Advertisement

Adipose-derived stem cells applied in skin diseases, wound healing and skin defects: a review

Open AccessPublished:September 15, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.08.005

      Abstract

      Adipose tissue presents a comparably easy source for obtaining stem cells, and more studies are increasingly investigating the therapeutic potential of adipose-derived stem cells. Wound healing, especially in chronic wounds, and treatment of skin diseases are some of the fields investigated. In this narrative review, the authors give an overview of some of the latest studies concerning wound healing as well as treatment of several skin diseases and concentrate on the different forms of application of adipose-derived stem cells.

      Key Words

      Introduction

      The influence of adipose-derived stem cells (ASCs) [
      • Bourin P.
      • et al.
      Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).
      ] in wound healing, particularly with regard to chronic wounds or diabetic patients, has been addressed in several studies [
      • Hassan W.U.
      • Greiser U.
      • Wang W
      Role of adipose-derived stem cells in wound healing.
      ,
      • Shingyochi Y.
      • Orbay H.
      • Mizuno H
      Adipose-derived stem cells for wound repair and regeneration.
      ,
      • Hassanshahi A.
      • et al.
      Adipose-derived stem cells for wound healing.
      ,
      • Moon K.C.
      • et al.
      Potential of Allogeneic Adipose-Derived Stem Cell-Hydrogel Complex for Treating Diabetic Foot Ulcers.
      ,
      • Klar A.S.
      • Zimoch J.
      • Biedermann T.
      Skin Tissue Engineering: Application of Adipose-Derived Stem Cells.
      ,
      • Gentile P.
      • Garcovich S.
      Systematic Review: Adipose-Derived Mesenchymal Stem Cells, Platelet-Rich Plasma and Biomaterials as New Regenerative Strategies in Chronic Skin Wounds and Soft Tissue Defects.
      ]. However, various studies have also been conducted concerning ASCs and their influence on different inflammatory skin diseases and appendages of the skin [
      • Barrera J.A.
      • et al.
      Adipose-Derived Stromal Cells Seeded in Pullulan-Collagen Hydrogels Improve Healing in Murine Burns.
      ]. In this narrative review, the authors give an overview of some of the studies that have used different forms of application of ASCs. After addressing ASCs involved in normal and aberrant wound healing, the authors will first focus on different formulations from a cell perspective, including decellularized adipose tissue (DAT), ASC-derived extracellular matrix, fibrin-embedded ASCs, pre-differentiated ASCs and the ASC secretome. The authors will then focus on different skin diseases, including diabetic ulcers, burn wounds, alopecia and atopic dermatitis (AD), and discuss the therapeutic approaches for each disease that include ASCs. These studies not only give insights into their respective fields but also help us to understand the paracrine and in vivo effects of ASCs and to possibly establish standardization in their clinical use.

      Methods

      For this narrative review, the authors searched the databases MEDLINE, CINAHL, Biological Abstracts, Allied and Complementary Medicine Database, Web of Knowledge, Scopus, SPORTDiscus, PubMed and Embase using the key words “adipose tissue,” “adipose-derived stem cells,” “stromal vascular fraction,” “skin,” “skin disease,” “burn wound,” “lupus erythematosus,” “atopic dermatitis,” “epidermolysis bullosa” and “diabetic ulcer.” The authors selected the most representative studies and used them to give an overview of the current status of skin ASC-related research.

      Role of extracellular matrix in skin wound healing

      Many different factors and processes orchestrate skin wound healing, and a good outcome depends strongly on their appropriate emergence and disappearance [
      • Gantwerker E.A.
      • Hom D.B.
      Skin: histology and physiology of wound healing.
      ]. In other words, the interplay of trophic factors as well as immigrating and differentiating cells directs the normal skin wound healing back to homeostasis [
      • Monavarian M.
      • Kader S.
      • Moeinzadeh S.
      • Jabbari E.
      Regenerative Scar-Free Skin Wound Healing.
      ]. The composition of the extracellular matrix (ECM) plays a non-negligible role not only in healing skin but also in the healthy normal state [
      • Hyldig K.
      • Riis S.
      • Pennisi C.P.
      • Zachar V.
      • Fink T
      Implications of Extracellular Matrix Production by Adipose Tissue-Derived Stem Cells for Development of Wound Healing Therapies.
      ].
      Skin is a connective tissue, and the ECM in healthy skin is predominantly composed of collagen I, with lower amounts of fibronectin, elastin, laminin and fibrin [
      • Deniz A.A.H.
      • et al.
      Zooming in across the Skin: A Macro-to-Molecular Panorama.
      ], enabling dynamic behavior and biomechanics during skin movements [
      • Wong R.
      • Geyer S.
      • Weninger W.
      • Guimberteau J.C.
      • Wong J.K
      The dynamic anatomy and patterning of skin.
      ]. Mediated by integrins [
      • Hegde S.
      • Raghavan S.
      A skin-depth analysis of integrins: role of the integrin network in health and disease.
      ], the ECM of the skin regulates cell behavior via attachment and through molecular signaling. In this way, ECM is able to influence cell proliferation as well as transformation, differentiation and apoptosis [
      • Giancotti F.G.
      • Ruoslahti E.
      Integrin signaling.
      ]. Furthermore, the expression of growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β), two major growth factors regulating skin homeostasis and healing, is modulated by the specific composition of the ECM. The ECM controls their release and degradation, in this way playing a basic role during the dynamics of healing. Proteins such as matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) help to maintain proper ECM composition by keeping degradation and expression in proper balance (remodeling) [
      • López-López N.
      • et al.
      Expression and vitamin D-mediated regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy skin and in diabetic foot ulcers.
      ].

      ASCs in skin wound healing

      Over the past several decades, ASCs have been investigated with respect to their impact on skin healing [
      • Huayllani M.T.
      • et al.
      Adipose-derived stem cells in wound healing of full-thickness skin defects: a review of the literature.
      ,
      • Vidor S.B.
      • et al.
      Adipose-derived stem cells improve full-thickness skin grafts in a rat model.
      ]. Basically, the effects of ASCs rely on their immunomodulatory [
      • Plock J.A.
      • Schnider J.T.
      • Solari M.G.
      • Zheng X.X.
      • Gorantla V.S
      Perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation.
      ] and pro-angiogenic characteristics [
      • Eke G.
      • Mangir N.
      • Hasirci N.
      • MacNeil S.
      • Hasirci V.
      Development of a UV crosslinked biodegradable hydrogel containing adipose derived stem cells to promote vascularization for skin wounds and tissue engineering.
      ] as well as their ability to promote the growth of keratinocytes and fibroblasts [
      • Hassan W.U.
      • Greiser U.
      • Wang W
      Role of adipose-derived stem cells in wound healing.
      ]. Moreover, ASCs have been shown to reduce scar formation [
      • Li Y.
      • et al.
      Adipose tissue-derived stem cells suppress hypertrophic scar fibrosis via the p38/MAPK signaling pathway.
      ,
      • Gentile P.
      New strategies in plastic surgery: autologous adipose-derived mesenchymal stem cells contained in fat grafting improves symptomatic scars.
      ,
      • Gentile P.
      • Sterodimas A.
      • Calabrese C.
      • Garcovich S.
      Systematic review: Advances of fat tissue engineering as bioactive scaffold, bioactive material, and source for adipose-derived mesenchymal stem cells in wound and scar treatment.
      ] and to mediate neovascularization through angiogenesis [
      • Scioli M.G.
      • et al.
      The biomolecular basis of adipogenic differentiation of adipose-derived stem cells.
      ]. Despite their promising and valuable effects, because of their potential pro-oncogenic role under certain circumstances, the interplay of ASCs and cancer cells should not be neglected [
      • Gentile P.
      • Garcovich S.
      Concise Review: Adipose-Derived Stem Cells (ASCs) and Adipocyte-Secreted Exosomal microRNA (A-SE-miR) Modulate Cancer Growth and proMote Wound Repair.
      ].
      In contrast to non-healing chronic wounds, during normal wound healing, the role of the ECM is to activate platelets that deposit fibronectin and fibrin after their aggregation. Furthermore, platelets secrete PDGF, a mitogenic and chemotactic growth factor [
      • Evrova O.
      • Buschmann J.
      In vitro and in vivo effects of PDGF-BB delivery strategies on tendon healing: a review.
      ] that attracts neutrophils and macrophages and supports their migration to the wound site. Macrophages are primarily of the M1 polarized type and lead to the release of pro-inflammatory cytokines, thus establishing the inflammatory phase in the wound healing cascade. Simultaneously, platelets also release TGF-β, which stimulates the transformation of monocytes to macrophages. As fibroblasts enter the wound site, the provisional ECM template of fibronectin [
      • Sakar M.S.
      • et al.
      Cellular forces and matrix assembly coordinate fibrous tissue repair.
      ] and fibrin is gradually replaced by collagen I, proteoglycans, glycoproteins and glycosaminoglycans, forming a granular preliminary tissue. After the establishment of this precursor ECM, endothelial cells migrate to the wound site and angiogenesis takes place, supported by hypoxic conditions that lead to the overexpression of hypoxia-inducible factor 1 [
      • Ke Q.
      • Costa M.
      Hypoxia-inducible factor-1 (HIF-1).
      ], vascular endothelial growth factor (VEGF) [
      • Apte R.S.
      • Chen D.S.
      • Ferrara N.
      VEGF in Signaling and Disease: Beyond Discovery and Development.
      ], basic fibroblast growth factor [
      • Zbinden A.
      • et al.
      Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.
      ] and TGF-β. Finally, transformation of fibroblasts into myofibroblasts leads to contraction of the wound site [
      • Hinz B.
      Myofibroblasts.
      ]. The ECM is remodeled by MMPs and TIMPs, and its appearance eventually turns from granulation tissue to normal, non-scarred, fully regenerated tissue. Keratinocytes come from the basement membrane and finally close the wound [
      • Werner S.
      • Krieg T.
      • Smola H
      Keratinocyte-fibroblast interactions in wound healing.
      ].
      By contrast, non-healing wounds show a prolonged inflammatory phase, where an important step—the resolution of inflammation accompanied by a switch in M1 macrophages to M2 polarization—does not take place properly. In other words, a chronic inflammatory state may be established in which pro-inflammatory cytokines such as IL-1β, IL-6 and tumor necrosis factor alpha [
      • Schulze-Tanzil G.
      • et al.
      The role of pro-inflammatory and immunoregulatory cytokines in tendon healing and rupture: new insights. Scand.
      ] are continually released and no resolving switch in the state of macrophages is achieved. This is the point where cell therapy with ASCs comes in. ASCs have been shown in vitro and in vivo to terminate a persistent inflammatory phase [
      • Manning C.N.
      • et al.
      Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro.
      ]. ASCs and their trophic factors are able to switch M1 macrophages to M2. Moreover, they support the switch in pathogenic T-helper 1 cells to the more beneficial T-helper 2 cell form [
      • Park J.E.
      • Barbul A.
      Understanding the role of immune regulation in wound healing.
      ], enabling and supporting pro-regenerative healing.
      In addition, non-healing wounds have been shown to be composed of fibroblasts that do not respond properly to TGF-β and thus do not migrate to the wound site, leading to decreased production of collagen I and to an overall dysregulated ECM. Again, ASCs counteract this insensitivity of the fibroblasts and enhance fibroblast proliferation and migration by paracrine actions. In this way, proper collagen I synthesis can take place and the ECM, which might otherwise become dysfunctional, can return to a more healthy state. One major problem of non-healing skin wounds is the increased proteolysis of their components caused by overexpression of MMP-1, MMP-2, MMP-3, MMP-8 and MMP-9, with decreased expression of TIMP-2, leading to an imbalanced ECM composition. As a result, devastating effects occur through impaired molecular signaling, including lowered cell migration to the wound, less proliferation and much less angiogenesis, leading to necrosis. With respect to remodeling, ASCs show positive effects. They inhibit ECM degradation by controlling MMPs and TIMPS, redirecting their otherwise detrimental influence to a normal state and inhibiting excessive proteolysis. Hence, the concept of ASC application to support wound healing is corroborated by a plethora of different beneficial, interdependent aspects.

      Perspective on Different Formulations That Include ASCs

      Experimental studies using ASCs and adipose derivatives to improve skin wound healing

      Decellularized adipose tissue

      In a study by Xia et al. [
      • Xia Z.
      • et al.
      The Application of Decellularized Adipose Tissue Promotes Wound Healing.
      ], adipose tissue was frozen, centrifuged, treated with 1% sodium dodecyl sulfate and dried, resulting in a powder. This powder was either dissolved in medium and then applied or used in combination with ASCs in a wound healing mouse model. In the combination, DAT served as a carrier for ASCs. The in vivo results showed the superiority of the DAT/ASC combination compared with the ASC-only group. The combination of DAT and ASCs promoted wound healing as well as better angiogenesis (higher CD31+ areas) and greater proliferation (higher proliferating cell nuclear antigen). Protein analysis under in vitro conditions confirmed the superiority observed in vivo, as it was determined that VEGF, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor (EGF) and PDGF-BB were expressed more in the co-culture of DAT and ASCs compared with the ASC culture.

      ASC-derived ECM

      There are many different types of advanced therapy medicinal products for tissue regeneration [
      • Gentile P.
      • et al.
      Systematic Review: Allogenic Use of Stromal Vascular Fraction (SVF) and Decellularized Extracellular Matrices (ECM) as Advanced Therapy Medicinal Products (ATMP) in Tissue Regeneration.
      ]. One ASC approach in skin wound healing involves the use of ECM as a scaffold after removal of the cells. Such acellular ASC-derived ECM exhibits different features compared with matrices derived from fibroblasts. ECM from bone marrow-derived stem cells is also composed of a slightly different set of components compared with ASC-derived ECM [
      • Ragelle H.
      • et al.
      Comprehensive proteomic characterization of stem cell-derived extracellular matrices.
      ]. For example, gene expression of fibronectin and extracellular matrix protein 2 is upregulated in ASC cultures, indicating that the ECM derived from these cells could enhance matrix molecule deposition and cell adhesion. Moreover, ECM from ASCs has been found to be enriched in different collagens as well as proteoglycans and MMPs, providing a good starting point for proper in vivo regeneration of the skin.
      It has to be emphasized that ECM harvested from ASCs differs from ECM harvested from other cells. In a proteomic approach, Ragelle et al. [
      • Ragelle H.
      • et al.
      Comprehensive proteomic characterization of stem cell-derived extracellular matrices.
      ] showed that bone marrow-derived mesenchymal stromal cells (MSCs) or neonatal fibroblasts displayed a specific and individual ECM matrisome that differed from ASC-derived ECM. In addition, when the conditioned medium (CM) of ASCs was compared with dermal fibroblasts by proteomics, it was found that the secretome of ASCs included factors important for ECM organization and immunological regulation, whereas the secretome of dermal fibroblasts contained factors important for epithelial development [
      • Niada S.
      • Giannasi C.
      • Magagnotti C.
      • Andolfo A.
      • Brini A.T.
      Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts.
      ]. Finally, culture conditions may affect the secretory composition, as impressively demonstrated by low-oxygen primary ASC cultures compared with normoxic cultures [
      • Frazier T.P.
      • Gimble J.M.
      • Kheterpal I.
      • Rowan B.G.
      Impact of low oxygen on the secretome of human adipose-derived stromal/stem cell primary cultures.
      ].
      Scleroderma is an autoimmune disease that affects the skin and is characterized by poor blood flow and thickening and stiffness of the skin. It has been reported that treatment with ASCs harvested from the abdominal fat of human patients and used in lipotransfer for localized scleroderma improved the survival rate of transplanted adipose tissue and skin texture in a nude mouse model [
      • Chen B.
      • et al.
      Supportive Use of Adipose-Derived Stem Cells in Cell-Assisted Lipotransfer for Localized Scleroderma.
      ]. In a study in which ASC-assisted, stromal vascular fraction (SVF)-assisted and normal autologous fat grafts were compared in the treatment of localized scleroderma, the ASC-assisted procedure resulted in the highest fat graft volume retention [
      • Wang C.
      • et al.
      A pilot study on ex vivo expanded autologous adipose-derived stem cells of improving fat retention in localized scleroderma patients.
      ].

