are new medical products based on genes (i.e., recombinant nucleic acids, gene therapy), cells (cell therapy), and/or tissue engineered products that contain or consist of engineered cells or tissues and are presented as having properties for, or are used in or administered to, human beings with a view of regenerating, repairing, or replacing human tissue [7, 8] . According to the Annex I to Directive 2001/83/EC, a somatic cell therapy medicinal product means a biological medicinal product that presents two main characteristics:

(a) Contains or consists of cells or tissues that have been subject to substantial manipulation so that biological characteristics, physiological functions, or structural properties relevant for the intended clinical use have been altered, or of cells or tissues that are not intended to be used for the same essential functions in the recipient and the donor; (b) Is presented as having properties for, or is used in or administered to, human beings with a view of treating, preventing, or diagnosing a disease through the pharmacological, immunological, or metabolic action of its cells or tissues. Regarding point (a), the European regulation has established which manipulations should not be considered as substantial manipulations. In this way, cellular products obtained by centrifugation, irradiation, cell separation, concentration, or purification proceedings are not considered as ATMPs. Thus, common cellular treatments for some forms of alopecia like platelet-rich plasma could not be included into the ATMPs. Autologous fat injections for the treatment of alopecia are considered as an ATMP because they are used for a different purpose than originally intended, although their use is currently only approved in the setting of clinical trials. However, its illegal use has become extended in several cosmetic medicine centers, which entails serious legal implications [9] .

ATMPs are complex products and risks may differ according to the type of product, nature/characteristics of the starting materials, and level of complexity of the manufacturing process. It is also acknowledged that the finished product may entail some degree of variability due to the use of biological materials and/or complex manipulation steps. In that way, ATMPs' quality plays a major role in their safety and efficacy profile. Compliance with Good Manufacturing Practice ("GMP") is an essential part of the pharmaceutical quality system and it is the responsibility of the ATMP manufacturer to ensure that appropriate measures are put in place to safeguard the quality of the product [10] .

ATMPs are usually well tolerated, but some side effects have been described, especially with intravenous human mesenchymal stem cell (hMSC) therapy. MSC-based products express variable levels of a highly procoagulant tissue factor (TF/CD142) that could lead to venous thrombosis and thromboembolism. Thirabanjasak et al. reported a lupus nephritis patient who had angio-myeloproliferative lesions after direct injection of stem cells into the renal parenchyma [11] . Thus, it is advisable to weigh the risks and benefits of this treatment in patients with procoagulant diseases and thrombophilias. Low-grade fever has also been associated with hMSC infusion [12, 13] . More data on the safety of stem cell application need to be collected.

ATMPs have numerous potential applications, especially in the field of regenerative medicine. With this background, the aim of this review is to outline the different forms of ATMPs and their application in the treatment of non-scarring alopecia forms such as alopecia areata and androgenic alopecia, by reviewing clinical and basic research studies as well as ongoing clinical trials.

HMSC-based therapies have been used in regenerative medicine in several medical areas such as orthopedics, neurology, cardiology, and dermatology. HMSCs originate from the mesoderm and share their origins with the skin. When these cells are implanted in a damaged tissue, they are likely to react better to paracrine factors; therefore, hMSCs are an ideal strategy for repairing and regenerating skin abnormalities ( Figure 2 ) [5, [13] [14] [15] .

When these cells are administered to an area affected by different diseases, hMSCs can be differentiated into injured tissue components. In addition, these cells have shown an important immunomodulatory activity and are able to secrete various cytokines and growth factors such as Il-6, Il-7, Il-8, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) that promote tissue regeneration [16] [17] [18] [19] . Although the main reserve of hMSCs is found in the bone marrow, adipose tissue has been identified as a source of these cells, displaying similar properties to those extracted from the bone marrow [20] [21] [22] .

HMSCs have been used in several medical and surgical disciplines in order to explore new therapeutic possibilities for diseases which current treatment modalities do not offer satisfactory results. For example, in the orthopedic field, hMSC have been successfully used for osteonecrosis, osteoarthritis, and to promote fracture healing [23] [24] [25] [26] . In dermatology, the main use of hMSCs has been focused on the treatment of wounds and skin ulcers. In that way, preclinical studies have shown that the application of hMSCs accelerates the re-epithelialization of skin wounds. This is due to the promoting action of dermal fibroblast proliferation by direct cellular contact and by transforming growth factor beta (TGFβ) and bFGF secretion. In addition, its ability to differentiate into adipocytes provides a supportive architecture for dermal regeneration and re-epithelialization [27] [28] [29] [30] [31] . When these cells are administered to an area affected by different diseases, hMSCs can be differentiated into injured tissue components. In addition, these cells have shown an important immunomodulatory activity and are able to secrete various cytokines and growth factors such as Il-6, Il-7, Il-8, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) that promote tissue regeneration [16] [17] [18] [19] . Although the main reserve of hMSCs is found in the bone marrow, adipose tissue has been identified as a source of these cells, displaying similar properties to those extracted from the bone marrow [20] [21] [22] .

