Mesenchymal Stem Cells for the Treatment of Skin Diseases

Mesenchymal stem cell (MSC)-based therapy involving both autologous and allogeneic MSCs shows great promise in treating several conditions. MSCs promote wound healing, and can differentiate into multiple cell lineages, including keratinocytes. Therefore, MSCs can be used for the treatment of congenital or acquired skin defects. Because of their immunomodulatory properties, MSCs may be useful for the treatment of inflammatory and autoimmune skin diseases. In particular, MSCs might be effective for the treatment of large vitiligo lesions as immunosuppressant or cultured grafts. MSCs can also be a novel cell source for regenerating hair in the treatment of scarring alopecia and androgenic alopecia. MSCs might also be an effective treatment for alopecia areata, which is associated with autoimmunity. Stem cell therapies with topical administration of MSCs and bone marrow transplantation were shown to alleviate recessive dystrophic epidermolysis bullosa in both animal models and human subjects. In addition to cell transplantation, the mobilization of endogenous MSCs has been attempted for skin regeneration. Overall, this review highlights the great potential of MSCs for the treatment of skin diseases in the near future.


Introduction
Regenerative medicine is a novel approach for the treatments of various diseases.Stem cells are a unique population of undifferentiated cells that can self-renew and differentiate into various cell lineages.These cells are expected to play a central role in the treatment of skin diseases as well as diseases of other organs.Stem cells are classified into embryonic stem cells (ESCs), induced pluripotent stem cells, and adult stem cells.The use of adult stem cells circumvents the ethical issues associated with the use of ESCs, and their generation does not require the introduction of exogenous transcription factors using viral vectors or other ex vivo manipulations into cells.Therefore, adult stem cells are thought to be very useful in regenerative medicine.
Adult stem cells include hematopoietic stem cells, mesenchymal stem cells (MSCs), and endothelial stem cells, among others.MSCs can be isolated from different tissues such as the bone marrow (BM-MSCs), umbilical cord (UC-MSCs), placenta (P-MSCs), umbilical cord blood (UCB-MSCs), amniotic fluid (AF-MSCs), fetal tissues, skin (dermal MSCs), and adipose tissues (ADSCs) [1][2][3][4][5].MSCs are multipotent stromal cells that can differentiate into various cell types such as adipocytes, osteoblasts, chondrocytes, fibroblasts, and myoblasts [6,7].MSCs were first discovered from the bone marrow, which showed the potential to differentiate into bone cells [8,9]; since then, these BM-MSCs have been the major source of MSCs for clinical use.However, UC-MSCs, UCB-MSCs, P-MSCs, and AF-MSCs can be collected in relatively larger quantities using less invasive techniques [10].Although dermal MSCs can be easily harvested, dermal stem cells only account for 0.3% of human dermal foreskin fibroblasts [2,11].ADSCs can be easily harvested in large quantities, and produce 500-times more colony-forming units than BM-MSCs [12].Therefore, ADSCs will be a practical and promising tool for regenerative medicine [13].donors with various matching levels of human leukocyte antigen (HLA) is a promising therapy.MSCs are immune-evasive cells because they do not express MHC class II antigens and co-stimulatory molecules such as CD40, CD80, and CD86, and minimally express MHC class I antigens [14].This lack of immunogenicity facilitates the clinical use of MSCs including for allogeneic cell transplantation [15].
The clinical utility of MSCs in wound healing is based on repairing and replacing cellular substrates, attenuation of inflammation, increasing angiogenesis, and enhancing the migration of reparative cells by cytokine/chemokine production.

