Mesenchymal Stromal Cell-Conditioned Medium for Skin Diseases: A Systematic Review

The skin is the largest organ of the human body, and its dysfunction is related to many diseases. There is a need to find new potential effective therapies for some skin conditions such as inflammatory diseases, wound healing, or hair restoration. Mesenchymal stromal cell (MSC)-conditioned medium (CM) provides a potential opportunity in the treatment of skin disease. Thus, the objective of this review is to evaluate the uses of MSC-CM for treating skin diseases in both animal and human models. A systematic review was conducted regarding the use of MSC-CM for treating skin conditions. One hundred one studies were analyzed. MSC-CM was evaluated in wound healing (55), hypertrophic scars (9), flap reperfusion (4), hair restoration (15), skin rejuvenation (15), and inflammatory skin diseases (3). MSC-CM was obtained from different MSC sources, mainly adipose tissue, bone marrow, and umbilical cord blood. MSC-CM was tested intravenously, intraperitoneally, subcutaneously, intradermally or intralesionally injected or topically applied. MSC-CM was used in both animals and humans. MSC-CM improved wound healing, hair restoration, skin rejuvenation, atopic dermatitis, and psoriasis in both animals and humans. MSC-CM also decreased hypertrophic scars and flap ischemia in animal models. In conclusion, MSC-CM is a promising therapy for skin conditions. Further studies are needed to corroborate safety and effectiveness and to standardize CM manufacturing.


INTRODUCTION
Mesenchymal stromal cells (MSCs) are a type of multipotent adult stem cells that have the potential to proliferate, selfregenerate, and differentiate into multiple cell lineages (Dominici et al., 2006). They can be isolated from several sources such as bone marrow (BM-MSCs), adipose tissue (AT-MSCs), umbilical cord (UC-MSCs), amnion, placenta, or dental pulp (Bogatcheva and Coleman, 2019). MSCs are considered to be one of the most promising therapeutic options in cell therapy and tissue engineering, as they can be used for treating skin, cardiovascular, hematological, neurological, bone, and cartilage diseases (Wong et al., 2015;Kim et al., 2017;Hu et al., 2018;Martinez-Lopez et al., 2020).
It has been observed that the beneficial effects of MSCs are due not only to their multipotent ability but also to their secreted cytokines and growth factors (Caplan, 2017). Cell-free preparations have several advantages over cell therapy, as they can be obtained more easily and more economically and can be manufactured, packaged, and transported straightforwardly (Yang et al., 2021). Moreover, cellfree preparations do not have adverse events associated with cell administration such as rejection, tumorigenic, thrombogenic, ossification, or calcification risk (Bogatcheva and Coleman, 2019;Yang et al., 2021). The risk of malignant transformation of MSCs is a great concern, as MSCs therapy involves ex vivo production and expansion of cell lines, although the spontaneous malignant transformation of human MSCs has not been completely proved and there are many studies that have demonstrated that MSCs, even after physical and chemical stress, undergo senescence rather than become tumorigenic (Caplan et al., 2019). MSCs activate the host innate immune systems and the coagulation, increasing the expression of procoagulant tissue factor and demonstrating a procoagulant effect after MSC contact with blood in in vitro investigations. Infusion reactions and thromboembolism have been reported when using intravascular MSC products. A possible proposed solution to this problem is diluting or treating the MSCs with tissue factor pathwayblocking reagents. Moreover, hemocompatibility testing and optimal product delivery are important for designing safer MSC therapies. Other experimental intravascular therapies, such as islets, hepatocytes, and products derived from MSCs, could also improve MSC safety (Moll et al., 2019). The activation of the immune response could also lead to rejection. There are many investigations that focus on strategies to evade immune recognition, such as human leukocyte antigen (HLA)-matched cells or pharmaceutical immunosuppression (Moll et al., 2019(Moll et al., , 2020. Cell-free preparation could help to reduce this risk of malignant transformation, thrombogenic risk, and rejection. The molecules secreted by stromal cells are referred to the stromal cell secretome and include proteins, microRNA, growth factors, antioxidants, proteasomes, and exosomes (Maguire, 2013). The stromal cell culture media that comprise the secretome are known as the conditioned media (CM), and they are considered to be an abundant resource of paracrine factors (Pawitan, 2014;Mizukami and Yagihashi, 2016). The paracrine factors secreted in vitro include vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulinlike growth factor-1 (IGF-1), IGF−2, and stromal cell-derived factor 1 (SDF−1) (Ratajczak et al., 2012;Deng et al., 2018). The administration of these factors to the site of an injured organ increases its metabolic activity and oxygen supply and remodels the extracellular matrix (Ratajczak et al., 2012).
The skin is the largest organ of the human body, and its dysfunction is linked to several diseases (Montero-Vilchez et al., 2021). MSCs provide a supply of new cells for epidermal homeostasis, hair cycling, and repairing injured tissue (Kim et al., 2017;Guo et al., 2020). MSCs-CM also provide a potential opportunity in the treatment of skin disease, and there is increased evidence justifying its use for the treatment of cutaneous conditions such as wound healing, hair growth, inflammatory skin diseases, or skin rejuvenation. Thus, the objective of this review is to evaluate the use of MSC-CM for treating skin diseases in both in animals and humans.