      Acellular dermal ECM

      In an experimental study by Mirzaei-Parsa et al. [
      • Mirzaei-Parsa M.J.
      • et al.
      Nanofiber-acellular dermal matrix as a bilayer scaffold containing mesenchymal stem cell for healing of full-thickness skin wounds.
      ], a decellularized rat dermal matrix was used as a skin substitute for healing a full-thickness skin wound. The rationale for this was to circumvent the two-step gold standard procedure in which a dermal substitute is placed on the wound for the first 3 weeks to enable vascularization, after which an autologous split-thickness graft is applied, which is otherwise prone to not being engrafted. In the researchers’ study, the acellular matrix was enriched and refined by the addition of several components. Each incremental step in the construction of the dermal substitute was tested in a rat model with excisional wounds on the back. ASCs were isolated from rat adipose tissue and characterized by fluorescence-activated cell sorting (strong expression of CD44, CD73 and CD90 and no expression of CD45), and induction toward the epidermal lineage was performed by medium supplementation (10 ng/mL EGF, 10 ng/mL keratinocyte growth factor and 50 µg/mL ascorbic acid). These cells were seeded on the acellular dermal matrix. In addition, a second layer was artificially produced by electrospinning the polymer polycaprolactone (PCL), which was tested separately and in combination with the dermal matrix (as a bilayer). Finally, the ASCs committed toward the epidermal lineage were seeded on the PCL layer with fibrinogen, which acted as a glue for cell adhesion. Wound closure experiments run for 2 weeks and 3 weeks clearly revealed the advantage of the cellular component. Regardless of whether they were seeded on the dermal acellular matrix alone, the synthetic PCL nanofiber mesh or the combination (bilayer matrix), the ASCs improved re-epithelization, angiogenesis and collagen remodeling compared with the corresponding cell-free matrices. Moreover, when the three scaffold materials were compared, it was the nanofibrous artificial PCL mesh that led to the best wound closure results. This is an interesting finding, as it suggests that in this specific setting, a synthetic and easily fabricated electrospun fiber mesh performs better than a dermal matrix of biological origin that has to be decellularized first. The pure PCL layer even performed better than the sophisticated bilayer construction.

      Fibrin gel and ASCs

      Other artificial skin substitutes have been investigated by combining a fibrin gel with ASCs from green fluorescent protein (GFP) transgenic mice [
      • Zeng R.X.
      • et al.
      Experimental study on repairing skin defect by tissue-engineered skin substitute compositely constructed by adipose-derived stem cells and fibrin gel.
      ]. A skin injury measuring 1 cm2 was covered with ASCs, fibrin gel or a combination of both and compared with wound closure with an autologous skin flap. After 1 week and 3 weeks, neovascularization and healing status were determined. With respect to blood flow, vessel density and skin flap survival as readouts, the combination of ASCs and fibrin gel performed best followed by ASCs, autologous flap and fibrin gel. As in the study by Mirzaei-Parsa et al. [
      • Mirzaei-Parsa M.J.
      • et al.
      Nanofiber-acellular dermal matrix as a bilayer scaffold containing mesenchymal stem cell for healing of full-thickness skin wounds.
      ], the more artificial skin substitute (here represented by a fibrin gel–ASC composite) supported skin healing better than a natural substitute (autologous skin flap).
      Furthermore, ASC treatment has been used for the healing of deep partial-thickness burn wounds [
      • Feng C.J.
      • Lin C.H.
      • Tsai C.H.
      • Yang I.C.
      • Ma H
      Adipose-derived stem cells-induced burn wound healing and regeneration of skin appendages in a novel skin island rat model.
      ]. Hot copper plates were used to induce wounds in a burn wound rat model. Either phosphate-buffered saline (PBS) or ASCs were injected intradermally (applied once), and wound healing was monitored longitudinally every week for up to 4 weeks. ASC-treated rats had higher vascular density and greater numbers of proliferating cells than the control.

      Pre-differentiation of ASCs

      Hur et al. [
      • Hur W.
      • et al.
      Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium.
      ] reported that ASCs differentiated toward the fibroblast lineage performed better than undifferentiated ASCs. For these experiments, human skin fibroblasts were cultured and the CM was harvested. ASCs were exposed to this fibroblast-CM (FCM) under in vitro conditions for 3 days, and pro-collagen type I levels were shown to be higher compared with those in ASC cultures not exposed to FCM. These findings accompanied an upregulation in Smad 2/3 protein expression related to the TGF-β/Smad signaling pathway. Furthermore, the researchers confirmed that ASCs had transdifferentiated to fibroblast-like cells. When used in a skin wound healing model in BALB/c nude mice, implanted FCM-treated ASCs performed better than conventional ASCs (receiving no transdifferentiation step) with respect to wound healing rate and size of the wound area on day 3, day 7 and day 10 after cell transplantation.

      SVF: a more complex and less defined approach

      Another study aiming to improve skin wound healing investigated the effects of ASCs and SVF [
      • Bi H.
      • et al.
      Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process.
      ]. SVF is derived from adipose tissue through digestion and centrifugation [
      • Gentile P.
      • Piccinno M.S.
      • Calabrese C.
      Characteristics and Potentiality of Human Adipose-Derived Stem Cells (hASCs) Obtained from Enzymatic Digestion of Fat Graft.
      ]. It contains ASCs, pericytes, lymphoid and myeloid cells and endothelial progenitors, and SVF cells secrete many paracrine factors. Compared with ASCs, SVF is fresh and not expanded, and its composition varies based on the different isolation protocols currently being used. Interestingly, researchers found beneficial effects for both expanded ASCs and fresh SVF when subcutaneously injected in full-thickness skin wounds of diabetic mice [
      • Bi H.
      • et al.
      Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process.
      ]. Both stimuli enhanced and promoted skin healing and angiogenesis. This was also supported by several in vitro assays. In a scratch test, for example, fibroblast migration was increased by co-culturing the fibroblasts with ASCs as well as SVF. Furthermore, SVF combined with a fat graft was safe when applied for clinical face rejuvenation and showed a higher soft tissue volume increase compared with a fat graft without SVF [
      • Gentile P.
      • et al.
      Regenerative application of stromal vascular fraction cells enhanced fat graft maintenance: clinical assessment in face rejuvenation.
      ].

      ASC secretome: paracrine impact on skin wound healing

      The use of ASCs as a cellular therapy has the disadvantage of requiring cell culture for expansion, which takes a long time and has to be coordinated with the operation date, when the cells are needed for transplantation. Another approach is to use the secretome of the ASCs, which can be stored and used as an “off-the-shelf” therapy that is available at any time, may it have been harvested from autologous or allogeneic ASCs before. Indeed, the positive impact of ASC treatment on skin wound healing is based not only on the cells being present at the wound site and capable of undergoing differentiation into, for example, adipocytes [
      • Kern S.
      • Eichler H.
      • Stoeve J.
      • Klüter H.
      • Bieback K.
      Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.
      ] or other cell types that serve as precursor cells but also to a great extent on their mediation capacity. In other words, the paracrine actions of ASCs that secrete growth factors, cytokines and extracellular vesicles, among them exosomes, are highly important for appropriate wound healing in skin injuries [
      • Maguire G.
      The Safe and Efficacious Use of Secretome From Fibroblasts and Adipose-derived (but not Bone Marrow-derived) Mesenchymal Stem Cells for Skin Therapeutics.
      ]. Two very important mechanisms of action are (i) the paracrine impact of ASCs on a tissue's resident fibroblasts, promoting the balance of regeneration and fibrosis toward regeneration by tuning the secretome of the fibroblasts and their microenvironment, and (ii) the paracrine impact on other cells in the surrounding tissue by stimulating their differentiation toward keratinocytes, adipocytes required for proper wound healing, anti-inflammatory M2 macrophages, etc.
      The secretome of ASCs consists of a large fraction of biomolecules associated with neovascularization, angiogenesis, ECM remodeling and epithelization [
      • Kapur S.K.
      • Katz A.J.
      Review of the adipose derived stem cell secretome.
      ]. Analyses by protein arrays and protein–protein interaction networks of differentially expressed proteins have been reported to demonstrate the promoting factors needed for skin wound healing [
      • Luo Y.
      • et al.
      Autograft microskin combined with adipose-derived stem cell enhances wound healing in a full-thickness skin defect mouse model.
      ]. Interestingly, in addition to typical angiogenic growth factors like VEGF, ASCs have also been shown to secrete IL-6 when co-cultured with pieces of micro-skin, which supports the general fact that an appropriate inflammatory phase is necessary for skin wound healing. However—and herein lies the crucial impact of the ASC secretome—many pro-resolving and anti-inflammatory factors are secreted, mediating the important switch toward the end of inflammation (resolution) and an appropriate remodeling phase.
      With respect to the release of angiogenic factors, ASCs cultured as spheroids have been shown to secrete higher amounts compared with ASCs cultured in two-dimensional cell cultures [
      • Lee J.S.
      • et al.
      Angiogenic factors secreted from human ASC spheroids entrapped in an alginate-based hierarchical structure via combined 3D printing/electrospinning system.
      ]. Moreover, spheroids of different size show upregulation of PECAM1 (CD31), VEGFA and HGF gene expression to different extents. Larger spheroids of ASCs exhibit mild hypoxic conditions inside, which resembles the natural tissue microenvironment [
      • Frazier T.P.
      • Gimble J.M.
      • Kheterpal I.
      • Rowan B.G.
      Impact of low oxygen on the secretome of human adipose-derived stromal/stem cell primary cultures.
      ]. In this way, the production of angiogenic factors is stimulated. Moreover, it has been reported that the culture time for spheroid formation impacts the gene expression of paracrine factors. When culture periods of 1 day, 3 days, 5 days and 7 days were compared, an overall best outcome was found for the 3-day cultivation time for 3000 cells per micro-tissue with respect to upregulation of angiogenic factors. Lee et al. [
      • Lee J.S.
      • et al.
      Angiogenic factors secreted from human ASC spheroids entrapped in an alginate-based hierarchical structure via combined 3D printing/electrospinning system.
      ] embedded spheroids in collagen or alginate hydrogels, emphasizing the importance of cell–material interactions. The researchers found alginate to be significantly superior to collagen with respect to the expression of angiogenesis-related genes. Finally, the fabrication of completely entrapped spheroids in which metabolic activity of cells within the scaffold material was restricted resulted in lower angiogenic gene expression compared with cells that were entrapped in porous electrospun fibers, allowing full metabolic activity.

      Modification of ASCs

      ASCs can be stimulated via modification of the culture medium, such as by supplementing with growth factors [
      • Orfei C.P.
      • et al.
      In Vitro Induction of Tendon-Specific Markers in Tendon Cells, Adipose- and Bone Marrow-Derived Stem Cells is Dependent on TGF3, BMP-12 and Ascorbic Acid Stimulation.
      ], ascorbic acid or insulin [
      • Zuk P.A.
      • et al.
      Multilineage cells from human adipose tissue: Implications for cell-based therapies.
      ]. By doing so, it has been shown that ASCs can be tuned to differentially express many ECM components, such as collagen I [
      • Yu J.
      • Tu Y.K.
      • Tang Y.B.
      • Cheng N.C.
      Stemness and transdifferentiation of adipose-derived stem cells using L-ascorbic acid 2-phosphate-induced cell sheet formation.
      ]. ASCs can also be forced to secrete even more beneficial wound healing factors than they otherwise would by transducing them for specific overexpression of target proteins. For example, the secretome of ASCs has been optimized via hypoxia-inducible factor 1 alpha overexpression and cultivation under low oxygen conditions—ideal conditions for promoting angiogenesis, which is partially impaired in conditions such as diabetes [
      • Xu J.
      • Liu X.
      • Zhao F.
      • Zhang Y.
      • Wang Z.
      HIF1α overexpression enhances diabetic wound closure in high glucose and low oxygen conditions by promoting adipose-derived stem cell paracrine function and survival.
      ]. In vitro experiments verified upregulation of VEGFA, fibroblast growth factor 2 and C-X-C motif chemokine ligand 12—factors that are otherwise inhibited by high glucose levels. The transduced ASCs were injected into a full-thickness skin defect diabetic mouse model. The approach resulted in faster wound closure, which was significant at 14 days.
      In addition, Bcl-2-modified ASCs are reported to act beneficially when applied to full-thickness skin defects of diabetic mice [
      • Ding S.
      • Xu Y.
      • Yan X.
      • Lin Y.
      • Tan Q.
      Effect of Collagen Scaffold With Bcl-2-Modified Adipose-Derived Stem Cells on Diabetic Mice Wound Healing.
      ]. Bcl-2 is an anti-apoptotic protein that inhibits cell death. As ASCs do not survive for a long time in the wound bed after transplantation, overexpression of Bcl-2 was hypothesized to prolong this time and therefore support wound healing. In addition, Bcl-2-modified ASCs were embedded in a collagen scaffold. The results showed that a combination of Bcl-2-modified ASCs and collagen was better with respect to wound healing compared with a non-modified ASC/collagen approach or collagen only or the control (no scaffold, no cells). Although healing rates of the Bcl-2-modified ASC/collagen and ASC/collagen groups differed dramatically at early time points, such as day 7, they performed similarly well at day 10 and day 14. These two groups, however, showed healing rates twice as high as those in the scaffold and control groups.

      How to apply ASCs

      The question of how to apply ASCs for skin wound healing was addressed by Kim et al. [
      • Kim H.
      • Hyun M.R.
      • Kim S.W.
      The Effect of Adipose-Derived Stem Cells on Wound Healing: Comparison of Methods of Application.
      ]. The researchers compared intravenous injection with intramuscular and topical application (ASCs mixed with a fibrin gel). For this purpose, GFP-labeled ASCs were used and applied in a dorsal full-thickness skin defect mouse model. Mice used as controls received saline via the same method. Regardless of how the ASCs were applied, skin wound healing was accelerated under all conditions. In particular, on day 3, there was a significant difference in wound size when ASC-treated groups were compared with the corresponding saline controls. ASC-treated wounds were retracting and closing from the beginning, whereas the saline-treated control wounds got larger before rapidly getting smaller. On day 14, at the end of the experiment, all groups showed complete wound closure. Interestingly, when tracking the GFP-labeled ASCs, it was noted that they migrated to the wound when injected intravenously, stayed in the muscle when injected intramuscularly and stayed in the wound after topical application but vanished as soon as wound closure was complete. In the second part of this review article, the authors will address typical skin diseases and sum up the main approaches for treating them when ASCs or their derivatives are involved.