HMSCs have been used in several medical and surgical disciplines in order to explore new therapeutic possibilities for diseases which current treatment modalities do not offer satisfactory results. For example, in the orthopedic field, hMSC have been successfully used for osteonecrosis, osteoarthritis, and to promote fracture healing [23] [24] [25] [26] . In dermatology, the main use of hMSCs has been focused on the treatment of wounds and skin ulcers. In that way, preclinical studies have shown that the application of hMSCs accelerates the re-epithelialization of skin wounds. This is due to the promoting action of dermal fibroblast proliferation by direct cellular contact and by transforming growth factor beta (TGFβ) and bFGF secretion. In addition, its ability to differentiate into adipocytes provides a supportive architecture for dermal regeneration and re-epithelialization [27] [28] [29] [30] [31] .

The hair follicle (HF) is a regenerating system, which physiologically undergoes cycles of growth (anagen), regression (catagen), and rest (telogen). HF has a niche for mature stem cells in the attachment region of arrector pili muscles, which contain epithelial and melanocyte stem cells. Another type of stem cells within the hair follicle is dermal papilla cells, probably originating from dermal condensation, which is the initial stage of hair follicle development [32, 33] . During adult HF cycling, the signal between epithelial keratinocytes and underlying specialized hair follicle and dermal papilla mesenchymal cells induces stem cell proliferation and initiates the cascade of cell differentiation into the hair follicle cell lineages [34] . These cells also take part in the regeneration of the sebaceous glands. The hair follicle (HF) is a regenerating system, which physiologically undergoes cycles of growth (anagen), regression (catagen), and rest (telogen). HF has a niche for mature stem cells in the attachment region of arrector pili muscles, which contain epithelial and melanocyte stem cells. Another type of stem cells within the hair follicle is dermal papilla cells, probably originating from dermal condensation, which is the initial stage of hair follicle development [32, 33] . During adult HF cycling, the signal between epithelial keratinocytes and underlying specialized hair follicle and dermal papilla mesenchymal cells induces stem cell proliferation and initiates the cascade of cell differentiation into the hair follicle cell lineages [34] . These cells also take part in the regeneration of the sebaceous glands.

Hair loss is determined by several factors such as hereditary conditions, hormonal disorders, autoimmunity diseases, nutritional deficiency, bacterial and fungal overgrowth, psychological factors, environmental elements, and aging. Some of these harming factors influence the hair cycle and reduce stem cell activity and HF recovery capacity [33] . Immunologic disturbances use HF as one of their main targets. When the HF immune privilege is collapsed, CD4 and CD8 T cells and natural killer (NK) cells accumulate around the autoantigens of the hair bulb and contribute to the development of AA [35, 36] . Multiple models have shown the association between the development of several types of alopecia with the disruption of certain cytokines and proteins. Th1 cytokines and chemokines, such as interferon-gamma (IFN-γ), CXCL9, and CXCL10, are predominantly detected in AA lesions and might induce the collapse of the hair follicle immune privilege [36] . CK15 immunoreactivity, which has been described as a marker of telogen, is decreased in people with active AA, whereas it is present in AHA. Hair follicles in the frontal parts of the scalp exhibit a deficit of CD34 in AHA, and its expression is preserved in hair follicles of the occipital region. Interfollicular injection of autologous CD34+ cell-containing PRP preparation has shown a positive therapeutic effect in AHA patients [37] [38] [39] . Moreover, CD200, another marker of matrix cells, is poorly expressed in patchy alopecia, which may be a sign of the disappearance of the immune privilege and can contribute to pathogenesis [40, 41] . The Wnt pathway and Wnt/beta-catenin signaling are known to increase mammalian hair growth. In that way, Leirós et al. demonstrated that androgens deregulated dermal papilla cell-secreted factors involved in normal HF stem cell differentiation via the inhibition of the Wnt pathway [42] .

Some preclinical studies have shown promising results by employing hMSCs in the treatment of AA and AHA (Table 1 ). Byun et al. developed a pilot study in order to demonstrate the immunomodulatory effect of MSC in AA. The investigators employed intravenous MSC in AA-induced C3H/HeJ mice on days 1 and 7, and after 15 weeks of follow-up, 23% of mice in the MSC-treated group showed AA incidence, whereas extensive hair loss was observed in 91% mice in the control group. Serum samples also showed decreased IFNγ, CXCL9, and CXCL10 concentrations in the treated group. Moreover, histological analysis demonstrated less inflammatory cellular infiltration around the dermal papilla [35] . Later, Kim et al. launched another in vitro study with the aim of assessing the effect of hMSCs on the viability and proliferation of human dermal papilla cells (hDPC) via the activation of the JAK/STAT and Wnt/beta-catenin signaling pathways in an AA-model. The investigators employed bone marrow-derived hMSCs that were co-cultured with hDPC, showing a 120% increased hDPC proliferation compared to hDPC cultured alone. In addition, hMSC treatment augmented beta-catenin levels and induced much higher phosphorylation of STAT1 and STAT3, proteins that are associated with a prolonged anagen phase. Finally, the investigators observed an immunomodulatory effect in the treatment with hMSC by restoring a HF privilege that induced re-entry signaling from the telogen to anagen phase [43] . Recently, Bak et al. conducted a mice-based study in order to evaluate the effect of umbilical cord blood hMSCs on reacquisition of hDPC conduction ability to induce hair growth. After 6 weeks of hMSC injection, a complete hair regrowth was observed. Moreover, histological analysis revealed that hMSC promoted hair follicle re-entry in the early and middle anagen phase prematurely compared with the minoxidil group. In addition, hMSC treatment up-regulated beta-catenin and AKT pathways and enhanced growth factors production [44] . 1-hUCB-MSCs can accelerate the initiation of the hair follicle telogen-anagen transition, increase the number of hairs in vivo, and enhance expression of proteins related to hair induction in vitro. 2-IGFBP-1 (assumed as the main secretory factor of hUCB-MSCs) restores and promotes the hair-induction ability of hDPCs via an IGF-1/IGFBP-1 co-localization. [44] In vitro analysis of the effect hUCB-MSCs co-cultured with human dermal papilla cells (hDPCs).