Differentiation of MSCs into Keratinocyte-like Cells
The ability of MSCs to differentiate into keratinocytes and melanocytes and to be bioengineered into hair follicles is important for their use in organ replacement therapy in dermatology.In particular, with respect to the differentiation potential of MSCs into keratinocytes, the cells responsible for wound re-epithelialization after wound formation are needed for the use of MSCs in skin regeneration.In addition, the differentiation of MSCs into ectoderm cells has been considered to be difficult.However, BM-MSCs were reported to have the ability to transdifferentiate into keratinocyte-like cells [36][37][38][39].
ADSCs can also differentiate into keratinocyte-like cells [40,41].In a previous study, ADSCs were co-cultured with normal human dermal fibroblasts on type IV collagen in Dulbecco's modified Eagle's medium containing all-trans retinoic acid (ATRA) and bone morphogenetic protein 4 (BMP4).
Next, the cells were co-cultured with fibroblasts in keratinocyte serum-free medium (KSFM) lacking ATRA and BMP4, followed by culturing in KSFM on type IV collagen in the absence of other cells.
These differentiated cells showed high expression of the keratinocyte markers desmoglein 3 and cytokeratin 5 [40].This fact suggested that ADSCs could possibly be used as keratinocyte progenitor cells in skin regeneration.

MSCs are a heterogeneous population of cells that have variable expression of markers depending
on the tissue source.The minimal criteria for defining human MSCs are: (1) plastic adherence when maintained in standard culture conditions; (2) expression of CD105, CD73, and CD90; lack of expression of CD45, CD34, CD14, or CD11b, CD79α, or CD19, and HLA-DR surface molecules; and (3) differentiation to osteoblasts, adipocytes, and chondroblasts in vitro [42].
Cell sorting, which is the method of separating cells according to their properties such as size, morphology, or surface protein expression, is thought to be useful to isolate putative stem cells.
Multilineage-differentiating stress enduring (Muse) cells are a new type of pluripotent stem cells derived from MSCs [43].Muse cells are isolated from human skin fibroblasts, ADSCs, and BM-MSCs, and can be identified by stage-specific embryonic antigen 3 as a marker.Muse cells were demonstrated to differentiate into functional melanocytes [44].Cell sorting techniques such as fluorescent-activated cell sorting and magnetic cell sorting will be important to increase the efficacy of stem cell therapy.

MSCs for the Treatment of Skin Diseases
Numerous studies have already shown the beneficial uses of MSCs in both preclinical and clinical trials, which have highlighted the potential of MSCs for the treatments of several disorders, including hematological disease, multiple sclerosis, diabetes, osteoarthritis, chronic spinal cord injury, and feline chronic kidney disease [45,46].In the dermatology field, MSCs may be applied in wound repair after a deep wound or burn.In addition, because of their immunomodulatory properties, MSCs have been used to successfully treat steroid-resistant acute graft-versus-host disease [26,[47][48][49][50][51].MSCs may be also of potential use for inflammatory skin diseases, including atopic dermatitis and psoriasis, and autoimmune skin diseases such as lupus erythematosus.
The human skin has two primary layers: the epidermis and dermis.The epidermis is an ectoderm-derived, keratinized, stratified layer with a unique cytokeratin expression pattern, while the dermis is a mesoderm-derived layer composed of connective tissue, nerves, vessels, and appendages.
The epidermis acts as a barrier against infection and regulates the amount of water released from the body.In a healthy state, the epidermis is a rapidly regenerating tissue that is maintained by the continuous transformation from transient amplifying cells and epidermal stem cells located in the basal layer of the interfollicular epidermis (interfollicular epidermal stem cells), bulge region of hair follicles (hair follicle stem cells), and sebaceous glands (sebaceous gland stem cells).Epidermal stem cells differentiate into keratinocytes that are directed to the upper layers of the epidermis and are involved in wound re-epithelialization. Hair follicle stem cells, which have the functions of hair follicle remodeling and epidermal regeneration, show potential for the treatment of patients with alopecia [52,53] and for inducing repigmentation in the patients with vitiligo [54,55].Sebaceous gland stem cells are able to differentiate into both sebocytes and the interfollicular epidermis [11].
Moreover, melanocytes are directly involved in pigmentation, and ultraviolet (UV) B exposure induces the differentiation of melanocyte stem cells into melanoblasts [56].Melanoblasts migrate from the neural crest to the hair follicles where they undergo proliferation, differentiation, and maturation into melanocytes [57][58][59].
However, mesenchymal compartments contain a greater number of adult stem cells than the epidermis.MSCs can differentiate into multiple cell lineages, including keratinocytes.Thus, MSCs can be used for the treatment of congenital or acquired skin defects, including burns, chronic ulcers, or deep wounds, owing to not only their wound-healing properties but also their differentiation potential into keratinocytes.
Skin aging is induced by a decrease or functional disorder of stem cells [2].Therefore, MSCs may induce anti-aging effects by promoting skin regeneration, maintaining skin homeostasis, and affecting the activity of various humoral factors.The anti-aging activity of MSCs should be determined before their clinical application for anti-aging therapy such as acne scars, wrinkles, and photoaging.