      Perspective on Different Skin Diseases and ASC Therapy

      Diabetic ulcers

      Diabetes mellitus is a common chronic disease. The prevalence of the condition in European countries was estimated to be around 8.5% in 2013, and numbers have increased in the last few years [
      • Tamayo T.
      • et al.
      Diabetes in Europe: An update.
      ]. The prevalence of type 1 and type 2 diabetes among US adults was estimated to be around 0.5% and 8.5%, respectively, in 2016 and 2017 [
      • Xu G.
      • et al.
      Prevalence of diagnosed type 1 and type 2 diabetes among US adults in 2016 and 2017: population based study.
      ].
      Diabetes mellitus is responsible for impaired or delayed wound healing. Chronic ulcers are a common problem among patients, who have a lifetime risk of approximately 25%, and lead to diminished quality of life, morbidity and death [
      • Lim J.Z.
      • Ng N.S.
      • Thomas C
      Prevention and treatment of diabetic foot ulcers.
      ,
      • Richard J.L.
      • Schuldiner S.
      Épidémiologie du pied diabétique.
      ]. The prevalence of diabetic ulcers is estimated to be around 6.3% globally and is higher in males and patients with type 2 diabetes. Other risk factors include older age, longer diabetes duration, higher body mass index, smoking and hypertension [
      • Zhang P.
      • et al.
      Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis.
      ].
      The pathophysiology of diabetic skin wounds involves multiple endogenous and exogenous factors. It is mostly attributed to peripheral neuropathy as well as microvascular and macrovascular disorders, but abnormal biomechanical loading with areas of high stress and infection also plays an important role [
      • Schaper N.C.
      • et al.
      Practical Guidelines on the prevention and management of diabetic foot disease (IWGDF 2019 update).
      ,
      • Falanga V.
      Wound healing and its impairment in the diabetic foot.
      ]. Diabetic wounds are associated with a disturbance and delay in the inflammatory and proliferation phases. A prolonged and increased presence of T-cell infiltration with an imbalance in CD4+/CD8+ cells as well as an increased number of macrophages has been observed in chronic diabetic wounds. Furthermore, excessive production of extracellular MMPs (e.g., MMP9) has been noted without removal of debris or tissue remodeling in a timely fashion, leading to impaired healing [
      • Falanga V.
      Wound healing and its impairment in the diabetic foot.
      ,
      • Loots M.A.M.
      • et al.
      Differences in Cellular Infiltrate and Extracellular Matrix of Chronic Diabetic and Venous Ulcers Versus Acute Wounds.
      ,
      • Dinh T.L.
      • Veves A.
      A Review of the Mechanisms Implicated in the Pathogenesis of the Diabetic Foot.
      ,
      • Signorelli S.S.
      • et al.
      Plasma levels and zymographic activities of matrix metalloproteinases 2 and 9 in type II diabetics with peripheral arterial disease.
      ]. Some of the resident cells show phenotypic drift. Fibroblasts from diabetic wounds demonstrate a decreased proliferation response to growth factors [
      • Loots M.A.M.
      • et al.
      Differences in Cellular Infiltrate and Extracellular Matrix of Chronic Diabetic and Venous Ulcers Versus Acute Wounds.
      ,
      • Loots M.A.M.
      • et al.
      Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls.
      ] and macrophages show a decrease in the release of cytokines [
      • Dinh T.L.
      • Veves A.
      A Review of the Mechanisms Implicated in the Pathogenesis of the Diabetic Foot.
      ,
      • Zykova S.N.
      • et al.
      Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.
      ].
      Current treatment strategies include control of glucose concentrations, pressure relief at the wound site, surgical debridement, vascular optimization and control of infection and edema. However, these methods are often not sufficient for adequate healing, with otherwise promising strategies using advanced therapy medicinal products [
      • Gentile P.
      • et al.
      Systematic Review: Allogenic Use of Stromal Vascular Fraction (SVF) and Decellularized Extracellular Matrices (ECM) as Advanced Therapy Medicinal Products (ATMP) in Tissue Regeneration.
      ]. The longer the healing is delayed, the greater is the possibility for serious complications (Figure 1) [
      • Schaper N.C.
      • et al.
      Practical Guidelines on the prevention and management of diabetic foot disease (IWGDF 2019 update).
      ,
      • Jeffcoate W.J.
      • et al.
      Unresolved issues in the management of ulcers of the foot in diabetes.
      ].
      Fig 1
      Fig. 1A 68-year-old patient with metabolic foot disorder after amputation of digits II and IV, with recurring ulcerations of the foot. Photos taken June 2021 and December 2021, respectively. Scale bar in centimeters.
      As an effort to reduce the time taken to complete wound closure, the following biologically active products such as ECM proteins, growth factors and bioengineered tissue have proven effective in different studies, including hyaluronic acid and platelet-rich plasma (PRP) [
      • De Angelis B.
      • et al.
      Wound Healing: In Vitro and In Vivo Evaluation of a Bio-Functionalized Scaffold Based on Hyaluronic Acid and Platelet-Rich Plasma in Chronic Ulcers.
      ], adipose tissue and PRP [
      • Cervelli V.
      • et al.
      P.R.L. platelet rich lipotransfert: our experience and current state of art in the combined use of fat and PRP.
      ], dermal substitutes commercially available like Integra (Integra Holding AG, Wallisellen, Switzerland) [
      • De Angelis B.
      • et al.
      One-Stage Reconstruction of Scalp after Full-Thickness Oncologic Defects Using a Dermal Regeneration Template (Integra).
      ] or Nevelia (Symatese, 69630 Chaponost, France) based on collagen [
      • Gentile P.
      • et al.
      Complex abdominal wall repair using a porcine dermal matrix.
      ,
      • De Angelis B
      • et al.
      Long-term follow-up comparison of two different bi-layer dermal substitutes in tissue regeneration: Clinical outcomes and histological findings.
      ]; however, they are not yet well supported for routine ulcer management [
      • Schaper N.C.
      • et al.
      Practical Guidelines on the prevention and management of diabetic foot disease (IWGDF 2019 update).
      ]. Bioengineered skin is envisioned to act through new matrix deposition, increased availability of growth factors and recruitment of stem and progenitor cells to the wound site [
      • Falanga V.
      • et al.
      Wounding of Bioengineered Skin: Cellular and Molecular Aspects After Injury.
      ].

      Preparation of cell sheets to address diabetic ulcers

      Various studies have investigated cell sheet engineering, which has previously been shown to preserve cell-to-cell connections and to improve cell survival rates. ASC sheets have also been used in previous studies to accelerate wound healing [
      • Cerqueira M.T.
      • et al.
      Human Adipose Stem Cells Cell Sheet Constructs Impact Epidermal Morphogenesis in Full-Thickness Excisional Wounds.
      ,
      • McLaughlin M.M.
      • Marra K.G.
      The use of adipose-derived stem cells as sheets for wound healing.
      ,
      • Lin Y.C.
      • et al.
      Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model.
      ].
      In a study of pressure ulcers in a mouse model, Alexandrushkina et al. [
      • Alexandrushkina N.
      • et al.
      Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization.
      ] found that cell sheets derived from ASCs improved healing by altering the pattern of vascularization. The secretome proteomics of ASC sheets revealed higher amounts of PDGF-BB, HGF and granulocyte colony-stimulating factor compared with monolayers of ASCs, which led to improved healing of pressure ulcers. Adipose SVF cells were also used for therapy of pressure ulcers and demonstrated more rapid wound closure compared with the cell-free approach [
      • Bukowska J.
      • et al.
      Safety of Human Adipose Stromal Vascular Fraction Cells Isolated with a Closed System Device in an Immunocompetent Murine Pressure Ulcer Model.
      ].
      Kato et al. [
      • Kato Y.
      • et al.
      Allogeneic Transplantation of an Adipose-Derived Stem Cell Sheet Combined With Artificial Skin Accelerates Wound Healing in a Rat Wound Model of Type 2 Diabetes and Obesity.
      ] showed that allogeneic transplantation of ASC sheets (8.76 × 105 cells) combined with artificial skin was able to accelerate wound healing in full-thickness wounds (1.5 × 1 cm) in 16-week-old Zucker diabetic fatty rats. The average wound area was significantly smaller in the ASC sheet transplantation group and the blood vessel density was significantly higher.
      In a similar diabetic mouse model, Hamada et al. [
      • Hamada M.
      • et al.
      Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes.
      ] investigated the xenogeneic transplantation of human ASC (hASC) sheets in a 1.5-cm full-thickness wound. Mice that underwent transplantation of hASC sheets (1.0 × 105 cells) with artificial skin demonstrated increased blood vessel density and dermal thickness and accelerated wound healing compared with the artificial skin-only control group.
      Moon et al. [
      • Moon K.C.
      • et al.
      Potential of Allogeneic Adipose-Derived Stem Cell-Hydrogel Complex for Treating Diabetic Foot Ulcers.
      ] studied a commercialized allogeneic ASC sheet (ALLO-ASC-SHEET; Anterogen, Seoul, South Korea) in patients with diabetic foot ulcers. Patients who had 5 × 5-cm2 hydrogel sheets containing 1 × 106 allogeneic ASCs placed on their wounds once a week for 12 weeks demonstrated a higher rate of wound closure than the control group receiving a polyurethane film. Dong et al. [
      • Dong Y.
      • et al.
      Acceleration of Diabetic Wound Regeneration using an In Situ–Formed Stem-Cell-Based Skin Substitute.
      ] studied the delivery of ASCs via an injectable polyethylene glycol (PEG)/gelatin-based hydrogel system compared with cells alone. In a diabetic mouse model, allogeneic ASCs from normoglycemic mice (3 × 105 ASCs per 0.6-cm diameter wound) were mixed with a gelling precursor and added to the wounds. The hydrogel delivery method showed a distinct advantage in ASC viability compared with cells alone up to 14 days post-injection. Improved stem cell retention and significant acceleration of wound closure were observed. The cell-only group also showed a better healing rate than the control group; however, this was observed only from day 9 onward. ASC/hydrogel-treated wounds showed decreased inflammatory cell infiltration, enhanced neovascularization and increased collagen type III:I ratio, indicating scarless tissue regeneration.
      In another diabetic mouse model, Feng et al. [
      • Feng J.
      • et al.
      An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells.
      ] investigated the therapeutic potential of ASCs cultured as micro-spheroids (20–120 μm) in three-dimensional (3D) culture in hyaluronic acid gel. These hASCs (6 × 105) were injected intradermally and showed faster wound epithelialization and thicker dermis compared with vehicle or cultured monolayer ASCs. The cultured spheroid ASCs promoted collagen deposition in the wound, and immunohistological analysis indicated healthier subcutaneous tissue. Moreover, more viable adipocytes and less inflammation were observed compared with two-dimensional cultured ASCs. Furthermore, Amos et al. [
      • Amos P.J.
      • et al.
      Human Adipose-Derived Stromal Cells Accelerate Diabetic Wound Healing: Impact of Cell Formulation and Delivery.
      ] showed in a diabetic mouse model (1-cm diameter full-thickness wound) that hASCs delivered as 3D multi-cellular aggregates using a hanging-drop cultivation method offered significantly improved wound healing compared with cell suspensions from monolayer culture. Multi-cellular aggregate culture (3.5 × 105 cells per wound) led to upregulation of wound healing-related genes and protein production, whereas inflammatory proteins were secreted in lower amounts. A low-dose experimental group (1.25 × 105 cells per wound) showed no significant positive effect after 12 days compared with a control group treated with PBS.

      What has to be favored? Allogeneic versus autologous ASCs

      Several studies have concentrated on the healing ability of diabetic ASCs in the treatment of diabetic wounds. A hind limb ischemia model demonstrated that ASCs from type 1 or type 2 diabetic rats had lower proliferation ability than ASCs from control rats and led to lower blood flow recovery [
      • Kim H.K.
      • et al.
      Alterations in the proangiogenic functions of adipose tissue-derived stromal cells isolated from diabetic rats.
      ]. Furthermore, diabetic ASCs showed lower levels of VEGF production, proliferation and tubulogenesis than young ASCs from healthy mice. Upregulation occurred when faced with hypoxia; however, the response was blunted compared with that observed in young controls [
      • El-ftesi S.
      • Chang E.I.
      • Longaker M.T.
      • Gurtner G.C
      Aging and Diabetes Impair the Neovascular Potential of Adipose-Derived Stromal Cells.
      ]. Subpopulations of murine ASCs characterized by high expression of genes known to be important for angiogenesis were diminished in type 1 and type 2 models of diabetes. Diabetic ASCs are compromised in their ability to establish a vascular network in vitro and in vivo and ineffective in promoting soft tissue neovascularization and wound healing [
      • Rennert R.C.
      • et al.
      Diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations.
      ].The expression of multiple growth factors and cytokines as well as their associated receptors was significantly decreased. In addition, hASCs isolated from the ischemic limbs of diabetic patients were found to be less potent when compared phenotypically and functionally with non-diabetic controls without limb ischemia [
      • Kočí Z.
      • et al.
      Characterization of human adipose tissue-derived stromal cells isolated from diabetic patient's distal limbs with critical ischemia.
      ].
      Cianfarani et al. [
      • Cianfarani F.
      • et al.
      Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.
      ] investigated the difference between autologous versus allogeneic ASCs and SVF cells in the treatment of diabetic ulcers. ASCs of diabetic rats showed reduced paracrine activity (e.g., HGF, VEGFA and insulin-like growth factor 1) as well as proliferation potential and migration. A reduction in stem cell marker-positive cells was also observed. ASCs of diabetic rats were still able to improve fibroblast proliferation and migration, although with reduced capability, and no influence on keratinocytes was observed. In an in vivo mouse wound model, autologous SVF cells showed less therapeutic potential compared with ASCs from healthy mice. By contrast, Nambu et al. [
      • Nambu M.
      • et al.
      Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissue-derived stromal cells combined with atelocollagen matrix.
      ] described significant improvement in the healing of full-thickness diabetic wounds using freshly isolated autologous ASCs on an atelocollagen matrix (4–6 × 105 ASCs per 1.5-cm diameter wound). However, the researchers did not compare the effect of non-diabetic ASCs and used an atelocollagen matrix alone as a control.
      A study by Gong et al. [
      • Gong J.H.
      • Dong J.Y.
      • Xie T.
      • Zhao Q.
      • Lu S.L.
      Different therapeutic effects between diabetic and non-diabetic adipose stem cells in diabetic wound healing.
      ] investigated the different effects of ASCs from diabetic and non-diabetic rats on wound healing in diabetic and non-diabetic mice. The researchers found that transplantation of diabetic ASCs promoted wound healing in only non-diabetic rats, whereas ASCs from non-diabetic rats promoted wound healing in both rat groups. ASCs from diabetic mice showed a longer generation time and more pre-apoptotic cells.
      With regard to immune rejection, autologous ASCs are generally considered the best source [
      • Hamada M.
      • et al.
      Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes.
      ]. However, considering the aforementioned findings, their effectiveness in diabetic patients seems to be impacted and their therapeutic potential reduced compared with non-diabetic ASCs; therefore, autologous administration should be carefully evaluated.

      Use of selected or modified ASCs

      Kinoshita et al. [
      • Kinoshita K.
      • et al.
      Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers.
      ] studied a selected population of ASCs that express stem cell marker stage-specific embryonic antigen 3-positive cells, also called Muse (multi-lineage differentiating stress-enduring) cells, in a diabetic immunodeficient mouse model. Muse cells with hyaluronic acid were locally injected subcutaneously into 0.6-cm diameter full-thickness wounds at four points (1 × 105 cells per mouse). Mice that received Muse-rich cells showed significantly better wound healing compared with mice treated with Muse-poor cells. The interval to wound closure was even faster than in healthy non-treated mice, and a significantly thicker epidermis was noted. In addition, more ASCs were detected in the dermis.
      Further improvement in diabetic wound healing was observed in a study by Di Rocco et al. [
      • Di Rocco G.
      • et al.
      Enhanced healing of diabetic wounds by topical administration of adipose tissue-derived stromal cells overexpressing stromal-derived factor-1: biodistribution and engraftment analysis by bioluminescent imaging.
      ], who genetically modified ASCs to overexpress stromal cell-derived factor 1 (SDF-1). Downregulation of SDF-1 plays a pivotal role in the pathophysiology of diabetic wounds. In this study, ASCs that overexpressed SDF-1 showed improved lesion repair potential compared with non-transduced ASCs. Moreover, wound closure was achieved earlier on and vessel formation was promoted (7.5 × 105 cells per mouse applied topically to 0.35-cm diameter full-thickness wound). Bioluminescence imaging showed better short-term engraftment and cell survival of ASCs overexpressing SDF-1.
      It is important to note that none of these studies reported clinical signs of immune rejection, infection or other complications. Studies involving human clinical trials have also not reported these complications, even those that were conducting follow-ups for up to 2 years [
      • Moon K.C.
      • et al.
      Potential of Allogeneic Adipose-Derived Stem Cell-Hydrogel Complex for Treating Diabetic Foot Ulcers.
      ,
      • Hamada M.
      • et al.
      Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes.
      ,
      • Dong Y.
      • et al.
      Acceleration of Diabetic Wound Regeneration using an In Situ–Formed Stem-Cell-Based Skin Substitute.
      ].
      Taken together, animal studies have shown the potential beneficial effect of the application of ASCs in diabetic wound healing. Cell sheets as well as other 3D culture methods have shown several advantages compared with single-cell transplantation. Autologous administration has been shown to have several disadvantages and should be carefully evaluated. Different application forms and cell isolation techniques have been investigated and shown to have specific benefits. Further animal studies should validate these results and identify the most effective application protocols for use in clinical studies.
      In searching ClinicalTrials.gov (key words “adipose stem cells” and “Diabetic Foot Ulcer”), the authors found several phase 1 and phase 2 studies as well as a phase 3 study (i.e., NCT03916211, NCT02831075, NCT03183726, NCT02394886, NCT03865394, NCT03754465, NCT04497805, NCT04466007, NCT02092870 and NCT03370874) (Table 1). It will be interesting to note the extent to which the results of these diabetic rodent models are applicable to human patients.
      Table 1Overview of phase 1, phase 2 and phase 3 clinical trials.
      Clinical trial, country of origin (ID)Disease indicationTherapeutic approachNumber of participantsEnrollment status
      Clinical Study of Adipose-Derived Stem Cells in the Treatment of Diabetic Foot, China (NCT03916211)Diabetic footIntramuscular injection of ASCs60Unknown
      A Clinical Study Using Adipose-Derived Stem Cells for Diabetic Foot, China

      (NCT02831075)
      Peripheral vascular disease

      Ischemia

      Diabetic foot
      ASCs240Recruiting
      Safety of ALLO-ASC-DFU in the Patients With Diabetic Foot Ulcers, Korea

      (NCT02394886)
      Diabetic foot ulcerALLO-ASC-DFU (hydrogel sheet containing allogeneic ASCs)5Completed, no results posted
      Treatment of Chronic Wounds in Diabetic Foot Syndrome With Allogeneic Adipose Derived Mesenchymal Stem Cells (1ABC), Poland

      (NCT03865394)
      Diabetic foot ulcerApplication of allogeneic ADSCs in fibrin gel46Completed, no results posted
      Clinical Study of ALLO-ASC-SHEET in Subjects With Diabetic Foot Ulcers, United States

      (NCT03754465)
      Diabetic foot ulcerALLO-ASC-DFU (hydrogel sheet containing allogeneic ASCs)56Recruiting
      Clinical Study of ALLO-ASC-SHEET in Subjects With Diabetic Wagner Grade II Foot Ulcers, United States

      (NCT04497805)
      Diabetic foot ulcerALLO-ASC-SHEET (hydrogel sheet containing allogeneic ASCs)64Recruiting
      Safety and Efficacy of Allogeneic Adipose Tissue Mesenchymal Stem Cells in Diabetic Patients With Critical Limb Ischemia, Spain

      (NCT04466007)
      Limb ischemia, diabetic footHigh and low doses of allogeneic ASCs administered intramuscularly90Recruiting
      Adipose Derived Regenerative Cellular Therapy of Chronic Wounds,

      United States

      (NCT02092870)
      Diabetic foot, venous ulcer, pressure ulcerMultiple injections of ASCs within and immediately surrounding the wound25Completed, no results posted
      Clinical Study to Evaluate Efficacy and Safety of ALLO-ASC-DFU in Patients With Diabetic Foot Ulcers,

      Korea

      (NCT03370874)
      Diabetic foot ulcerALLO-ASC-DFU

      (hydrogel sheet containing allogeneic ASCs)
      164Active, not recruiting
      ID, identifier.