Co-culture of 1 × 10 5 hDPCs with 2 × 10 5 hUCB-MSCs.

1-hUCB-MSCs co-culture enhanced hDPC proliferation and restored ALP activity after 5 days, both of which are anagen markers of the hair cycle.

2-β-catenin, AKT, and GSK3β, which are proteins contained in the pathway related to cell growth and proliferation, were up-regulated in hDPCs by co-culture with hUCB-MSCs. 3-IGFBP-1 and VEGF were upregulated in the medium of the hDPC plus hUCB-MSCs group compared to the hDPC group.

Few clinical studies with advanced hMSC therapies have been conducted for several forms of alopecia, probably because of ethical considerations and the high cost of launching these studies (Table 2) . Firstly, a group of Egyptian authors published clinical data on the use of hMSCs in the treatment of AA and AHA. In 2011, this group conducted a pilot study in eight AA patients in order to evaluate the effect of cultured and enhanced follicular stem cells (FSC) in the treatment of this condition. The FSC were extracted from a 4mm skin punch biopsy from the scalp and the FSC were injected once in the affected areas. At the end of the 6-month evaluation period, 5/8 patients (62.5%) achieved an excellent response, graded as an improvement of 50% or more. Two patients developed a good response (10-50% of improvement), whereas one patient showed poor response [45] . Later, Elmaadawy et al. developed a blinded randomized clinical trial where the purpose was to evaluate the safety and efficacy of the use of autologous bone marrow-derived mononuclear cells (including hMSCs) obtained without substantial manipulation compared to advanced FSC treatment for the management of resistant cases of AA and AHA. Patients were divided into four groups: groups 1 (10 resistant AA patients) and 3 (10 resistant AHA patients) received a single injection of mononuclear cells and groups 2 (10 AA patients) and 4 (10 AHA patients) received a single injection of FSC. After six months of clinical, dermoscopic, and histopathologic follow up, all the patients showed very good or excellent responses, especially in female patients, although 45% of AA patients suffered recurrence after one year of follow up. Moreover, no significant differences in dermoscopic or histopathologic analysis were found between the two studied treatments. Those data showed promising clinical results with advanced hMSC therapies for AA and AHA, but the frequent relapses after one injection revealed the need of multiple sessions, especially in AA patients [46] . In 2015, a Chinese group conducted a phase 1/phase 2, open-label clinical trial with human cord blood stem cells (CB-SC) in nine patients with established AA. All the patients received a single treatment of intravenous CB-SC combined with a Stem Cell Educator (Tianhe Stem Cell Biotechnologies ® , Jinan, China). In order to carry out this Stem Cell Education Therapy, the patient's blood was passed through a Blood Cell Separator MCS+ (Haemonetics ® , Braintree, MA, USA) for 6 to 7 h to isolate mononuclear cells in accordance with the manufacturer's recommended protocol. The collected mononuclear cells were transferred into the device for exposure to allogeneic CB-SCs, and other blood components were automatically returned to the patient. After 2 to 3 h in the device, CB-SC-treated mononuclear cells were returned to the patient's circulation. The authors stated that all the patients tolerated the treatment well with a lack of serious adverse effects. Hair regrowth was noted after 4 weeks in patients with patchy and total AA. Moreover, 3/4 of the universal AA patients showed short vellus hairs on the scalp in week 12. After a two-year follow-up, two patients achieved complete hair regrowth, with no relapses. Only one patient with universal AA failed to show a response with this therapy [47] . Table 2 . Clinical studies of MSCs as advanced cell therapy for non-scarring alopecia.

Trial Title

Autologous hair follicle stem cells from the lower bulge areas N = 8 patients with Alopecia Areata (AA).

One millilitre (in a density of 10 5 cells/mL) was injected intradermally once per centimetre square using a 23-gauge needle. 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.

The extent of affection ranged from 25 to 70% (mean: 48 ± 15%). Patients age ranged from 6 to 17 years. (mean: 10.12 ± 3.68 years).

The duration of disease ranged from 1 to 4 years (mean: 2.31 ± 0.96 years) 1-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. 2-After 6 months, 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. 3-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.

60% of the patients (the excellent responders) reported improved quality of life. [45] [ [49] [50] [51] [52] Regarding autologous fat injection obtained by lipoaspiration, a 9-patient case series was published in 2017. In this study, the investigators employed a scalp injection of adipose cells obtained by a liposuction surgical technique enriched with a stromal vascular fraction (SVF) in patients with AHA. After 24 weeks of follow up, an increase in the number of hairs/cm 2 measured by TrichoScan was noted [48] . Later, another group employed autologous adipose-derived stromal vascular cells (ADSVCs) in twenty patients with AA. In this paper, a significant increase in hair density and hair diameter was noted after a 6-month follow-up. Moreover, a significant decrease in the number of extracted hairs measured by a pull test was also found [49] . Finally, an anecdotical case report has shown the possible positive effect of autologous fat injections for scarring alopecia [50] .