Vitiligo
Vitiligo vulgaris is characterized by the development of white patches on various parts of the body.
Although vitiligo is not a contagious or life-threatening disease, it significantly reduces the quality of life of patients with widespread lesions.Vitiligo is clinically divided into two types: the generalized and segmental type [60,61].Although the pathogenesis of vitiligo is unclear, autoimmunity against melanocytes and melanins, and impaired peripheral nerve function are suggested to play a role in vitiligo development.Therefore, UV therapy and topical steroid therapy are the first-line treatments for both types of vitiligo, based on their immunosuppression effect against autoimmunity.Intractable and stable vitiligo lesions can be treated by autologous epidermal transplantation, including suction blister grafting and mini-grafting [62][63][64].
Among MSCs, Muse cells were reported to have the ability to differentiate into melanocytes [44,65].For the treatment of large vitiligo lesions, autologous MSCs might be useful as an immunosuppressant or in cultured autologous grafting, although no clinical reports of these applications have been reported to date.

Alopecia
Scarring alopecia develops as a result of injury or autoimmune skin diseases such as discoid lupus erythematosus of the scalp.Patients with scarring alopecia show destruction of the hair follicles indicating the need for excision and plasty or hair transplantation.Androgenic alopecia (male pattern alopecia) is currently treated using drugs and hair transplantation.However, the drug treatment exerts temporary effects, and the total transplantable hair number of the donor site is limited.To overcome these problems, in vitro culturing and implantation of hair follicles using autologous stem cells may be useful for treating scarring and androgenic alopecia.Yoo et al. [66] showed that dermal papilla-like tissues formed using BM-MSCs and UC-MSCs induced the growth of new hair follicles in mice.Thus, autologous MSCs are expected to be a novel cell source for regenerating hair in the treatment of scarring alopecia and androgenic alopecia.
Conversely, alopecia areata is characterized by round or oval patches of non-scarring hair loss across the entire body.In severe cases, the hair loss may progress to complete scalp hair loss.Although the pathogenesis of alopecia areata is unclear, autoimmunity is thought to be involved in its development [67][68][69].Therefore, alopecia areata is treated with topical steroids, steroid injections, UV therapies, and immunosuppressants.Because some cases are intractable, the immunomodulatory and anti-inflammatory effects of autologous MSCs could be considered as one of the options for the treatment of alopecia areata.In animal models, alopecia areata-induced mice were successfully treated with MSCs [70].However, no successful strategies for treating scarring alopecia, androgenic alopecia, and alopecia areata using MSCs have been reported in human studies.