      Burn wounds

      Several studies have investigated the application of ASCs in burn wounds. A persistent inflammatory response in burn injuries leads to greater susceptibility to infections and sepsis—a risk that is further increased by a delay in wound healing. The risk of mortality in burn patients is related to augmented numbers of pro-inflammatory cytokines [
      • Finnerty C.C.
      • Przkora R.
      • Herndon D.N.
      • Jeschke M.G.
      Cytokine expression profile over time in burned mice.
      ,
      • Jeschke M.G.
      • et al.
      Survivors versus nonsurvivors postburn: differences in inflammatory and hypermetabolic trajectories.
      ]. Fire-related burns alone account for over 180 000 deaths per year—with some sources documenting up to 300 000 deaths per year—with additional deaths being attributed to other forms of burns [
      • Abdul Kareem N.
      • Aijaz A.
      • Jeschke M.G.
      Stem Cell Therapy for Burns: Story so Far.
      ,
      • Mock C.
      A WHO plan for burn prevention and care.
      ].
      Burn wounds, which are mostly caused by heat but can also be caused by electricity, radiation, radioactivity or chemicals, are classified based on the depth of the injury. First-degree burns are superficial, involve only the epidermis and heal without scars. Second-degree burns consist of partial-thickness injuries affecting the epidermis and upper layer of the dermis, leading to blistering and pain, as well as deeper burns extending to the deeper dermis (Figure 2). Third-degree burns are full-thickness wounds that reach all layers of the epidermis and dermis, including hair follicles and nerve endings. Fourth-degree burns involve deep tissues like muscle and bone [
      • Evers L.H.
      • Bhavsar D.
      • Mailänder P.
      The biology of burn injury.
      ].
      Fig 2
      Fig. 2A 28-year-old patient with second-degree burn wound of the left hand.
      The goal of burn healing is to re-establish skin integrity; however, extensive injuries as well as full-thickness burns do not heal without medical interventions and end in scar formation. This means that there is a high demand for medical interventions that improve healing and decrease scar formation [
      • Rohani Ivari J.
      • Mahdipour E
      Adipose tissue versus stem cell-derived small extracellular vesicles to enhance the healing of acute burns.
      ], such as nanofat procedures and, particularly, interventions that involve the use of SVF [
      • Gentile P.
      • Scioli M.G.
      • Bielli A.
      • Orlandi A.
      • Cervelli V.
      Comparing different nanofat procedures on scars: role of the stromal vascular fraction and its clinical implications.
      ].
      Although superficial second-degree burns possess stem cells within the hair follicles to help skin regeneration, deep second-degree and third-degree burns result in reduced healing capability, re-epithelialization and regeneration [
      • Levy V.
      • Lindon C.
      • Zheng Y.
      • Harfe B.D.
      • Morgan B.A
      Epidermal stem cells arise from the hair follicle after wounding.
      ]. Treatment of these types of burns includes debridement of necrotic tissue, with the gold standard consisting of an autologous split-thickness skin graft to cover the wound bed. This is not always feasible and is associated with various disadvantages (e.g., prolonged immobility and donor site pain), and different studies have concentrated on the application of stem cells in burn wounds [
      • Atiyeh B.S.
      • Costagliola M.
      Cultured epithelial autograft (CEA) in burn treatment: three decades later.
      ,
      • Condé-Green A.
      • et al.
      Fat Grafting and Adipose-Derived Regenerative Cells in Burn Wound Healing and Scarring: A Systematic Review of the Literature.
      ].
      Several animal studies have investigated different methods of ASC application in second- or third-degree burn wounds, mostly in rodent models. None of the studies reported the occurrence of adverse events after the application of ASCs.
      A search on ClinicalTrials.gov (key words “burns or burn injury” and “adipose stem cells or ASC”) through January 24, 2022, showed a completed phase 1 study as well as a follow-up study (NCT02394873, NCT03183622) investigating the use of ASCs in human burn patients; however, no results have yet been provided. In an in vitro experiment, Collawn et al. [
      • Collawn S.S.
      • Banerjee N.S.
      • de la Torre J.
      • Vasconez L.
      • Chow L.T
      Adipose-derived stromal cells accelerate wound healing in an organotypic raft culture model.
      ] showed that organotypic human skin equivalent cultures with laser burn wounds inflicted to the dermis treated with ASCs showed faster healing than those cultures treated without.

      Allogeneic versus autologous: one single application

      Feng et al. [
      • Feng C.J.
      • Lin C.H.
      • Tsai C.H.
      • Yang I.C.
      • Ma H
      Adipose-derived stem cells-induced burn wound healing and regeneration of skin appendages in a novel skin island rat model.
      ] studied the effect of a single intradermal application of 5 × 105 allogeneic ASCs in second-degree burn wounds (1 × 1 cm) in a rat model. This showed improved wound healing compared with a control group as well as improved hair growth and enhanced angiogenesis. A similar positive effect was observed by Bliley et al. [
      • Bliley J.M.
      • et al.
      Administration of adipose-derived stem cells enhances vascularity, induces collagen deposition, and dermal adipogenesis in burn wounds.
      ] in full-thickness wounds (1-cm diameter) in a mouse model after a single subcutaneous injection of 6.8 × 106 hASCs. A reduction in wound area and enhanced vascularity, collagen deposition and dermal adipogenesis were noted. However, in contrast to other studies, no significant difference in total time to wound closure was observed. Loder et al. [
      • Loder S.
      • et al.
      Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts.
      ] showed that local injection of 1 × 106 allogeneic ASCs improved healing of second-degree burn wounds in a mouse model, covering 30 % of the surface area compared with the saline control. Decreased wound depth, wound area and apoptotic activity were observed. Interestingly, the adipose tissue fat fraction had a comparable effect on wound healing. However, in contrast to other studies, an observation period of 2 weeks did not show a significant improvement in vascularization.
      Using a rat model, Chang et al. [
      • Chang Y.W.
      • et al.
      Autologous and not allogeneic adipose-derived stem cells improve acute burn wound healing.
      ] showed that autologous ASCs (5 × 106 cells) led to faster healing of acute full-thickness 0.4-cm radius burn wounds after 15 days compared with allogeneic ASCs. In addition, injections 0.5 cm from the wound edge showed significantly improved wound healing compared with injections in the wound center. In contrast to observations in other studies, rats receiving allogeneic ASCs did not show significantly faster wound healing compared with the control group receiving cell injection medium (K-NAC). However, it should be noted that this study used a small design (three rats per experiment) and a rather small wound area. A study by Riccobono et al. [
      • Riccobono D.
      • et al.
      Application of adipocyte-derived stem cells in treatment of cutaneous radiation syndrome.
      ] reported similar results. The researchers studied the effect of autologous and allogeneic ASCs on the treatment of cutaneous radiation syndrome in mini-pigs. Autologous and allogeneic ASCs (50 × 106 ASCs per injection and 1 × 106 ASCs/cm2, respectively) were injected intradermally four times over a 3-month period. Injection of autologous but not allogeneic ASCs promoted superior burn wound healing without necrosis and decreased pain compared with the control. Hence, autologous ASCs may be judged superior overall to allogeneic ASCs.
      By contrast, a study by Franck et al. [
      • Franck C.L.
      • et al.
      Influence of Adipose Tissue-Derived Stem Cells on the Burn Wound Healing Process.
      ] of abdominal full-thickness (third-degree) burn wounds (4.84 cm2) in a rat model showed that an intradermal ASC injection of 3.2 × 106 allogeneic ASCs on the day of the burn and 4 days later led to a reduction in scar tissue area and increased deposition of collagen type III. However, it is interesting to note that similar to Loder et al. [
      • Loder S.
      • et al.
      Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts.
      ], the researchers did not find a quantitative increase in blood vessels after 14 days [
      • Franck C.L.
      • et al.
      Influence of Adipose Tissue-Derived Stem Cells on the Burn Wound Healing Process.
      ]. Furthermore, Hanson et al. [
      • Hanson S.E.
      • et al.
      Local delivery of allogeneic bone marrow and adipose tissue-derived mesenchymal stromal cells for cutaneous wound healing in a porcine model.
      ] showed that 1 × 106 allogeneic ASCs injected intradermally could improve the healing of partial-thickness cutaneous wounds in a porcine model. Wounds injected with MSCs were associated with improved appearance and faster re-epithelialization compared with saline controls. Another study showed that a single intramuscular or intravenous injection of 1 × 106 autologous ASCs favored wound healing in a mouse model of radiation burn (20 Gy) [
      • Ebrahimian T.G.
      • et al.
      Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing.
      ].
      Karimi et al. [
      • Karimi H.
      • Soudmand A.
      • Orouji Z.
      • Taghiabadi E.
      • Mousavi S.J.
      Burn wound healing with injection of adipose-derived stem cells: a mouse model study.
      ] investigated different methods of preparing ASCs and their efficacy in a third-degree burn mouse model (1 × 106 cells per wound applied to 1.5 × 1.5-cm dorsal wound). Enzymatically purified syngeneic ASCs were compared with mechanically prepared adipose tissue. Wounds treated with ASCs or adipose tissue showed a smaller wound surface area and eschar thickness throughout the study period, and collagen synthesis and remodeling were more favorable. However, no significant difference was observed in ASC-treated mice compared with a control group receiving no treatment.

      Repetitive injections

      Zhou et al. [
      • Zhou X.
      • et al.
      Multiple Injections of Autologous Adipose-Derived Stem Cells Accelerate the Burn Wound Healing Process and Promote Blood Vessel Regeneration in a Rat Model.
      ] used autologous ASCs in a third-degree burn model (2 cm2) in 1-year-old rats. Injection of 2 × 106 ASCs in the wound center and edges compared with a PBS control showed improved wound healing. Not surprisingly, repetitive injections (three times on day 0, day 4 and day 8) accelerated the wound healing process even more effectively than a single injection.

      Application of SVF to treat burns

      The application of SVF also delivered positive results in some burn wound studies. Atalay et al. [
      • Atalay S.
      • Coruh A.
      • Deniz K.
      Stromal vascular fraction improves deep partial thickness burn wound healing.
      ] studied the effect of autologous SVF in a deep partial-thickness burn wound rat model. The group found that an immediate intradermal application of 4 × 106 SVF in 1.5-cm radius second-degree burn wounds resulted in a positive effect on wound healing compared with physiological serum as a control. Increased expression of VEGF and reduced inflammation of burn wounds were observed, whereas vascularization and fibroblastic activity were increased and accelerated. Foubert et al. [
      • Foubert P.
      • et al.
      Uncultured adipose-derived regenerative cells (ADRCs) seeded in collagen scaffold improves dermal regeneration, enhancing early vascularization and structural organization following thermal burns.
      ] studied the effect of clinical-grade SVF on a collagen-based matrix (CBM). Uncultured autologous porcine adipose-derived regenerative cells (ADRCs) were seeded onto CBM (2.5 × 105 cells/cm2) and applied in a full-thickness thermal burn wound model (10 cm2) in mini-pigs. SVF-treated wounds showed accelerated maturation of wound bed tissue and a significant increase in the depth of the wound bed and collagen deposition as well as blood vessel density, blood vessel lumen area and the state of maturation within 3 weeks. Blood vessel density was increased by CBM alone as well as in the SVF group compared with control.

      Different hydrogels/carriers/spheroids

      Lu et al. [
      • Lu T.Y.
      • et al.
      Enzyme-Crosslinked Gelatin Hydrogel with Adipose-Derived Stem Cell Spheroid Facilitating Wound Repair in the Murine Burn Model.
      ] used a gelatin/microbial transglutaminase hydrogel as a carrier for hASC spheroids (1 × 106 cells) and cell suspension. Partial-thickness burn wounds (1-cm diameter) showed significantly improved healing when treated with hydrogel and ASC spheroids compared with hydrogel and cell suspension. Smaller wound area at day 10, less discoloration and scar development, thicker epidermis and more effective angiogenesis were observed.
      Motamed et al. [
      • Motamed S.
      • et al.
      Cell-based skin substitutes accelerate regeneration of extensive burn wounds in rats.
      ] compared the effect of amniotic membrane combined with ASCs of human fetal fibroblasts on burn wound healing in rats. Large third-degree burn wounds (11–18 cm2) showed faster wound closure when treated with hASCs or fetal fibroblasts seeded on human amniotic membrane (5 × 105 cells/cm2), especially within the first 14 days.
      In another experiment, Chung et al. [
      • Chung E.
      • et al.
      Fibrin-based stem cell containing scaffold improves the dynamics of burn wound healing.
      ] used ASCs seeded on a 3D PEGylated fibrin gel scaffold in a second-degree burn wound rat model. The researchers found that gels containing 4 × 105 ASCs improved wound healing in 1.7-cm diameter third-degree burn wounds in rats and enhanced vascularization early on.
      Oryan et al. [
      • Oryan A.
      • Alemzadeh E.
      • Mohammadi A.A.
      • Moshiri A
      Healing potential of injectable Aloe vera hydrogel loaded by adipose-derived stem cell in skin tissue-engineering in a rat burn wound model.
      ] studied an injectable aloe vera hydrogel loaded with allogeneic ASCs in 1-cm diameter full-thickness circular burn wounds in a rat model. The ASC-containing aloe vera hydrogel (50% aloe vera) was applied topically and injected intradermally around the wound, and in one group, wounds were additionally dressed with a demineralized bone matrix. The demineralized bone matrix/aloe vera/ASC group (7 × 105 cells injected and 3 × 105 cells applied topically) showed enhanced wound healing, lower inflammatory cell count and higher levels of angiogenesis and re-epithelialization as well as decreased scar formation.
      Burmeister et al. [
      • Burmeister D.M.
      • et al.
      Delivery of Allogeneic Adipose Stem Cells in Polyethylene Glycol-Fibrin Hydrogels as an Adjunct to Meshed Autografts After Sharp Debridement of Deep Partial Thickness Burns.
      ] studied the efficacy and feasibility of ASCs in PEGylated fibrin hydrogels added to meshed split-thickness skin grafts in a porcine model. The study showed that the PEGylated fibrin hydrogel was able to prevent the wound contraction seen after meshing of split-thickness skin grafts and that allogeneic ASCs (5 × 104 ASCs/mL, 2.5 × 105 ASCs/mL and 5 × 105 ASCs/mL) increased blood vessel size and accelerated angiogenesis in deep partial-thickness burn wounds (7 cm2) in a dose-dependent manner.