Recently, some authors have published their experience with Rigenera ® technology in patients with AHA. After extracting three 3-mm scalp samples, the tissues were disaggregated by employing Rigeneracons ® and, after that, the solution was rotated in the Rigenera machine allowing them to obtain the micrografts. This treatment was directly infiltrated into the scalp of the 100 included patients as a mesotherapy and, after a two-month follow-up, TrichoScan ® revealed an increase in hair density and total hair count [51] .

Some other clinical trials with advanced hMSC and cellular therapy have been carried out in the last decade. In the beginning of the 2010s, a multicentric North American phase 2 study was conducted in order to evaluate and compare the efficacy of injections of ex vivo cultured and enhanced occipital autologous dermal and epidermal cells vs. dermal cells alone-two non-HMSC cellular therapies-in patients with hair loss. However, no further results of this study have been published yet [52] . Currently, a Lebanese group is recruiting patients for an open-label non-randomized clinical trial aiming to assess the effect of adipose-derived cultured hMSCs obtained by lipoaspiration and to compare their efficacy with non-cultured hMSCs. This group has experience with the employment of adipose-derived hMSCs in patients with AA, a therapy which has shown good clinical results [49, 53] . Moreover, some other clinical trials are also evaluating the effectiveness and safety of autologous ex vivo expanded dermal and epidermal cultured cells in subjects with AA and AHA. Those trials are still in a pre-therapeutic phase and no results are available [54] [55] [56] .

Gene therapy is a new approach to discover and treat many diseases, including alopecia. Gene-based therapeutics are broadly defined as using a vector to introduce nucleic acids into cells with the aim of altering gene expression to prevent, halt, or reverse a pathological process [57] . The skin was one of the first targets for experimental gene transfer, as it is a superficial organ, easy to manipulate and observe [58] . In gene therapy techniques, genetic material is usually transferred using modified vectors directly into a subject's epidermal tissue (in vivo) or indirectly (ex vivo). When ex vivo techniques are used, cells are removed from the host, they are genetically manipulated, and then, reconstituted into the subject's skin [59] .

Gene therapy through RNA interference has been considered one of the most recent and revolutionary approaches used in individualized therapy. In fact, gene silencing and knockdown by topical siRNA has rapidly developed in recent years and its application in gene therapy has become an attractive alternative for drug development [60] . RNA interference by small interfering RNA (siRNA) is a technique to suppress the expression of certain genes with a high specificity [61] .

The use of topical siRNA is limited because of its low permeability through the epidermis, its high molecular weight, its negative charge, and its susceptibility to degradation by endogenous enzymes [62] [63] [64] . For this reason, efforts in topical siRNA delivery are being focused on chemical methods to prepared carrier molecules able to mask siRNA-negative charges, compress the siRNA molecule to make it smaller, and protect siRNA from degradation as well as the use of physical methods [65] .

Regarding chemical methods, currently, carriers of siRNA can be classified into two categories: viral and non-viral. Non-viral vectors are preferred because they have less toxicity, less immunogenicity, and easier preparation, but they present a low-efficiency transient gene expression [66, 67] . Non-viral carriers typically involve complexing of siRNA with different compounds such as polymers, cationic lipids, cell penetration peptides nanocarriers, or others. Using nanocarriers has been shown to efficiently encapsulate siRNA, providing protection against degradation and greatly improving the efficiency of delivery [68] .

Besides the advancement in nanocarriers systems, the release of the siRNA into the cellular cytoplasm (site of biological activity) remains low range [69] . As an alternative, different physical methods have been proposed to facilitate molecular permeability through non-endosomal cellular penetration, transferring the siRNA directly to the cellular cytosol facilitating the endosomal escape of the siRNA [70] . Iontophoresis, microneedle array devices, and ultrasound technique are the physical methods most employed for improving efficient delivery of either naked or loaded siRNA into the skin and promotion of gene silencing [60] .

Nakumura et al. reported the effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of alopecia areata and demonstrated the specific inhibition of target gene expression, resulting in a restoration of hair shaft elongation (Table 3) . They firstly proved the dominant role of Th1 cells in the alopecic areas, as the infiltrating CD4 T lymphocytes around the hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells. After that, they tried to reveal the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant Il-4 and neutralizing anti-interferon antibody local injections. They found that both effectively treated alopecia in in C3H/HeJ mice and demonstrated that intralesional injection of Il-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon mRNA expression in the affected alopecic skin. The siRNA used in this work was targeted to T-box21 (Tbox transcription factor), in order to inhibit the transcription factor responsible for the cytokine Th1 production. Therefore, Th1 T-box21 siRNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. In that way, they demonstrated the use of gelatin-small interfering RNA conjugates as an efficient and safe tool for alopecia areata [71] . the hair growth index more efficiently than the control rat IgG (Group 5). There was no disappearance of hair shafts from these mice during a 2-month observation period after the cessation of antibody application. 4-Antisense Tbx21 oligonucleotide (Group 6) was significantly more effective for alopecia than non-sense oligonucleotide (Group 7). 5-Cationized gelatin-conjugated Tbx21 siRNA injections (Group 8) were more effective than naked Tbx21 siRNA injections (Group 9) or non-sense siRNA conjugated with cationized gelatin (Group 10). There was no recurrence of alopecia in the mice during a 2-month observation period after the cessation of Tbx21 siRNA application. Four microRNAs-miR-221, miR-125b, miR-106a, and miR-410-were proved to be upregulated in balding papilla cells. In that way, they could participate in the pathogenesis of male pattern baldness. Therefore, it was suggested these microRNAs were possible candidates for a gene therapy regarding the strong therapeutic potential of microRNAs and the easy accessibility of hair follicles for gene therapy [72] .