Epidermolysis Bullosa
Epidermolysis bullosa (EB) is a group of genetic skin fragility disorders characterized by blisters and skin erosions in response to minor injury.Recessive dystrophic EB (RDEB) is one of the more severe forms of EB.The pathogenesis of RDEB involves mutations in COL7A1 that encodes type VII collagen (Col7), the main constituent of the anchoring fibrils that anchor the epidermal basement membrane to the papillary dermis [71], resulting in blistering and repeated wounding of the skin, oral mucosa, and gastrointestinal tract components [72,73].In these cases, persistent skin erosion frequently results in intractable ulcers.As the ulcers heal, they result in severe scarring, fusion of the fingers and toes, loss of nails, and contractures of the finger joints.
Various biological dressings, including autologous and allogeneic cultured skin substitutes, have been used for intractable ulcers in patients with RDEB [74][75][76][77][78][79][80].However, allogeneic cultured skin substitutes cannot be permanently adopted.Autologous cultured skin can serve as a permanent covering, but their use does not alleviate the effects of COL7A1 mutations.Thus, both autologous and allogeneic cultured skin substitutes are only used only as temporary biological dressings.
Stem cell therapy involving autologous and allogeneic MSCs may be used for EB, including RDEB.
Previous studies have shown that stem cell therapies using adult stem cells, including topical administration of MSCs and bone marrow transplantation, could alleviate RDEB in both animal models and human subjects [81][82][83][84][85][86][87][88].To determine the utility of MSCs from a healthy donor in an allogeneic transplantation for repairing basement membrane alterations in patients with RDEB, it is important to examine Col7 expression in these cells because patients with RDEB have mutations in Col7.Thus, allogeneic MSCs may enhance wound healing, correct Col7 insufficiency, repair defective/reduced anchoring fibrils, and improve skin integrity in patients with RDEB.However, it is necessary to consider the presence of anti-Col7 antibodies, which are relatively common in patients with RDEB [89].Junctional EB (JEB) is another major form of EB, and severe generalized JEB is the most severe type of EB that leads to the early death of patients, usually within the first few months of life [72,90,91].
Severe generalized JEB is caused by mutations in the laminin-332-encording genes LAMA3, LAMB3, and LAMC2.Autologous epidermal stem cells were reported to be useful in gene therapy for the treatment of JEB [92].Epidermal stem cells from a patient affected by LAMB3-deficient JEB were transfected with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-β3), and were successfully used to prepare genetically corrected cultured epidermal grafts.Autologous MSCs might be used similarly in gene therapy for the treatment of various types of EB.
In addition to the transplantation of autologous or allogeneic MSCs, mobilization of endogenous MSCs from the bone marrow to the affected site has been attempted for skin regeneration.
High-mobility group box 1 (HMGB1) is a non-histone nuclear protein that regulates chromatin structure remodeling [93].In injured tissues, HMGB1 is secreted by macrophages and dendritic cells and is released from necrotic cells to induce tissue remodeling and regeneration by activating inflammatory reactions and angiogenesis [94][95][96][97][98][99][100].HMGB1 is also a strong chemoattractant for mesoangioblasts and endothelial precursor cells [101,102].Intravenously administered recombinant HMGB1 was shown to mobilize endogenous platelet-derived growth factor receptor α-positive mesenchymal cells, which are epithelial progenitors, from the bone marrow in mice [103].HMGB1 could be used to stimulate endogenous mesenchymal cells to the site epithelial injury such as in RDEB patients.This method is more convenient than bone marrow transplantation.

Delivery Route
Systemic delivery routes such as intravenous injection or organ-specific local delivery methods should be examined for MSC administration.Systemic administration would be preferable over topical administration when MSCs are used for achieving anti-inflammation and immunosuppression effects.Although intravenous delivery is a simple route of administration, this method can possibly cause pulmonary embolism, which has not been recorded in humans.Since dermal substitutes are an appropriate environment for MSCs [104], local injection and the use of suitable scaffolds, including commercial dermal substitutes, in combination with MSCs may also be used for skin diseases, especially for the purpose of organ replacement.It is also necessary to establish an optimal delivery dose for MSC administration, together with additional preclinical and clinical studies.Because cells can be directly administered to the skin, the skin is suitable for practical application in regenerative medicine as well.

Conclusion
This review describes the potential use of MSCs in cell-based therapies for the clinical management of various dermatoses.The work conducted thus far shows the potential of MSCs for treating skin diseases owing to their immunomodulatory effect and transdifferentiation capacities.
MSCs have proven to be useful in skin regeneration.Among the different types of MSCs, the advantages of using ADSCs include their abundance in donors and their convenient isolation by less invasive methods such as liposuction.Although the optimal method for administering MSCs is still debatable, MSCs show the possibility to be widely used for treating skin diseases in the near future.
of stem cells for tissue engineering has greatly progressed in recent years.MSC-based therapy involving autologous and allogeneic MSCs isolated from related or unrelated 106 AIMS Cell and Tissue Engineering Volume 1, Issue 2, 104-117.