      Induction and cultivation

      Chen et al. [
      • Chen Y.W.
      • et al.
      The Effects of Adipose-Derived Stem Cell-Differentiated Adipocytes on Skin Burn Wound Healing in Rats.
      ] investigated the effect of adipogenic inducers like 3-isobutyl-1-methylxanthine (IBMX) and insulin on the wound healing ability of ASCs in burn wounds. One million induced ASCs were injected subcutaneously in 3-cm intermediate burn wounds in a rat model. ASCs pre-treated with IBMX for a short time and insulin achieved similar wound healing results compared with ASC and fat injections; however, an acceleration of early healing was observed. Reduced fibrosis and mild inflammatory infiltration limited to the superficial dermis were observed. However, prolonged treatment with IBMX (11 days) resulted in cell deterioration and mitotic inhibition.
      Kaita et al. [
      • Kaita Y.
      • et al.
      Sufficient therapeutic effect of cryopreserved frozen adipose-derived regenerative cells on burn wounds.
      ] studied whether differences existed between fresh human ADRCs (hADRCs) (5 × 104) and frozen hADRCs (5 × 104) cultured over an artificial dermis. Their results indicated a significant difference in the percentage of wound closure observed after day 12 post-treatment between the hADRC and control groups but higher expression of type I and type III collagen when frozen hADRCs were used. The higher synthesis of collagen type I, in particular, was presumed to be due to higher expression levels of EGF and fibroblast growth factor 2—known to stimulate collagen type I production in fibroblasts—in frozen ADRCs. The researchers surmised that purification of ADRCs took place during cryopreservation and thawing and was responsible for the observed difference.
      These studies show the positive effect of different applications of ASCs, ASC-CM and SVF on burn wound healing. Autologous ASCs, repetitive injections and different carrier materials have an additional positive impact and should be further investigated. In reviewing these studies, a lack of standardization of source, dose, timing, delivery method and control stood out, which made comparison difficult. Larger standardized studies will help to further establish optimal treatment.

      Hair loss: alopecia

      Another topic investigated in recent years is hair loss (alopecia) and hair regeneration. Female and male pattern hair loss, which is characterized by a thinning of the scalp hair, affects approximately 50% of women by the age of 50 and up to 50% of men before the age of 50 [
      • Rhodes T.
      • et al.
      Prevalence of male pattern hair loss in 18-49 year old men.
      ].
      The cyclic growth of hair follicles is divided into three phases: growth phase (anagen, 2–6 years), transitional phase (catagen, 2–4 weeks) and mitotic quiescence phase (telogen, 2–3 months) [
      • Limat A.
      • et al.
      Soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of outer root sheath cells.
      ,
      • Juárez-Rendón K.J.
      • et al.
      Alopecia Areata. Current situation and perspectives.
      ]. Different forms of hair loss exist, the most common in both men and women being androgenetic alopecia (AGA) [
      • Qi J.
      • Garza L.A
      An overview of alopecias. Cold Spring Harb.
      ]. Treatment options for alopecia include medical therapy (e.g., finasteride, minoxidil, prostaglandins), hair transplant surgery [
      • Gentile P.
      • et al.
      AIRMESS - Academy of International Regenerative Medicine & Surgery Societies: recommendations in the use of platelet-rich plasma (PRP), autologous stem cell-based therapy (ASC-BT) in androgenetic alopecia and wound healing.
      ], low-level laser therapy [
      • Gentile P.
      • Garcovich S.
      The Effectiveness of Low-Level Light/Laser Therapy on Hair Loss.
      ] and PRP application [
      • York K.
      • Meah N.
      • Bhoyrul B.
      • Sinclair R
      A review of the treatment of male pattern hair loss.
      ,
      • Gentile P.
      • et al.
      Impact of the Different Preparation Methods to Obtain Autologous Non-Activated Platelet-Rich Plasma (A-PRP) and Activated Platelet-Rich Plasma (AA-PRP) in Plastic Surgery: Wound Healing and Hair Regrowth Evaluation.
      ]. However, not all therapies are effective or approved in some patients, and some show negative side effects, such as changes in prostate-specific antigen levels and sexual dysfunction (finasteride) or allergic or irritant contact dermatitis and hypertrichosis in some areas (minoxidil) [
      • Fukuoka H.
      • Suga H.
      Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms.
      ,
      • Blumeyer A.
      • et al.
      Evidence-based (S3) guideline for the treatment of androgenetic alopecia in women and in men.
      ]. Both in vitro and animal models have shown that hair growth is regulated by systemic effects as well as by intercellular communication through multiple growth factors from the surrounding cells [
      • Limat A.
      • et al.
      Soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of outer root sheath cells.
      ]. Although hair follicle MSCs have been shown to act beneficially in alopecia [
      • Gentile P.
      Autologous Cellular Method Using Micrografts of Human Adipose Tissue Derived Follicle Stem Cells in Androgenic Alopecia.
      ,
      • Gentile P.
      • Scioli M.G.
      • Cervelli V.
      • Orlandi A.
      • Garcovich S.
      Autologous Micrografts from Scalp Tissue: Trichoscopic and Long-Term Clinical Evaluation in Male and Female Androgenetic Alopecia.
      ,
      • Gentile P.
      • Scioli M.G.
      • Bielli A.
      • Orlandi A.
      • Cervelli V.
      Stem cells from human hair follicles: first mechanical isolation for immediate autologous clinical use in androgenetic alopecia and hair loss.
      ,
      • Gentile P.
      • Garcovich S.
      Advances in Regenerative Stem Cell Therapy in Androgenic Alopecia and Hair Loss: Wnt pathway, Growth-Factor, and Mesenchymal Stem Cell Signaling Impact Analysis on Cell Growth and Hair Follicle Development.
      ], ASCs can be harvested in much higher numbers and more easily [
      • Buschmann J.
      • et al.
      Yield and proliferation rate of adipose-derived stem cells as a function of age, BMI and harvest site: Increasing the yield by using adherent and supernatant fractions?.
      ]. Considering that ASCs secrete various growth factors that activate neighboring cells, including VEGF, insulin-like growth factor, HGF and PDGF, their therapeutic potential in hair loss has been investigated by several studies [
      • Park B.S.
      • et al.
      Hair growth stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: evidence of increased growth factor secretion.
      ,
      • Festa E.
      • et al.
      Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.
      ]. It is believed that the hair regeneration effect of ASCs takes place via paracrine effects, i.e. via anti-inflammatory, anti-androgen and pro-angiogenic secreted factors [
      • Festa E.
      • et al.
      Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.
      ,
      • Epstein G.K.
      • Epstein J.S.
      Mesenchymal Stem Cells and Stromal Vascular Fraction for Hair Loss: Current Status.
      ].

      In vitro and animal studies

      In a 2010 in vitro study, Won et al. [
      • Won C.H.
      • et al.
      Hair growth promoting effects of adipose tissue-derived stem cells.
      ] showed that human dermal papilla cells (hDPCs) treated with ASC-CM significantly enhanced the proliferation of hDPCs through modulation of the cell cycle (increase in S phase, upregulated expression of cyclin D and cyclin-dependent kinase 2) and earlier conversion of telogen to anagen. In a mouse model, ASC-CM administered subcutaneously also showed induction of the anagen phase and increased hair regeneration as well as proliferation of hDPCs and human epithelial keratinocytes via a paracrine mechanism. Hypoxic culture conditions further increased hair regrowth [
      • Park B.S.
      • et al.
      Hair growth stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: evidence of increased growth factor secretion.
      ,
      • Park B.S.
      • et al.
      Hair growth stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: evidence of increased growth factor secretion.
      ]. Jeong et al. [
      • Jeong Y.M.
      • et al.
      Ultraviolet B preconditioning enhances the hair growth-promoting effects of adipose-derived stem cells via generation of reactive oxygen species.
      ] showed that low-dose ultraviolet B radiation (10–20 mJ/cm2) had an effect on the survival and regenerative potential of ASCs similar to that of hypoxia and a positive effect on hair growth in a mouse model. Ultraviolet B radiation could potentially be a new ASC pre-conditioning method for hair regeneration.
      Evin et al. [
      • Evin N.
      • et al.
      Effects of Adipose-Derived Stem Cells and Platelet-Rich Plasma for Prevention of Alopecia and Other Skin Complications of Radiotherapy.
      ] showed in a mouse model that a single injection of 0.6 × 106 ASCs was able to help prevent radiotherapy-induced alopecia. A combination of ASCs and PRP showed an even better effect. Xiao et al. [
      • Xiao S.
      • et al.
      Promotion of Hair Growth by Conditioned Medium from Extracellular Matrix/Stromal Vascular Fraction Gel in C57BL/6 Mice.
      ] compared CM from an ECM/SVF gel with SVF-CM in a mouse model. Three weekly applications of ECM/SVF-CM stimulated hair growth more than SVF-CM by promoting the proliferation of dermal papilla and bulge cells as well as neovascularization and anagen induction.

      ASC-conditioned medium

      Some clinical studies with small patient groups have reported a positive effect, with an increase in the number of hairs as well as noticeable better hair quality [
      • Fukuoka H.
      • Suga H.
      Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms.
      ,
      • Shin H.
      • Won C.H.
      • Chung W.K.
      • Park B.S
      Up-to-date Clinical Trials of Hair Regeneration Using Conditioned Media of Adipose-Derived Stem Cells in Male and Female Pattern Hair Loss.
      ,
      • Narita K.
      • Fukuoka H.
      • Sekiyama T.
      • Suga H.
      • Harii K
      Sequential Scalp Assessment in Hair Regeneration Therapy Using an Adipose-Derived Stem Cell-Conditioned Medium.
      ], even 1 year after performing the study [
      • Shin H.
      • Won C.H.
      • Chung W.K.
      • Park B.S
      Up-to-date Clinical Trials of Hair Regeneration Using Conditioned Media of Adipose-Derived Stem Cells in Male and Female Pattern Hair Loss.
      ]. ASC-CM and SVF have been used in several studies. For example, Fukuoka et al. [
      • Fukuoka H.
      • Narita K.
      • Suga H.
      Hair Regeneration Therapy: Application of Adipose-Derived Stem Cells.
      ] treated 21 patients with AGA or female pattern hair loss (FPHL) with six sessions of ASC-CM (0.02 mL/cm2) every 3–5 weeks for 3 months. After the first treatment, an increase in the number of hairs was observed. Patients noticed positive changes after four to five treatments, and there was a significant difference between the treatment and placebo groups in comparative studies.
      A retrospective case study conducted by Shin et al. [
      • Shin H.
      • Ryu H.H.
      • Kwon O.
      • Park B.S.
      • Jo S.J.
      Clinical use of conditioned media of adipose tissue-derived stem cells in female pattern hair loss: a retrospective case series study.
      ] analyzed the effect of ASC-CM on FPHL. After 12 weeks of therapy, increased hair density and hair thickness were observed in 27 patients. Narita et al. [
      • Narita K.
      • Fukuoka H.
      • Sekiyama T.
      • Suga H.
      • Harii K
      Sequential Scalp Assessment in Hair Regeneration Therapy Using an Adipose-Derived Stem Cell-Conditioned Medium.
      ] additionally evaluated the effect of ASC-CM on the interfollicular scalp in 40 patients with AGA or FPHL (Hamilton–Norwood grade II–VI, Ludwig grade I–II). Patients treated with ASC-CM injection every month for 6 months showed—in addition to increased hair density—an increase in dermal thickness and echogenicity as well as a gradual increase in transepidermal water loss value at 4 months and 6 months after the initial treatment, suggesting regenerative changes in the skin of the scalp.
      Lee et al. [
      • Lee Y.I.
      • Kim J.
      • Kim J.
      • Park S.
      • Lee J.H
      The Effect of Conditioned Media From Human Adipocyte-Derived Mesenchymal Stem Cells on Androgenetic Alopecia After Nonablative Fractional Laser Treatment.
      ] used a slightly different application method in their randomized, double-blind and placebo-controlled trial. After a single fractional laser treatment followed by self-applied micro-needling to increase the absorbance of ASC-CM, ASC-CM was topically administered to the scalp of 30 patients with AGA. The treatment group showed significantly higher hair density compared with the placebo group after weekly application of ASC-CM for 12 weeks.

      Application of SVF

      Other studies have concentrated on the application of SVF in small groups of patients with alopecia [
      • Anderi R.
      • Makdissy N.
      • Azar A.
      • Rizk F.
      • Hamade A
      Cellular therapy with human autologous adipose-derived adult cells of stromal vascular fraction for alopecia areata.
      ,
      • Perez-Meza D.
      • et al.
      Hair follicle growth by stromal vascular fraction-enhanced adipose transplantation in baldness.
      ,
      • Stevens H.P.
      • Donners S.
      • de Bruijn J.
      Introducing Platelet-Rich Stroma: Platelet-Rich Plasma (PRP) and Stromal Vascular Fraction (SVF) Combined for the Treatment of Androgenetic Alopecia.
      ,
      • Kuka G.
      • et al.
      Cell Enriched Autologous Fat Grafts to Follicular Niche Improves Hair Regrowth in Early Androgenetic Alopecia.
      ]. Anderi et al. [
      • Anderi R.
      • Makdissy N.
      • Azar A.
      • Rizk F.
      • Hamade A
      Cellular therapy with human autologous adipose-derived adult cells of stromal vascular fraction for alopecia areata.
      ] performed a retrospective study on autologous adipose-derived SVF in 20 patients with grade I and II alopecia areata on the Ludwig scale. A single injection of 4–4.7 × 106 cells (0.2 mL per injection, 25 spots, separated by 1 cm) led to increased hair growth 3 months and 6 months after treatment. Another small study in nine patients with Norwood–Hamilton grade II–VI or Ludwig grade I–III FPHL, in which autologous SVF was used with autologous fat injection (1.0 mL/cm2 scalp), also reported favorable results. Increased hair density was observed after 6 months of follow-up. Injection of fat alone also led to an increase—albeit a smaller one—in hair density [
      • Perez-Meza D.
      • et al.
      Hair follicle growth by stromal vascular fraction-enhanced adipose transplantation in baldness.
      ]. Another study in 71 patients with early AGA showed a positive effect on hair count in men with Norwood–Hamilton grade III hair loss who received Puregraft fat with low-dose SVF (0.5 × 106/cm2) compared with a control group; however, a higher dose (1.0 × 106/cm2) did not provide a better outcome [
      • Kuka G.
      • et al.
      Cell Enriched Autologous Fat Grafts to Follicular Niche Improves Hair Regrowth in Early Androgenetic Alopecia.
      ].
      Stevens et al. [
      • Stevens H.P.
      • Donners S.
      • de Bruijn J.
      Introducing Platelet-Rich Stroma: Platelet-Rich Plasma (PRP) and Stromal Vascular Fraction (SVF) Combined for the Treatment of Androgenetic Alopecia.
      ] evaluated injections of SVF in combination with PRP in 10 male patients with grade II–III alopecia areata on the Norwood–Hamilton scale. A single intradermal injection of autologous SVF and PRP (5 mL PRP and 1 mL SVF with 0.53–3.15 × 106 cells) at the level of the hair follicles (0.01 mL, 0.4 cm apart, 100 cm2) led to increased hair density after 6 weeks and 12 weeks.
      Zanzottera et al. [
      • Zanzottera F.
      • Lavezzari E.
      • Trovato L.
      • Icardi A.
      • Graziano A.
      Adipose Derived Stem Cells and Growth Factors Applied on Hair Transplantation. Follow-Up of Clinical Outcome.
      ] studied the effect of applying an autologous cell suspension containing ASCs, epithelial cells, immature adipocytes and their secretome additionally to hair transplantation (three patients). Treated patients demonstrated faster healing of the micro-wound and continuous growth of the transplanted hair even after 2 months.