Tissue engineering is an interdisciplinary field combining scaffolds, cells, and biomolecular signals to treat skin lesions. Its main challenge is the reconstitution of fully organized and functional organ systems from dissociated cells that have been propagated under a defined tissue culture condition. This strategy may contribute to the treatment of deep skin injuries and to the understanding of skin regeneration. Many dermal-epidermal composites or skin equivalents have been described to use in the clinic but the inability of these skin constructs to regenerate epidermal appendages, such as hair follicles, sebaceous, and sweat glands remains a major challenge [73] [74] [75] .

Lee et al. developed a simplified procedure to reconstitute hair-producing skin (Table 4 ). They obtained epidermal and dermal cells from newborn mice, and mixed them in different ratios. A high-density cellular suspension was prepared in drops of minimal volume on tissue culture inserts or wells. They allowed the cells to settle until a gel consistency was obtained and they seeded the cells on the collagen beside a porous matrix of crosslinked bovine tendon collagen and glycosaminoglycan and a silicone layer. Both constructs were grafted in full thickness skin wounds generated on the back of athymic mice. Hair germ started to appear eight days after grafting, progressed to hair peg, and complete hair follicles were observed four days later. The skin was in good condition after a twelve-month follow-up [76] . Furthermore, two reports showed hair follicle neogenesis using hair germs obtained by the previously described "organ germ method" [77] . Asakawa et al. generated bioengineered hair germs mixing epithelial and mesenchymal cells derived from mouse embryos within a collagen gel drop, generating a structure (called "hair germ") with two cellular layers separated by a translucent region (Table 5 ). They observed the presence of mature hair follicles when these hair germs were transplanted ectopically into subrenal capsules. These bioengineered hair follicles had a normal histological structure with outer root sheath, inner root sheath, dermal papilla, hair matrix, and sebaceous glands and they were connected to the arrector pili muscle and nerve fibers and were able to produce hair shafts [78] . Toyoshima et al. also demonstrated fully functional orthotopic hair regeneration via intracutaneous transplantation of bioengineered hair follicle germs generated by epithelial and mesenchymal cells derived from mouse embryos [79] . Later, Qiao et al. showed a more developed morphological structure, called "proto hairs". It presented hair-like characteristics as an inner mass of cells similar to the dermal papilla structure, surrounded by matrix-like keratinocytes and a partially keratinized substance that could produce a hair shaft [80] . 1-Human and rat vibrissa DP cells were able to reorganize the collagen lattices within the first 48 h. Contraction was significantly stronger with rat vibrissa DP cells than with human DP cells (60 and 40%, respectively). 2-Addition of epidermal keratinocytes enhanced contraction in both cases (75 and 56%, respectively). 3-After 1 week, rat vibrissa DP cells cultured together with keratinocytes totally disaggregated and lysed collagen lattices. 4-Human DP cells reorganized collagen matrix but were unable to disintegrate it. 5-After 10 days, DP cells embedded into collagen gel, and keratinocytes seeded on the top of the gel formed tubular structures.

tube-like outgrowths in the culture of epidermal keratinocytes [81] [84]

Mouse dermal and human epidermal cells.

Patch assay

Mouse dermal and epidermal cells were freshly isolated from C57BL/6 used for control experiments. Human dermal papilla (DP) spheres (10 4 cells) were prepared from two-dimensional (2D) cultured DP cells using either low cell-binding plate or hydrocell plate and combined with freshly isolated mouse epidermal cells for implantation A total of 200 DP spheres (2 × 10 6 cells) prepared from human DP cells were mixed with fresh mouse epidermal cells (2 × 10 6 cells) and implanted. 50 DP spheres (5 × 10 5 cells) prepared from human DP cells were mixed with fresh mouse epidermal cells (5 × 10 5 cells) and implanted. Mice were killed 2 weeks after cell implantation in order to verify hair follicle induction.

1-Hair follicle was observed in positive control experiments with mouse dermal and epidermal cells. 2-Hair follicle formation was observed when human DP spheres from various passages of culture were mixed with new born mouse epidermal cells. 3-Hair follicles were never observed when 2D cultures from the same population were use 4-The morphology and size of hair follicles induced by human DP spheres resembled the ones induced by mouse dermal cells.

1-Using a reconstitution assay, sphere formation increases the ability of cultured human DP cells to induce hair follicles from mouse epidermal cells [85] In vivo study -Nude mice Human DP cells were isolated from temporal scalp dermis. DECs were constructed by combining DP cells with rat tail collagen type 1, adding NFKs on top and bringing the constructs to the air-liquid interface for 2 days before grafting onto female nude mice.