      Dystrophic epidermolysis bullosa

      Dystrophic epidermolysis bullosa (DEB) is a rare hereditary mechanobullous disease. It is characterized by skin fragility and the development of blisters after minor stress to the skin, but extracutaneous (e.g., gastrointestinal) manifestations can also occur. Recessive and dominant forms exist, with further division into clinical subtypes. In DEB, mutations in the COL7A1 gene result in dysfunction of type VII collagen, a structural component of anchoring fibrils at the dermal–epidermal junction. The resulting skin wounds, which are most often at sites of mechanical stress (i.e., knees, feet, hands and elbows), are often associated with complications (e.g., infection or extensive scarring) and lead to chronic wounds [
      • Uitto J.
      • Has C.
      • Vahidnezhad H.
      • Youssefian L.
      • Bruckner-Tuderman L.
      Molecular pathology of the basement membrane zone in heritable blistering diseases:: The paradigm of epidermolysis bullosa.
      ,
      • Fine J.D.
      • et al.
      The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB.
      ].
      A case study by Maseda et al. [
      • Maseda R.
      • et al.
      Beneficial Effect of Systemic Allogeneic Adipose Derived Mesenchymal Cells on the Clinical, Inflammatory and Immunologic Status of a Patient With Recessive Dystrophic Epidermolysis Bullosa: A Case Report.
      ] demonstrated the effect of intravenous ASC application in a patient with recessive DEB with long-lasting refractory oral ulcers. Three intravenous applications of 1 × 106 allogeneic ASCs every 21 days, concomitant with the established symptomatic treatment, led to an improvement in wound healing, itching, pain and quality of life. The maximum effect was observed 6–9 months post-treatment, with an effect remaining noticeable for up to 2 years.

      Atopic dermatitis

      Another skin disease in which the application of ASCs has been investigated is AD, which is one of the most common chronic inflammatory skin diseases and manifests most often in childhood. AD is accompanied by xerosis, eczematous lesions and severe pruritus [
      • Leung D.Y.
      • et al.
      Disease management of atopic dermatitis: an updated practice parameter. Joint Task Force on Practice Parameters.
      ].
      AD is a T-cell-mediated immune disorder characterized by an excessive T-helper 2 cell-mediated inflammatory response. B lymphocytes lead to an increase in IgE level and subsequent mast cell (MC) degranulation, release of various inflammatory cytokines (e.g., IL-5, IL-17, macrophage inflammatory protein 1 beta) and recruitment of lymphocytes and eosinophils into the lesion [
      • Simon D.
      • Braathen L.R.
      • Simon H.U.
      Eosinophils and atopic dermatitis.
      ,
      • Kim M.
      • et al.
      Human Adipose Tissue-Derived Mesenchymal Stem Cells Attenuate Atopic Dermatitis by Regulating the Expression of MIP-2, miR-122a-SOCS1 Axis, and Th1/Th2 Responses.
      ]. It has been suggested that dysregulation of MSCs might be involved early on in the pathogenesis of AD [
      • Orciani M.
      • et al.
      T helper (Th)1, Th17 and Th2 imbalance in mesenchymal stem cells of adult patients with atopic dermatitis: at the origin of the problem.
      ]. Current clinical management of AD includes prevention of trigger factors, topical corticosteroids and systemic immunosuppressants as well as treatment with biologics [
      • Eichenfield L.F.
      • et al.
      Guidelines of care for the management of atopic dermatitis: section 2. Management and treatment of atopic dermatitis with topical therapies.
      ,
      • Montes-Torres A.
      • Llamas-Velasco M.
      • Pérez-Plaza A.
      • Solano-López G.
      • Sánchez-Pérez J.
      Biological Treatments in Atopic Dermatitis.
      ]. However, these treatments have several side effects, including skin atrophy (topical steroids), nephrotoxicity and malignancy (cyclosporine) and hepatotoxicity and infections (azathioprine) and show limited and non-uniform efficacy. MSCs have been studied as a potential alternative [
      • Snast I.
      • et al.
      Are Biologics Efficacious in Atopic Dermatitis? A Systematic Review and Meta-Analysis.
      ].
      Although several studies with AD mouse models have been performed to investigate the potential of MSCs in AD treatment and have shown beneficial effects, few studies have used ASCs [
      • Yang J.W.
      • et al.
      Extracellular Vesicles from SOD3-Transduced Stem Cells Exhibit Improved Immunomodulatory Abilities in the Murine Dermatitis Model.
      ,
      • Sah S.K.
      • et al.
      Enhanced therapeutic effects of human mesenchymal stem cells transduced with superoxide dismutase 3 in a murine atopic dermatitis-like skin inflammation model.
      ,
      • Li Y.
      • Ye Z.
      • Yang W.
      • Zhang Q.
      • Zeng J.
      An Update on the Potential of Mesenchymal Stem Cell Therapy for Cutaneous Diseases.
      ,
      • Na K.
      • et al.
      Bone marrow-derived clonal mesenchymal stem cells inhibit ovalbumin-induced atopic dermatitis.
      ]. The positive therapeutic effect is thought to be through regulation of B-cell-mediated IgE production [
      • Shin T.H.
      • et al.
      Human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis via regulation of B lymphocyte maturation.
      ].
      A study by Shin et al. [
      • Kim M.
      • et al.
      Human Adipose Tissue-Derived Mesenchymal Stem Cells Attenuate Atopic Dermatitis by Regulating the Expression of MIP-2, miR-122a-SOCS1 Axis, and Th1/Th2 Responses.
      ] in an AD mouse model showed that mice having been given 1 × 106 hASCs intravenously exhibited improved clinical symptoms compared with a control group where human dermal fibroblasts were injected. Clinical symptoms were attenuated and number of degranulated MCs, amount of histamine released, prostaglandin E2 level and expression levels of cytokines and chemokines (e.g., IL-5, macrophage inflammatory protein 1 beta, macrophage inflammatory protein 2, C-C motif chemokine ligand 5 and IL-17) were decreased. This effect seemed to be dose-dependent. Another study in an AD mouse model showed that a lower dose of ASCs (2 × 105) injected intravenously resulted in no significant clinical improvement [
      • Shin T.H.
      • et al.
      Human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis via regulation of B lymphocyte maturation.
      ]. However, a dose-dependent reduction in serum IgE levels as well as attenuation of epidermal hyperplasia and lymphocyte infiltration was observed. In vitro experiments showed that hASCs significantly inhibited the proliferation and maturation of B lymphocytes via cyclooxygenase 2 signaling and suppressed MC degranulation.
      A study by Cho et al. [
      • Cho B.S.
      • Kim J.O.
      • Ha D.H.
      • Yi Y.W.
      Exosomes derived from human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis.
      ] using ASC-derived exosomes demonstrated a therapeutic effect in an AD mouse model that was comparable to prednisolone treatment. Intravenous and subcutaneous applications of ASC-derived exosomes (0.14 µg, 1.4 µg or 10 µg) three times weekly for 4 weeks showed a dose-dependent positive effect on clinical score as well as decreased levels of IgE and eosinophils in the blood and reduced infiltration of MCs and CD86+ and CD206+ cells and decreased inflammatory cytokine levels in skin lesions.
      Villatoro et al. [
      • Villatoro A.J.
      • et al.
      Allogeneic adipose-derived mesenchymal stem cell therapy in dogs with refractory atopic dermatitis: clinical efficacy and safety.
      ] showed the safety and efficacy of allogeneic canine ASCs in dogs with refractory AD. One intravenous injection of 1.5 × 106 canine ASCs/kg resulted in improved symptoms and clinical scores, with no adverse events seen at 6-month follow-up.
      In summary, it can be said that the intravenous application of ASCs and ASC-derived exosomes has shown a positive therapeutic effect in AD animal models. Additionally, a positive dose-dependent effect has been described.

      Systemic lupus erythematosus

      Systemic lupus erythematosus (SLE) is a rare heterogeneous autoimmune disease that affects both sexes and all races, with a higher prevalence in black populations and women [
      • Pons-Estel G.J.
      • Alarcón G.S.
      • Scofield L.
      • Reinlib L.
      • Cooper G.S
      Understanding the epidemiology and progression of systemic lupus erythematosus.
      ]. In SLE, antigen–antibody complexes are produced and lodge in small vessels, the basement membrane of the skin and kidneys and various other organ systems. Genetic predisposition and environmental factors such as stress, infection and hormone levels as well as immunoregulatory factors appear to play a role in the pathogenesis of SLE; however, the exact mechanism is unknown. B-cell over-reactivity and subsequent overproduction of pathogenic autoantibodies also seem to play an important role in the pathogenesis of SLE [
      • Lai N.S.
      • Koo M.
      • Yu C.L.
      • Lu M.C.
      Immunopathogenesis of systemic lupus erythematosus and rheumatoid arthritis: the role of aberrant expression of non-coding RNAs in T cells.
      ,
      • Tsokos G.C.
      Systemic lupus erythematosus.
      ]. Abnormalities in MSCs themselves have been shown to be involved in the disease process of SLE [
      • Geng L.
      • et al.
      Association of TNF-α with impaired migration capacity of mesenchymal stem cells in patients with systemic lupus erythematosus.
      ]. MSCs from SLE patients show enhanced senescence and apoptosis [
      • Li X.
      • et al.
      Enhanced apoptosis and senescence of bone-marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus.
      ] as well as abnormal gene expression [
      • Tang Y.
      • et al.
      Gene expression profile reveals abnormalities of multiple signaling pathways in mesenchymal stem cell derived from patients with systemic lupus erythematosus.
      ] and migration [
      • Geng L.
      • et al.
      Association of TNF-α with impaired migration capacity of mesenchymal stem cells in patients with systemic lupus erythematosus.
      ], and autologous MSCs fail to maintain the balance among immune cells [
      • Carrion F.
      • et al.
      Autologous mesenchymal stem cell treatment increased T regulatory cells with no effect on disease activity in two systemic lupus erythematosus patients.
      ]. The skin is the second most commonly affected organ after the musculoskeletal system and is involved in over 80% of SLE patients (Figure 4) [
      • Aringer M.
      • Johnson S.R.
      Classifying and diagnosing systemic lupus erythematosus in the 21st century.
      ,
      • Cervera R.
      • et al.
      Systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1,000 patients. The European Working Party on Systemic Lupus Erythematosus.
      ].
      Current therapy for SLE includes immunosuppressive drugs such as corticosteroids, cyclophosphamide, azathioprine and methotrexate as well as new agents such as rituximab and abatacept, and many other biologics are in clinical trials [
      • Tanaka Y.
      State-of-the-art treatment of systemic lupus erythematosus.
      ]. However, not all patients respond to treatment, and some experience severe side effects; for example, corticosteroids can lead to hyperglycemia, infection and osteoporosis [
      • Wang Q.
      • et al.
      Combined transplantation of autologous hematopoietic stem cells and allogenic mesenchymal stem cells increases T regulatory cells in systemic lupus erythematosus with refractory lupus nephritis and leukopenia.
      ,
      • Popa R.
      • et al.
      Therapy Side Effects in Systemic Lupus Erythematosus.
      ].
      Because of their immunomodulatory properties, MSCs have been investigated as a potential treatment alternative. Several pre-clinical phase 1 and phase 2 studies have demonstrated improvements in SLE through application of allogeneic MSCs; however, long-term follow-up studies are needed [
      • Tang W.Y.
      • et al.
      Functional Characteristics and Application of Mesenchymal Stem Cells in Systemic Lupus Erythematosus.
      ]. The application of ASCs has been tested in several murine models of SLE [
      • Wei S.
      • et al.
      Allogeneic adipose-derived stem cells suppress mTORC1 pathway in a murine model of systemic lupus erythematosus.
      ,
      • Zhang W.
      • Feng Y.L.
      • Pang C.Y.
      • Lu F.A.
      • Wang Y.F.
      Transplantation of adipose tissue-derived stem cells ameliorates autoimmune pathogenesis in MRL/lpr mice: Modulation of the balance between Th17 and Treg.
      ,
      • Choi E.W.
      • Lee M.
      • Song J.W.
      • Shin I.S.
      • Kim S.J.
      Mesenchymal stem cell transplantation can restore lupus disease-associated miRNA expression and Th1/Th2 ratios in a murine model of SLE.
      ,
      • Choi E.W.
      • et al.
      Comparative Efficacies of Long-Term Serial Transplantation of Syngeneic, Allogeneic, Xenogeneic, or CTLA4Ig-Overproducing Xenogeneic Adipose Tissue-Derived Mesenchymal Stem Cells on Murine Systemic Lupus Erythematosus.
      ,
      • He X.
      • et al.
      Suppression of interleukin 17 contributes to the immunomodulatory effects of adipose-derived stem cells in a murine model of systemic lupus erythematosus.
      ]; however, only one study has reported the effect on skin lesions.
      Choi et al. [
      • Choi E.W.
      • et al.
      Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Prevents the Development of Lupus Dermatitis.
      ] performed a study on MRL-lpr mice, which exhibit lupus-like symptoms. Mice subjected to 10 applications of 1 × 106 ASCs starting at an early stage (week 5) did not show dermatitis upon gross examination and demonstrated significant improvement in hyperkeratosis, acanthosis and inflammatory cell infiltration scores on histopathology. Cytotoxic T lymphocyte-associated protein 4 immunoglobulin-overexpressing ASCs showed results similar to those of unselected ASCs, and no adverse effects were observed. These results seem promising, and further studies are needed to confirm the positive effect and identify the most effective treatment protocols.

      Final Remarks and Limitations

      The authors are aware that the field of ASC application in skin diseases is quite large and comprehensiveness cannot be achieved in one review article. For example, the authors excluded fat and nanofat applications [
      • Gentile P.
      • Scioli M.G.
      • Bielli A.
      • Orlandi A.
      • Cervelli V.
      Comparing different nanofat procedures on scars: role of the stromal vascular fraction and its clinical implications.
      ] in skin diseases, which potentially have positive effects that are caused by ASCs, as ASCs are a non-negligible component in these tissues. In addition, exosomes harvested from ASCs—a topic on which the authors touched only marginally—are currently being widely investigated [
      • Gentile P.
      • Garcovich S.
      Concise Review: Adipose-Derived Stem Cells (ASCs) and Adipocyte-Secreted Exosomal microRNA (A-SE-miR) Modulate Cancer Growth and proMote Wound Repair.
      ]. Finally, the application of ASCs in the prevention of skin photoaging, including wrinkle formation and hyperpigmentation, has not been further addressed [
      • Gentile P.
      • Garcovich S.
      Adipose-Derived Mesenchymal Stem Cells (AD-MSCs) against Ultraviolet (UV) Radiation Effects and the Skin Photoaging.
      ]. Nevertheless, the authors utilized two main sections to cover the topic, the first of which dealt with different formulations of ASCs (laboratory perspective) and the second of which dealt with different skin diseases (clinic perspective). For the second section, the authors summarized the most important examples of current treatments and the potential future application of ASCs (Figure 5A,B) [
      • Fanouriakis A.
      • et al.
      2019 update of the EULAR recommendations for the management of systemic lupus erythematosus.
      ,
      • Fanouriakis A.
      • Tziolos N.
      • Bertsias G.
      • Boumpas D.T
      Update οn the diagnosis and management of systemic lupus erythematosus.
      ]. Because of the still sparsely available data on ASC use in clinics, it remains difficult to undertake balanced risk–benefit considerations with respect to the potential future application of ASCs.
      ASCs are easily accessible and occur in large numbers compared with other sources of stem cells, which makes them highly attractive [
      • Buschmann J.
      • et al.
      Yield and proliferation rate of adipose-derived stem cells as a function of age, BMI and harvest site: Increasing the yield by using adherent and supernatant fractions?.
      ]. The authors have demonstrated that their pro-angiogenic and immunomodulatory effects support skin wound healing and also have positive effects in a diverse set of skin diseases, such as diabetic ulcers and burn wounds. Therefore, ASCs should be applied more often in daily clinical practice [
      • Gentile P.
      • Scioli M.G.
      • Bielli A.
      • Orlandi A.
      • Cervelli V.
      Concise Review: The Use of Adipose-Derived Stromal Vascular Fraction Cells and Platelet Rich Plasma in Regenerative Plastic Surgery.
      ], as this type of stem cell offers a viable option and as well as alternative/add-on to regular medication. In particular, burn wounds should be treated with ASCs more often by clinicians because many pre-clinical models have proven their advantage over conventional therapy options. In addition, promising data are available regarding their application in diabetic ulcers. The results of ongoing clinical trials should ensure more widespread use in the treatment of refractory chronic diabetic wounds. Other skin diseases such as DEB and AD are less investigated and need more pre-clinical trials, especially with regard to the mode of action and potential side effects of systemic application, before larger clinical studies can be performed (Figure 3).
      Fig 3
      Fig. 3A 22-year-old patient with alopecia areata.
      Fig 4
      Fig. 4A 21-year-old patient with SLE with skin manifestation.
      Fig 5
      Fig. 5Overview of current therapies and potential application of ASCs. (A) Summary for SLE, diabetic ulcer and alopecia. Examples of adverse reactions are shown in parentheses. (B) Summary for burn wounds, AD and DEB. Examples of adverse reactions are shown in parentheses. AZA, azathioprine; BEL, belimumab; CNI, calcineurin inhibitor; CYC, cyclophosphamide; GC, glucocorticoid; HCQ, hydroxychloroquine; MMF, mycophenolate mofetil (teratogenic); MTX, methotrexate; RTX, rituximab 198,199.

      Funding

      No funding was received.

      Author Contributions

      Conception and design of the study: I.S. and J.B.. Acquisition of data: I.S. and J.B.. Drafting or revising the manuscript: I.S. and J.B.. All authors have approved the final article.

      Declaration of Competing Interest

      The authors have no commercial, proprietary or financial interest in the products or companies described in this article.

      References

        • Bourin P.
        • et al.
        Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).
        Cytotherapy. 2013; 15: 641-648
        • Hassan W.U.
        • Greiser U.
        • Wang W
        Role of adipose-derived stem cells in wound healing.
        Wound Repair Regen. 2014; 22: 313-325
        • Shingyochi Y.
        • Orbay H.
        • Mizuno H
        Adipose-derived stem cells for wound repair and regeneration.
        Expert Opin. Biol. Ther. 2015; 15: 1285-1292
        • Hassanshahi A.
        • et al.
        Adipose-derived stem cells for wound healing.
        J. Cell. Physiol. 2019; 234: 7903-7914
        • Moon K.C.
        • et al.
        Potential of Allogeneic Adipose-Derived Stem Cell-Hydrogel Complex for Treating Diabetic Foot Ulcers.
        Diabetes. 2019; 68: 837-846
        • Klar A.S.
        • Zimoch J.
        • Biedermann T.
        Skin Tissue Engineering: Application of Adipose-Derived Stem Cells.
        Biomed Res Int. 2017; 20179747010
        • Gentile P.
        • Garcovich S.
        Systematic Review: Adipose-Derived Mesenchymal Stem Cells, Platelet-Rich Plasma and Biomaterials as New Regenerative Strategies in Chronic Skin Wounds and Soft Tissue Defects.
        Int. J. Mol. Sci. 2021; 22: 1538-1551
        • Barrera J.A.
        • et al.
        Adipose-Derived Stromal Cells Seeded in Pullulan-Collagen Hydrogels Improve Healing in Murine Burns.
        Tissue Eng Part A. 2021; 27: 844-856
        • Gantwerker E.A.
        • Hom D.B.
        Skin: histology and physiology of wound healing.
        Facial Plast. Surg. Clin. North Am. 2011; 19: 441-453
        • Monavarian M.
        • Kader S.
        • Moeinzadeh S.
        • Jabbari E.
        Regenerative Scar-Free Skin Wound Healing.
        Tissue engineering. Part B, Reviews. 2019; 25: 294-311
        • Hyldig K.
        • Riis S.
        • Pennisi C.P.
        • Zachar V.
        • Fink T
        Implications of Extracellular Matrix Production by Adipose Tissue-Derived Stem Cells for Development of Wound Healing Therapies.
        Int. J. Mol. Sci. 2017; 18: 1167-1177
        • Deniz A.A.H.
        • et al.
        Zooming in across the Skin: A Macro-to-Molecular Panorama.
        Adv. Exp. Med. Biol. 2020; 1247: 157-200
        • Wong R.
        • Geyer S.
        • Weninger W.
        • Guimberteau J.C.
        • Wong J.K
        The dynamic anatomy and patterning of skin.
        Exp. Dermatol. 2016; 25: 92-98
        • Hegde S.
        • Raghavan S.
        A skin-depth analysis of integrins: role of the integrin network in health and disease.
        Cell communication & adhesion. 2013; 20: 155-169
        • Giancotti F.G.
        • Ruoslahti E.
        Integrin signaling.
        Science. 1999; 285: 1028-1032
        • López-López N.
        • et al.
        Expression and vitamin D-mediated regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy skin and in diabetic foot ulcers.
        Arch. Dermatol. Res. 2014; 306: 809-821
        • Huayllani M.T.
        • et al.
        Adipose-derived stem cells in wound healing of full-thickness skin defects: a review of the literature.
        J. Plast. Surg. Hand Surg. 2020; 54: 263-279
        • Vidor S.B.
        • et al.
        Adipose-derived stem cells improve full-thickness skin grafts in a rat model.
        Res. Vet. Sci. 2018; 118: 336-344
        • Plock J.A.
        • Schnider J.T.
        • Solari M.G.
        • Zheng X.X.
        • Gorantla V.S
        Perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation.
        Front. Immunol. 2013; 4: 7
        • Eke G.
        • Mangir N.
        • Hasirci N.
        • MacNeil S.
        • Hasirci V.
        Development of a UV crosslinked biodegradable hydrogel containing adipose derived stem cells to promote vascularization for skin wounds and tissue engineering.
        Biomaterials. 2017; 129: 188-198
        • Li Y.
        • et al.
        Adipose tissue-derived stem cells suppress hypertrophic scar fibrosis via the p38/MAPK signaling pathway.
        Stem Cell. Res. Ther. 2016; 7: 102
        • Gentile P.
        New strategies in plastic surgery: autologous adipose-derived mesenchymal stem cells contained in fat grafting improves symptomatic scars.
        Front Biosci (Landmark Ed). 2021; 26: 255-257
        • Gentile P.
        • Sterodimas A.
        • Calabrese C.
        • Garcovich S.
        Systematic review: Advances of fat tissue engineering as bioactive scaffold, bioactive material, and source for adipose-derived mesenchymal stem cells in wound and scar treatment.
        Stem Cell. Res. Ther. 2021; 12: 318
        • Scioli M.G.
        • et al.
        The biomolecular basis of adipogenic differentiation of adipose-derived stem cells.
        Int. J. Mol. Sci. 2014; 15: 6517-6526
        • Gentile P.
        • Garcovich S.
        Concise Review: Adipose-Derived Stem Cells (ASCs) and Adipocyte-Secreted Exosomal microRNA (A-SE-miR) Modulate Cancer Growth and proMote Wound Repair.
        Journal of clinical medicine. 2019; 8: 855-867
        • Evrova O.
        • Buschmann J.
        In vitro and in vivo effects of PDGF-BB delivery strategies on tendon healing: a review.
        Eur. cells mat. 2017; 34: 15-39
        • Sakar M.S.
        • et al.
        Cellular forces and matrix assembly coordinate fibrous tissue repair.
        Nature communications. 2016; 7: 11036
        • Ke Q.
        • Costa M.
        Hypoxia-inducible factor-1 (HIF-1).
        Mol. Pharmacol. 2006; 70: 1469-1480
        • Apte R.S.
        • Chen D.S.
        • Ferrara N.
        VEGF in Signaling and Disease: Beyond Discovery and Development.
        Cell. 2019; 176: 1248-1264
        • Zbinden A.
        • et al.
        Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.
        Biomater Sci. 2018; 6: 1076-1083
        • Hinz B.
        Myofibroblasts.
        Exp. Eye Res. 2016; 142: 56-70
        • Werner S.
        • Krieg T.
        • Smola H
        Keratinocyte-fibroblast interactions in wound healing.
        J. Invest. Dermatol. 2007; 127: 998-1008
        • Schulze-Tanzil G.
        • et al.
        The role of pro-inflammatory and immunoregulatory cytokines in tendon healing and rupture: new insights. Scand.
        J. Med. Sci. Sports. 2011; 21: 337-351
        • Manning C.N.
        • et al.
        Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro.
        Stem Cell. Res. Ther. 2015; 6: 1-14
        • Park J.E.
        • Barbul A.
        Understanding the role of immune regulation in wound healing.
        The American Journal of Surgery. 2004; 187: S11-S16
        • Xia Z.
        • et al.
        The Application of Decellularized Adipose Tissue Promotes Wound Healing.
        Tissue Eng Regen Med. 2020;
        • Gentile P.
        • et al.
        Systematic Review: Allogenic Use of Stromal Vascular Fraction (SVF) and Decellularized Extracellular Matrices (ECM) as Advanced Therapy Medicinal Products (ATMP) in Tissue Regeneration.
        Int. J. Mol. Sci. 2020; 21: 4982-4995
        • Ragelle H.
        • et al.
        Comprehensive proteomic characterization of stem cell-derived extracellular matrices.
        Biomaterials. 2017; 128: 147-159
        • Niada S.
        • Giannasi C.
        • Magagnotti C.
        • Andolfo A.
        • Brini A.T.
        Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts.
        J. Proteomics. 2021; 232104069
        • Frazier T.P.
        • Gimble J.M.
        • Kheterpal I.
        • Rowan B.G.
        Impact of low oxygen on the secretome of human adipose-derived stromal/stem cell primary cultures.
        Biochimie. 2013; 95: 2286-2296
        • Chen B.
        • et al.
        Supportive Use of Adipose-Derived Stem Cells in Cell-Assisted Lipotransfer for Localized Scleroderma.
        Plast. Reconstr. Surg. 2018; 141: 1395-1407
        • Wang C.
        • et al.
        A pilot study on ex vivo expanded autologous adipose-derived stem cells of improving fat retention in localized scleroderma patients.
        Stem Cells Transl Med. 2021; 10: 1148-1156
        • Mirzaei-Parsa M.J.
        • et al.
        Nanofiber-acellular dermal matrix as a bilayer scaffold containing mesenchymal stem cell for healing of full-thickness skin wounds.
        Cell Tissue Res. 2019; 375: 709-721
        • Zeng R.X.
        • et al.
        Experimental study on repairing skin defect by tissue-engineered skin substitute compositely constructed by adipose-derived stem cells and fibrin gel.
        Eur. Rev. Med. Pharmacol. Sci. 2017; 21: 1-5
        • Feng C.J.
        • Lin C.H.
        • Tsai C.H.
        • Yang I.C.
        • Ma H
        Adipose-derived stem cells-induced burn wound healing and regeneration of skin appendages in a novel skin island rat model.
        J. Chin. Med. Assoc. 2019; 82: 635-642
        • Hur W.
        • et al.
        Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium.
        Stem Cell. Res. Ther. 2017; 8: 92
        • Bi H.
        • et al.
        Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process.
        Stem Cell. Res. Ther. 2019; 10: 302
        • Gentile P.
        • Piccinno M.S.
        • Calabrese C.
        Characteristics and Potentiality of Human Adipose-Derived Stem Cells (hASCs) Obtained from Enzymatic Digestion of Fat Graft.
        Cells. 2019; 8: 282-301
        • Gentile P.
        • et al.
        Regenerative application of stromal vascular fraction cells enhanced fat graft maintenance: clinical assessment in face rejuvenation.
        Expert Opin. Biol. Ther. 2020; 20: 1503-1513
        • Kern S.
        • Eichler H.
        • Stoeve J.
        • Klüter H.
        • Bieback K.
        Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.
        Stem Cells. 2006; 24: 1294-1301
        • Maguire G.
        The Safe and Efficacious Use of Secretome From Fibroblasts and Adipose-derived (but not Bone Marrow-derived) Mesenchymal Stem Cells for Skin Therapeutics.
        J. Clin. Aesthet. Dermatol. 2019; 12: E57-e69
        • Kapur S.K.
        • Katz A.J.
        Review of the adipose derived stem cell secretome.
        Biochimie. 2013; 95: 2222-2228
        • Luo Y.
        • et al.
        Autograft microskin combined with adipose-derived stem cell enhances wound healing in a full-thickness skin defect mouse model.
        Stem Cell. Res. Ther. 2019; 10: 279
        • Lee J.S.
        • et al.
        Angiogenic factors secreted from human ASC spheroids entrapped in an alginate-based hierarchical structure via combined 3D printing/electrospinning system.
        Biofabrication. 2020; 12045028
        • Orfei C.P.
        • et al.
        In Vitro Induction of Tendon-Specific Markers in Tendon Cells, Adipose- and Bone Marrow-Derived Stem Cells is Dependent on TGF3, BMP-12 and Ascorbic Acid Stimulation.
        Int. J. Mol. Sci. 2019; 20: 1-15
        • Zuk P.A.
        • et al.
        Multilineage cells from human adipose tissue: Implications for cell-based therapies.
        Tissue Eng. 2001; 7: 211-228
        • Yu J.
        • Tu Y.K.
        • Tang Y.B.
        • Cheng N.C.
        Stemness and transdifferentiation of adipose-derived stem cells using L-ascorbic acid 2-phosphate-induced cell sheet formation.
        Biomaterials. 2014; 35: 3516-3526
        • Xu J.
        • Liu X.
        • Zhao F.
        • Zhang Y.
        • Wang Z.
        HIF1α overexpression enhances diabetic wound closure in high glucose and low oxygen conditions by promoting adipose-derived stem cell paracrine function and survival.
        Stem Cell. Res. Ther. 2020; 11: 148
        • Ding S.
        • Xu Y.
        • Yan X.
        • Lin Y.
        • Tan Q.
        Effect of Collagen Scaffold With Bcl-2-Modified Adipose-Derived Stem Cells on Diabetic Mice Wound Healing.
        The international journal of lower extremity wounds. 2020; 19: 139-147
        • Kim H.
        • Hyun M.R.
        • Kim S.W.
        The Effect of Adipose-Derived Stem Cells on Wound Healing: Comparison of Methods of Application.
        Stem Cells Int. 2019; 20192745640
        • Tamayo T.
        • et al.
        Diabetes in Europe: An update.
        Diabetes Res. Clin. Pract. 2014; 103: 206-217
        • Xu G.
        • et al.
        Prevalence of diagnosed type 1 and type 2 diabetes among US adults in 2016 and 2017: population based study.
        BMJ. 2018; 362: k1497
        • Lim J.Z.
        • Ng N.S.
        • Thomas C
        Prevention and treatment of diabetic foot ulcers.
        J. R. Soc. Med. 2017; 110: 104-109
        • Richard J.L.
        • Schuldiner S.
        Épidémiologie du pied diabétique.
        La Revue de Médecine Interne. 2008; 29: S222-S230
        • Zhang P.
        • et al.
        Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis.
        Ann. Med. 2017; 49: 106-116
        • Schaper N.C.
        • et al.
        Practical Guidelines on the prevention and management of diabetic foot disease (IWGDF 2019 update).
        Diabetes Metab. Res. Rev. 2020; 36: e3266
        • Falanga V.
        Wound healing and its impairment in the diabetic foot.
        The Lancet. 2005; 366: 1736-1743
        • Loots M.A.M.
        • et al.
        Differences in Cellular Infiltrate and Extracellular Matrix of Chronic Diabetic and Venous Ulcers Versus Acute Wounds.
        J. Invest. Dermatol. 1998; 111: 850-857
        • Dinh T.L.
        • Veves A.
        A Review of the Mechanisms Implicated in the Pathogenesis of the Diabetic Foot.
        The international journal of lower extremity wounds. 2005; 4: 154-159
        • Signorelli S.S.
        • et al.
        Plasma levels and zymographic activities of matrix metalloproteinases 2 and 9 in type II diabetics with peripheral arterial disease.
        Vasc. Med. 2005; 10: 1-6
        • Loots M.A.M.
        • et al.
        Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls.
        Eur. J. Cell Biol. 2002; 81: 153-160
        • Zykova S.N.
        • et al.
        Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.
        Diabetes. 2000; 49: 1451-1458
        • Jeffcoate W.J.
        • et al.
        Unresolved issues in the management of ulcers of the foot in diabetes.
        Diabet. Med. 2008; 25: 1380-1389
        • De Angelis B.
        • et al.
        Wound Healing: In Vitro and In Vivo Evaluation of a Bio-Functionalized Scaffold Based on Hyaluronic Acid and Platelet-Rich Plasma in Chronic Ulcers.
        Journal of clinical medicine. 2019; 8: 1486-1499
        • Cervelli V.
        • et al.
        P.R.L. platelet rich lipotransfert: our experience and current state of art in the combined use of fat and PRP.
        Biomed Res Int. 2013; 2013434191
        • De Angelis B.
        • et al.
        One-Stage Reconstruction of Scalp after Full-Thickness Oncologic Defects Using a Dermal Regeneration Template (Integra).
        Biomed Res Int. 2015; 2015698385
        • Gentile P.
        • et al.