1-Alkaline phosphatase activity was variable between samples, with cells from 3 of the donors showing alkaline phosphatase activity in more than 50% of the cells. 2-8 weeks after grafting, hair follicles (HFs) were observed in mice grafted with the 3 human DP cells with higher alkaline phosphatase activity. 3-HFs had a bulb, dermal sheath, hair matrix and cortex 4-Cells in the region of the DP and displayed alkaline phosphatase activity, normal reactivity with specific antibodies to human nestin and versican. 5-Basal layer of the outer root sheath was immunoreactive for keratin 15.

1-Cultured specialized human cells such as DP cells can induce complete pilosebaceous units in vivo in the grafted DEC model. [86] After that, Osada et al. artificially prepared dermal papilla cells spheres by aggregation of mouse vibrissae follicles in a round-bottom 96-well low-binding plate ( Table 6 ). These spheres expressed higher amounts of versican, an anagen dermal papilla marker, than dermal papilla cells in monolayer cultures and they induced hair follicle neogenesis for at least twenty-six passages. However, these methods obtain variable sizes of microtissues and cell number content. To resolve this problem, substratum materials to enhance dermal papillae cells aggregation, such as polyethylene-co-vinyl alcohol or polyvinyl alcohol membranes, were employed, inducing hair follicular neogenesis [87] [88] [89] . 3-Proto-hairs could undergo further maturation in vivo. 4-Within 2 weeks, black-pigmented hair fibres appeared.

implantation, approximately 50-60% of implants developed follicles with pigmented hair shafts. 6-Hairs developed from implanted proto-hairs were well anchored in the skin and persisted for at least 6 months 7-Implanted proto-hairs were able to maintain their growth for many months. [80]

DP cells were dissected from mouse vibrissae follicles (versican-GFP-Tg) and 10 4 DP cells were aggregated to form one spherical structure and maintained for 2 to 12 days. Epidermal and DP cells were dissected from C57BL/6J mouse. Fifty of the DP spheres or 5 × 10 5 of the dissociated DP cells were combined with 5 × 10 5 epidermal cells. Cells were injected subcutaneously into athymic nude mice. Mice were killed 2 weeks after cellimplantation to verify hair follicle induction Moreover, Chermnykh et al. showed that human keratinocytes from outer root sheath and dermal papillae cells cultured in a 3D network of extracellular matrix proteins and collagen formed tubule-like structures in this skin-equivalent in vitro [81] , but did not form complete hair follicles. Similarly, Sriwiriyanont et al. observed neofollicle formation in nude mice grafted with engineered skin substitutes containing murine dermal papillae cells and human keratinocytes in a collagen-glycosaminoglycan matrix, but not in those containing human dermal papillae cells and human keratinocytes [83] .

Ehama et al. showed the formation of hair follicle-like structures using human primary cultures of foreskin-or adult-derived epidermal cells co-grafted with murine dermal papillae cells by a chamber assay. The innermost regions were similar to the hair cortex and medulla of mature human follicles but they did not show a bulge region, all the follicular epithelial layers, and versican was not expressed, suggesting that the differentiation process was altered [84] . At the same time, Kang et al. obtained spheroid microtissues by culturing human dermal papillae cells in a 96-well low-binding plate, and implanted them intradermically into nude mice using the "patch assay", generating new hair follicles. However, these hair follicles were not observed when monolayer dermal papillae cell cultures were used, concluding that cultured human dermal papillae cells do not induce hair neogenesis unless changes in the culture conditions were made [85] . Later, Higgins et al. found that human hair follicle dermal cells can be interchanged with interfollicular fibroblasts and used as an alternative cell source for establishing the dermal component of engineered skin, both in vitro and in vivo. These authors established some in vitro skin constructs by incorporating into the collagenous dermal compartment: primary interfollicular dermal fibroblasts, hair follicle dermal papilla cells, or hair follicle dermal sheath cells. In vivo skins were established by mixing dermal cells and keratinocytes in chambers on top of immunologically compromised mice. They found that all fibroblast subtypes were capable of supporting the growth of overlying epithelial cells, both in vitro and in vivo, being hair follicle dermal sheath cells superior to fibroblasts in their capacity to influence the establishment of a basal lamina. They also evaluated the human dermal papilla cells' transcriptome, observing that monolayer dermal papillae cells cultures showed the most important changes immediately after early outgrowths from dermal papillae explants, which suggests that human dermal papillae cells spheroids were able to initiate hair follicle morphogenesis, but the production of a complete hair follicle required additional signals [90, 91] . Others investigators produced dermal papillae cell spheroids from cultured dermal papillae cells on a Matrigel™ (Corning Life Sciences, Corning, NY, USA) scaffold, in order to restore dermal papillae signature gene expression of NCAM, versican, and α-smooth muscle actin, markers that are lost during the monolayer culture. They also observed that these dermal papillae cell-spheres, combined with hair germinal matrix cells onto Matrigel-coated plates, produced colorless fiber-like structures in vitro [82] .