        Complex abdominal wall repair using a porcine dermal matrix.
        Surg. Innov. 2013; 20: Np12-Np15
        • De Angelis B
        • et al.
        Long-term follow-up comparison of two different bi-layer dermal substitutes in tissue regeneration: Clinical outcomes and histological findings.
        Int Wound J. 2018; 15: 695-706
        • Falanga V.
        • et al.
        Wounding of Bioengineered Skin: Cellular and Molecular Aspects After Injury.
        J. Invest. Dermatol. 2002; 119: 653-660
        • Cerqueira M.T.
        • et al.
        Human Adipose Stem Cells Cell Sheet Constructs Impact Epidermal Morphogenesis in Full-Thickness Excisional Wounds.
        Biomacromolecules. 2013; 14: 3997-4008
        • McLaughlin M.M.
        • Marra K.G.
        The use of adipose-derived stem cells as sheets for wound healing.
        Organogenesis. 2013; 9: 79-81
        • Lin Y.C.
        • et al.
        Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model.
        Acta Biomater. 2013; 9: 5243-5250
        • Alexandrushkina N.
        • et al.
        Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization.
        Int. J. Mol. Sci. 2020; 21: 5567-5587
        • Bukowska J.
        • et al.
        Safety of Human Adipose Stromal Vascular Fraction Cells Isolated with a Closed System Device in an Immunocompetent Murine Pressure Ulcer Model.
        Stem Cells Dev. 2020; 29: 452-461
        • Kato Y.
        • et al.
        Allogeneic Transplantation of an Adipose-Derived Stem Cell Sheet Combined With Artificial Skin Accelerates Wound Healing in a Rat Wound Model of Type 2 Diabetes and Obesity.
        Diabetes. 2015; 64: 2723-2734
        • Hamada M.
        • et al.
        Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes.
        Regenerative Therapy. 2017; 6: 65-73
        • Dong Y.
        • et al.
        Acceleration of Diabetic Wound Regeneration using an In Situ–Formed Stem-Cell-Based Skin Substitute.
        Advanced Healthcare Materials. 2018; 71800432
        • Feng J.
        • et al.
        An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells.
        Sci. Rep. 2017; 7: 1548
        • Amos P.J.
        • et al.
        Human Adipose-Derived Stromal Cells Accelerate Diabetic Wound Healing: Impact of Cell Formulation and Delivery.
        Tissue Engineering Part A. 2009; 16: 1595-1606
        • Kim H.K.
        • et al.
        Alterations in the proangiogenic functions of adipose tissue-derived stromal cells isolated from diabetic rats.
        Stem Cells Dev. 2008; 17: 669-680
        • El-ftesi S.
        • Chang E.I.
        • Longaker M.T.
        • Gurtner G.C
        Aging and Diabetes Impair the Neovascular Potential of Adipose-Derived Stromal Cells.
        Plast. Reconstr. Surg. 2009; 123: 475-485
        • Rennert R.C.
        • et al.
        Diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations.
        Stem Cell. Res. Ther. 2014; 5: 79
        • Kočí Z.
        • et al.
        Characterization of human adipose tissue-derived stromal cells isolated from diabetic patient's distal limbs with critical ischemia.
        Cell Biochem. Funct. 2014; 32: 597-604
        • Cianfarani F.
        • et al.
        Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.
        Wound Repair Regen. 2013; 21: 545-553
        • Nambu M.
        • et al.
        Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissue-derived stromal cells combined with atelocollagen matrix.
        Ann. Plast. Surg. 2009; 62: 317-321
        • Gong J.H.
        • Dong J.Y.
        • Xie T.
        • Zhao Q.
        • Lu S.L.
        Different therapeutic effects between diabetic and non-diabetic adipose stem cells in diabetic wound healing.
        J. Wound Care. 2021; 30: S14-s23
        • Kinoshita K.
        • et al.
        Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers.
        Stem Cells Transl Med. 2015; 4: 146-155
        • Di Rocco G.
        • et al.
        Enhanced healing of diabetic wounds by topical administration of adipose tissue-derived stromal cells overexpressing stromal-derived factor-1: biodistribution and engraftment analysis by bioluminescent imaging.
        Stem Cells Int. 2010; 2011304562
        • Finnerty C.C.
        • Przkora R.
        • Herndon D.N.
        • Jeschke M.G.
        Cytokine expression profile over time in burned mice.
        Cytokine. 2009; 45: 20-25
        • Jeschke M.G.
        • et al.
        Survivors versus nonsurvivors postburn: differences in inflammatory and hypermetabolic trajectories.
        Ann. Surg. 2014; 259: 814-823
        • Abdul Kareem N.
        • Aijaz A.
        • Jeschke M.G.
        Stem Cell Therapy for Burns: Story so Far.
        Biologics. 2021; 15: 379-397
        • Mock C.
        A WHO plan for burn prevention and care.
        WHO AIDS Tech. Bull. 2008; 23
        • Evers L.H.
        • Bhavsar D.
        • Mailänder P.
        The biology of burn injury.
        Exp. Dermatol. 2010; 19: 777-783
        • Rohani Ivari J.
        • Mahdipour E
        Adipose tissue versus stem cell-derived small extracellular vesicles to enhance the healing of acute burns.
        Regen. Med. 2021; 16: 629-641
        • Gentile P.
        • Scioli M.G.
        • Bielli A.
        • Orlandi A.
        • Cervelli V.
        Comparing different nanofat procedures on scars: role of the stromal vascular fraction and its clinical implications.
        Regen. Med. 2017; 12: 939-952
        • Levy V.
        • Lindon C.
        • Zheng Y.
        • Harfe B.D.
        • Morgan B.A
        Epidermal stem cells arise from the hair follicle after wounding.
        FASEB J. 2007; 21: 1358-1366
        • Atiyeh B.S.
        • Costagliola M.
        Cultured epithelial autograft (CEA) in burn treatment: three decades later.
        Burns. 2007; 33: 405-413
        • Condé-Green A.
        • et al.
        Fat Grafting and Adipose-Derived Regenerative Cells in Burn Wound Healing and Scarring: A Systematic Review of the Literature.
        Plast. Reconstr. Surg. 2016; 137: 302-312
        • Collawn S.S.
        • Banerjee N.S.
        • de la Torre J.
        • Vasconez L.
        • Chow L.T
        Adipose-derived stromal cells accelerate wound healing in an organotypic raft culture model.
        Ann. Plast. Surg. 2012; 68: 501-504
        • Bliley J.M.
        • et al.
        Administration of adipose-derived stem cells enhances vascularity, induces collagen deposition, and dermal adipogenesis in burn wounds.
        Burns. 2016; 42: 1212-1222
        • Loder S.
        • et al.
        Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts.
        J Burn Care Res. 2015; 36: 70-76
        • Chang Y.W.
        • et al.
        Autologous and not allogeneic adipose-derived stem cells improve acute burn wound healing.
        PLoS One. 2018; 13e0197744
        • Riccobono D.
        • et al.
        Application of adipocyte-derived stem cells in treatment of cutaneous radiation syndrome.
        Health Phys. 2012; 103: 120-126
        • Franck C.L.
        • et al.
        Influence of Adipose Tissue-Derived Stem Cells on the Burn Wound Healing Process.
        Stem Cells Int. 2019; 20192340725
        • Hanson S.E.
        • et al.
        Local delivery of allogeneic bone marrow and adipose tissue-derived mesenchymal stromal cells for cutaneous wound healing in a porcine model.
        J. Tissue Eng. Regen. Med. 2016; 10: E90-e100
        • Ebrahimian T.G.
        • et al.
        Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing.
        Arterioscler. Thromb. Vasc. Biol. 2009; 29: 503-510
        • Karimi H.
        • Soudmand A.
        • Orouji Z.
        • Taghiabadi E.
        • Mousavi S.J.
        Burn wound healing with injection of adipose-derived stem cells: a mouse model study.
        Ann Burns Fire Disasters. 2014; 27: 44-49
        • Zhou X.
        • et al.
        Multiple Injections of Autologous Adipose-Derived Stem Cells Accelerate the Burn Wound Healing Process and Promote Blood Vessel Regeneration in a Rat Model.
        Stem Cells Dev. 2019; 28: 1463-1472
        • Atalay S.
        • Coruh A.
        • Deniz K.
        Stromal vascular fraction improves deep partial thickness burn wound healing.
        Burns. 2014; 40: 1375-1383
        • Foubert P.
        • et al.
        Uncultured adipose-derived regenerative cells (ADRCs) seeded in collagen scaffold improves dermal regeneration, enhancing early vascularization and structural organization following thermal burns.
        Burns. 2015; 41: 1504-1516
        • Lu T.Y.
        • et al.
        Enzyme-Crosslinked Gelatin Hydrogel with Adipose-Derived Stem Cell Spheroid Facilitating Wound Repair in the Murine Burn Model.
        Polymers (Basel). 2020; 12: 2997-3009
        • Motamed S.
        • et al.
        Cell-based skin substitutes accelerate regeneration of extensive burn wounds in rats.
        Am. J. Surg. 2017; 214: 762-769
        • Chung E.
        • et al.
        Fibrin-based stem cell containing scaffold improves the dynamics of burn wound healing.
        Wound Repair Regen. 2016; 24: 810-819
        • Oryan A.
        • Alemzadeh E.
        • Mohammadi A.A.
        • Moshiri A
        Healing potential of injectable Aloe vera hydrogel loaded by adipose-derived stem cell in skin tissue-engineering in a rat burn wound model.
        Cell Tissue Res. 2019; 377: 215-227
        • Burmeister D.M.
        • et al.
        Delivery of Allogeneic Adipose Stem Cells in Polyethylene Glycol-Fibrin Hydrogels as an Adjunct to Meshed Autografts After Sharp Debridement of Deep Partial Thickness Burns.
        Stem Cells Transl Med. 2018; 7: 360-372
        • Chen Y.W.
        • et al.
        The Effects of Adipose-Derived Stem Cell-Differentiated Adipocytes on Skin Burn Wound Healing in Rats.
        J Burn Care Res. 2017; 38: 1-10
        • Kaita Y.
        • et al.
        Sufficient therapeutic effect of cryopreserved frozen adipose-derived regenerative cells on burn wounds.
        Regen Ther. 2019; 10: 92-103
        • Rhodes T.
        • et al.
        Prevalence of male pattern hair loss in 18-49 year old men.
        Dermatol. Surg. 1998; 24: 1330-1332
        • Limat A.
        • et al.
        Soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of outer root sheath cells.
        Arch. Dermatol. Res. 1993; 285: 205-210
        • Juárez-Rendón K.J.
        • et al.
        Alopecia Areata. Current situation and perspectives.
        Arch. Argent. Pediatr. 2017; 115: e404-e411
        • Qi J.
        • Garza L.A
        An overview of alopecias. Cold Spring Harb.
        Perspect. Med. 2014; 4: a013615-a013628
        • Gentile P.
        • et al.
        AIRMESS - Academy of International Regenerative Medicine & Surgery Societies: recommendations in the use of platelet-rich plasma (PRP), autologous stem cell-based therapy (ASC-BT) in androgenetic alopecia and wound healing.
        Expert Opin. Biol. Ther. 2021; 21: 1443-1449
        • Gentile P.
        • Garcovich S.
        The Effectiveness of Low-Level Light/Laser Therapy on Hair Loss.
        Facial Plast Surg Aesthet Med. 2021; 0: 8
        • York K.
        • Meah N.
        • Bhoyrul B.
        • Sinclair R
        A review of the treatment of male pattern hair loss.
        Expert Opin. Pharmacother. 2020; 21: 603-612
        • Gentile P.
        • et al.
        Impact of the Different Preparation Methods to Obtain Autologous Non-Activated Platelet-Rich Plasma (A-PRP) and Activated Platelet-Rich Plasma (AA-PRP) in Plastic Surgery: Wound Healing and Hair Regrowth Evaluation.
        Int. J. Mol. Sci. 2020; 21: 431-439
        • Fukuoka H.
        • Suga H.
        Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms.
        Eplasty. 2015; 15: e10
        • Blumeyer A.
        • et al.
        Evidence-based (S3) guideline for the treatment of androgenetic alopecia in women and in men.
        J Dtsch Dermatol Ges. 2011; 9: S1-57
        • Gentile P.
        Autologous Cellular Method Using Micrografts of Human Adipose Tissue Derived Follicle Stem Cells in Androgenic Alopecia.
        Int. J. Mol. Sci. 2019; 20: 3446-3462
        • Gentile P.
        • Scioli M.G.
        • Cervelli V.
        • Orlandi A.
        • Garcovich S.
        Autologous Micrografts from Scalp Tissue: Trichoscopic and Long-Term Clinical Evaluation in Male and Female Androgenetic Alopecia.
        Biomed Res Int. 2020; 20207397162
        • Gentile P.
        • Scioli M.G.
        • Bielli A.
        • Orlandi A.
        • Cervelli V.
        Stem cells from human hair follicles: first mechanical isolation for immediate autologous clinical use in androgenetic alopecia and hair loss.
        Stem Cell Investig. 2017; 4: 58
        • Gentile P.
        • Garcovich S.
        Advances in Regenerative Stem Cell Therapy in Androgenic Alopecia and Hair Loss: Wnt pathway, Growth-Factor, and Mesenchymal Stem Cell Signaling Impact Analysis on Cell Growth and Hair Follicle Development.
        Cells. 2019; 8: 466-486
        • Buschmann J.
        • et al.
        Yield and proliferation rate of adipose-derived stem cells as a function of age, BMI and harvest site: Increasing the yield by using adherent and supernatant fractions?.
        Cytotherapy. 2013; 15: 1098-1105
        • Park B.S.
        • et al.
        Hair growth stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: evidence of increased growth factor secretion.
        Biomed. Res. 2010; 31: 27-34
        • Festa E.
        • et al.
        Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.
        Cell. 2011; 146: 761-771
        • Epstein G.K.
        • Epstein J.S.
        Mesenchymal Stem Cells and Stromal Vascular Fraction for Hair Loss: Current Status.
        Facial Plast. Surg. Clin. North Am. 2018; 26: 503-511
        • Won C.H.
        • et al.
        Hair growth promoting effects of adipose tissue-derived stem cells.
        J. Dermatol. Sci. 2010; 57: 134-137
        • Park B.S.
        • et al.
        Hair growth stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: evidence of increased growth factor secretion.
        Biomedical research (Tokyo, Japan). 2010; 31: 27-34
        • Jeong Y.M.
        • et al.
        Ultraviolet B preconditioning enhances the hair growth-promoting effects of adipose-derived stem cells via generation of reactive oxygen species.
        Stem Cells Dev. 2013; 22: 158-168
        • Evin N.
        • et al.
        Effects of Adipose-Derived Stem Cells and Platelet-Rich Plasma for Prevention of Alopecia and Other Skin Complications of Radiotherapy.
        Ann. Plast. Surg. 2021; 86: 588-597
        • Xiao S.
        • et al.
        Promotion of Hair Growth by Conditioned Medium from Extracellular Matrix/Stromal Vascular Fraction Gel in C57BL/6 Mice.
        Stem Cells Int. 2020; 20209054514
        • Shin H.
        • Won C.H.
        • Chung W.K.
        • Park B.S
        Up-to-date Clinical Trials of Hair Regeneration Using Conditioned Media of Adipose-Derived Stem Cells in Male and Female Pattern Hair Loss.
        Curr. Stem Cell Res. Ther. 2017; 12: 524-530
        • Narita K.
        • Fukuoka H.
        • Sekiyama T.
        • Suga H.
        • Harii K
        Sequential Scalp Assessment in Hair Regeneration Therapy Using an Adipose-Derived Stem Cell-Conditioned Medium.
        Dermatol. Surg. 2020; 46: 819-825