Other groups have cultured dermal mesenchymal cells in 3D conditions as the organotypic method of culturing cells inside a scaffold. This gel was seeded with keratinocytes from interfollicular skin, superior outer root sheath, or inferior outer root sheath obtaining an in vitro bi-layered skin. Nevertheless, only the constructs containing superior outer root sheath keratinocytes showed hair follicle-like structures [92] . Thangapazham et al. used a dermal equivalent composed of dermal papillae cells from human scalp contained in a collagen-I gel. Eight weeks after grafting onto nude mice, these constructs presented hair follicles showing bulb, dermal sheath, hair matrix, and cortex. Histological analysis showed concentric layers of inner root sheath and outer root sheath, sebaceous glands, and hair shaft. Immunohistochemistry assays revealed that both epithelial and dermal cells from neofollicles were of human origin, and dermal papillae cells and dermal sheath cells expressed human nestin and versican [86] . Leirós et al. showed that both epithelial and dermal cultured cells from adult human scalp in a dermal scaffold were able to produce in vivo structures that recapitulate embryonic hair development. In fourteen days, histological structures reminiscent of many different stages of embryonic hair follicle development were observed in the grafted area. These structures showed concentric cellular layers of human origin, and expressed K6hf, a keratin present in epithelial cells of the companion layer. However, the presence of fully mature hair follicles was not observed [93] .

Recently, Abaci et al. regenerated for the first time a truly functional human skin in an entirely ex vivo context that incorporated hair follicles from cultured human cells. They used a biomimetic approach for generation of human hair follicles within human skin constructs by recapitulating the physiological 3D organization of cells in the hair follicle microenvironment using 3D-printed molds. Overexpression of Lef-1 in dermal papilla cells restored the intact dermal papilla cells' transcriptional signature and enhanced the efficiency of hair follicle differentiation in human skin constructs. After that, vascularization of hair follicle-bearing human skin constructs increased graft survival and enabled efficient human hair growth in mice. They were able to generate 225 hair follicles, starting from only one hair follicles donor tissue, which was not possible with previous techniques [94] .

Some clinical trials with tissue engineered therapies for alopecia have been developed in recent years. A Taiwanese group designed an observational study with 400 healthy adults that were going to receive cosmetic surgery such as removal of moles. The aim of this study was to note if a sample of in vitro cultured dermal papilla cells was able to induce hair follicle formation after maintaining their spherical structure before transplanting into dermis in vivo. To date, the authors have not published the results of this study [95] .

Alopecia areata and androgenic alopecia are still challenging conditions for dermatologists, with a lack of highly effect treatments. With this background, advanced therapies are a promising therapeutic option that have shown good results in preclinical studies. However, more clinical studies are needed to verify if ATMPs can be a safe and effective treatment for diverse forms of non-scarring alopecia.

Do Dermatologic Diagnosis Change in Hot vs Cold Periods of The Year? A Sub-Analysis of the DIADERM National Sample

Association Between Alopecia Areata, Anxiety, and Depression: A Systematic Review and Meta-analysis

Safety concerns when using novel medications to treat alopecia

Cicatricial (Scarring) Alopecias: An overview of pathogenesis, classification, diagnosis, and treatment

Stem Cells and Tissue Engineering: Regeneration of the Skin and Its Contents

Advanced therapies in current Medicine

White paper on how to go forward with cell-based advanced therapies in Europe

of the European Parliament and of the Council of 13 November 2007 on Advanced Therapy Medicinal Products and Amending Directive 2001/83/EC and Regulation

The regenerative medicine and stem cell business: Confusion with legal implications

Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products

Angiomyeloproliferative lesions following autologous stem cell therapy

MSC Therapies for COVID-19: Importance of Patient Coagulopathy, Thromboprophylaxis, Cell Product Quality and Mode of Delivery for Treatment Safety and Efficacy. Front

Therapeutic Potential of Umbilical Cord Mesenchymal Stromal Cells Transplantation for Cerebral Palsy: A Case Report

Concise Review: Role of Mesenchymal Stem Cells in Wound Repair

Exosomes Secreted from Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Accelerate Skin Cell Proliferation

Multipotent stromal cells/mesenchymal stem cells and fibroblasts combine to minimize skin hypertrophic scarring

Key transcription factors in the differentiation of mesenchymal stem cells

The promotion of chondrogenesis, osteogenesis, and adipogenesis of human mesenchymal stem cells by multiple growth factors incorporated into nanosphere-coated microspheres

Regulatory Mechanism of The Induction of Regulatory T Cells through Growth Factors Released by Human Mesenchymal Stem Cells

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue

Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue, and Lung Tissue Differentially Mitigate Lung and Distal Organ Damage in Experimental Acute Respiratory Distress Syndrome

Bone marrow or adipose tissue mesenchymal stem cells: Comparison of the therapeutic potentials in mice model of acute liver failure

The Use of Growth Factors and Mesenchymal Stem Cells in Orthopaedics: In particular, their use in Fractures and Non-Unions: A Systematic Review

Exosomes Secreted from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Prevent Osteonecrosis of the Femoral Head by Promoting Angiogenesis

Adipose-Derived Mesenchymal Stem Cells: Are They a Good Therapeutic Strategy for Osteoarthritis?

In Vivo Models for the Evaluation of the Osteogenic Potency of Bone Substitutes Seeded with Mesenchymal Stem Cells of Human Origin: A Concise Review

Mesenchymal stromal cells: Properties and role in management of cutaneous diseases

Skin mesenchymal stem cells: Prospects for clinical dermatology

Role of Mesenchymal Stem Cells in Dermal Repair in Burns and Diabetic Wounds

Mesenchymal stem cells: Potential for therapy and treatment of chronic non-healing skin wounds

Human umbilical cord mesenchymal stem cells improve irradiation-induced skin ulcers healing of rat models

A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration

Therapeutic Potential of Stem Cells in Follicle Regeneration

Stem cells from human hair follicles: First mechanical isolation for immediate autologous clinical use in androgenetic alopecia and hair loss

Bone marrow-derived mesenchymal stem cells prevent alopecia areata development through the inhibition of NKG2D expression: A pilot study

Increased Expression of Cxcr3 and Its Ligands, Cxcl9 and Cxcl10, during the Development of Alopecia Areata in the Mouse

Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells

The effect of CD34+ cell-containing autologous platelet-rich plasma injection on pattern hair loss: A preliminary study

Th1/Th2 PB balance and CD200 expression of patients with active severe alopecia areata

Involvement of the bulge region with decreased expression of hair follicle stem cell markers in senile female cases of alopecia areata

Hair follicle stem cell differentiation is inhibited through cross-talk between Wnt/β-catenin and androgen signalling in dermal papilla cells from patients with androgenetic alopecia

Effects of mesenchymal stem cell therapy on alopecia areata in cellular and hair follicle organ culture models

Human umbilical cord blood mesenchymal stem cells engineered to overexpress growth factors accelerate outcomes in hair growth

Autologous progenitor cell implantation as a novel therapeutic intervention for alopecia areata

Stem cell therapy as a novel therapeutic intervention for resistant cases of alopecia areata and androgenetic alopecia

Hair regrowth in alopecia areata patients following Stem Cell Educator therapy

Hair follicle growth by stromal vascular fraction-enhanced adipose transplantation in baldness

Cellular therapy with human autologous adipose-derived adult cells of stromal vascular fraction for alopecia areata

Autologous adipose transplantation an effective method to treat alopecia after trauma: A case report

Progenitor-cell-enriched micrografts as a novel option for the management of androgenetic alopecia

A Study to Evaluate and Compare Injections of Autologous Dermal and Epidermal Cells into the Balding Scalp of Subjects with Hair Loss (CA-0002899)

Autologous Adipose-Derived Adult Stromal Vascular Cell Transplantation for Alopecia

AGA Biocellular Stem/Stromal Hair Regenerative Study

Regenerative Treatment Scaring Alopecia and Alopecia Areata

Nanofat Grafting for Treatment of Androgenetic Alopecia

State-of-the-art gene-based therapies: The road ahead

Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles

Gene Therapy for Skin Diseases. Cold Spring Harb

Current Non-viral siRNA Delivery Systems as a Promising Treatment of Skin Diseases

Unlocking the potential of the human genome with RNA interference

Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier

Penetration of nanoparticles and nanomaterials in the skin: Fiction or reality?

Formulation challenges for 21st century topical and transdermal delivery systems

Suppression of tumor metastasis by NK4 plasmid DNA released from cationized gelatin

Dendrimers for siRNA Delivery

CXCL1 gene silencing in skin using liposome-encapsulated siRNA delivered by microprojection array

Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape

Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine

Controlled Delivery of T-box21 Small Interfering RNA Ameliorates Autoimmune Alopecia (Alopecia Areata) in a C3H/HeJ Mouse Model

Differential expression analysis of balding and nonbalding dermal papilla microRNAs in male pattern baldness with a microRNA amplification profiling method

Hyaluronic acid biomaterial for human tissue-engineered skin substitutes: Preclinical comparative in vivo study of wound healing

Management of burn injuries of various depths

A review of tissue-engineered skin bioconstructs available for skin reconstruction

A Simplified Procedure to Reconstitute Hair-Producing Skin

The development of a bioengineered organ germ method

Hair organ regeneration via the bioengineered hair follicular unit transplantation

Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches

Hair morphogenesis in vitro: Formation of hair structures suitable for implantation

Dermal papilla cells induce keratinocyte tubulogenesis in culture

Controllable Production of Transplantable Adult Human High-Passage Dermal Papilla Spheroids Using 3D Matrigel Culture

Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells

Hair Follicle Regeneration Using Grafted Rodent and Human Cells

Sphere Formation Increases the Ability of Cultured Human Dermal Papilla Cells to Induce Hair Follicles from Mouse Epidermal Cells in a Reconstitution Assay

Dissociated human dermal papilla cells induce hair follicle neogenesis in grafted dermal-epidermal composites

Long-Term Culture of Mouse Vibrissal Dermal Papilla Cells and De Novo Hair Follicle Induction

Self-assembly of dermal papilla cells into inductive spheroidal microtissues on poly (ethylene-co-vinyl alcohol) membranes for hair follicle regeneration

Hair?inducing ability of human dermal papilla cells cultured under Wnt/??-catenin signalling activation

Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth

Multifaceted role of hair follicle dermal cells in bioengineered skins

Hair follicle reformation induced by dermal papilla cells from human scalp skin

Dermal Papilla Cells Improve the Wound Healing Process and Generate Hair Bud-Like Structures in Grafted Skin Substitutes Using Hair Follicle Stem Cells

Tissue engineering of human hair follicles using a biomimetic developmental approach

Tissue Engineering for Hair Follicle Regeneration-Full Text View-ClinicalTrials.gov

We gratefully acknowledge financial support from Instituto de Salud Carlos III through the projects PI-17/02083 (co-funded by European Regional Development Fund "A way to make Europe") and Ministry of Health of the Andalusian Regional Government (SAS PI-0458-2016 and AML PIGE-0249-2019).

The authors declare no conflict of interest.