Inclusion and Exclusion Criteria
The search was limited to (i) human or animal data, (ii) in vivo studies, (iii) using MSC-CM for skin conditions, and (iv) articles written in English or Spanish.
All types of epidemiological studies (clinical trials, cohort studies, case-control studies, and cross-sectional studies) regarding MSC-CM use for skin conditions were included and analyzed. Reviews, guidelines, protocols, and conference abstracts were excluded.

Study Selection
Two researchers (TMV and AML) independently reviewed the titles and abstracts of the articles obtained in the first search to assess relevant studies. The full texts of all articles meeting the inclusion criteria were reviewed, and their bibliographic references were checked for additional sources. The articles considered relevant by both researchers were included in the analysis. Disagreements about inclusion or exclusion of articles were discussed until a consensus was reached. If no consensus was reached, resolution was achieved by discussion with a third researcher (SAS). route of administration, sample size, assessment, outcomes, and adverse events.

RESULTS
Our literature search identified 1,422 references, 757 after removing duplicates. After the title and abstract were screened, 197 records underwent full-text review. A total of 96 records were excluded because they did not investigate MSC-CM treatment in vivo skin conditions. Other reasons for exclusion along with the flowchart are shown in Figure 1. Ultimately, 101 studies met the eligible criteria.
Mesenchymal stromal cell-CM has been tested in several skin conditions: wound healing, hypertrophic scars, flap and
Different MSC sources and CM delivery were also compared. Allogeneic MSC-CM showed better healing rate than xenogeneic MSC-CM treatment (Joseph et al., 2020). The wound closure rate was higher in animals treated with carrageenan-embedded CM than mice treated with polyvinyl alcohol-embedded CM (Robert et al., 2019). Moreover, CM supplemented with selenium and basic fibroblast growth factor (bFGF) showed better results in wound healing than CM alone or CM only supplemented with bFGF or selenium, as complete wound closure after 11 days' follow-up was only observed in CM supplemented with both growth factors (Park et al., 2018). Hypoxic conditions also improved wound healing, accelerating wound closure (Chen et al., 2014;Jun et al., 2014;Sun et al., 2014;Du et al., 2017). TNFαand TFG-β1-implemented CM also accelerated wound healing as compared with non-supplemented MSC-CM (Cho et al., 2010;Heo et al., 2011). CM derived from Wnt7a-transduced MSCs also showed higher closures rates than did the MSC-CM group . No adverse events were reported in these studies.
Different ways of CM implementation have been also evaluated. CM supplemented with deferoxamine, a hypoxic mimetic agent, had a more potent effect on recovering skin vasculatures than non-supplemented CM (De Gregorio et al., 2020). DMBT1 expression also improves wound healing (Chen C. Y. et al., 2018). Moreover, CM cultured on polydopamine-modified three-dimensional-printed bioceramic (DOPA-BC) scaffolds showed a higher healing rate than CM cultured on tissue culture plates (TCPs) or pure threedimensional-printed bioceramic (pure BC) (Li T. et al., 2019). After 14 days' follow-up, the remaining wound area was 7.1 ± 3.4% in the DOPA-BC-CM group, 15.2 ± 6.6% in the BC-CM group, and 21.2 ± 11.3% in TCP-CM group (Li T. et al., 2019).
Twenty-eight mice with burn wounds were treated with MSC-CM with a mean follow-up of 25.67 days (from 21 to 28 days). MSC-CM was tested using subcutaneous (Li J. Y. et al., 2019) or intraperitoneal (Aryan et al., 2019) injections or topically applied (Zhou et al., 2019). In burn wounds, MSC-CM showed faster wound healing, increased re-epithelialization, and vascularization compared with controls (unconditioned medium, PBS, or DMEM).
Moreover, 72 mice infected with methicillin-resistant Staphylococcus aureus were treated with MSC-CM or MSC-CM plus photobiomodulation (PBM). MSC-CM plus PBM decreased colony-forming units and the number of inflammatory cells Kouhkheil et al., 2019).
Twelve mice with radiation-induced skin injuries were also treated with MSC-CM. Topical application of MSC-CM in hydrogel accelerated wound closure and enhanced the wound healing quality (Sun et al., 2019).

Combined therapies using mesenchymal stromal cell-conditioned medium
Five studies evaluated the effects of MSC-CM combined with PBM or pulsed wave low-level laser therapy (PWLLLT) for treating diabetic wounds and infected wounds (Pouriran et al., 2016;Amini et al., 2018;Bagheri et al., 2018;Fridoni et al., 2019;Kouhkheil et al., 2019) (Supplementary Table 1). Regarding diabetic wounds, the results are controversial (Pouriran et al., 2016;Amini et al., 2018;Bagheri et al., 2018). Two studies showed that MSC-CM, PBM, and the combined therapy improved wound healing as compared with control group (Pouriran et al., 2016;Bagheri et al., 2018). Moreover, it was found that the extent of healing was significantly greater in the MSC-CM+PBM group . On the other hand, it was showed that PWLLLT and MSC-CM, alone or in combination, improved biomechanical parameters in the wound but that PWLLLT was more effective compared with MSC-CM (Pouriran et al., 2016). Concerning infected wounds, it was observed that both PBM+MSC-CM and PBM groups decreased colony-forming units and hastened wound healing process as compared with controls, while it did not happen when using MSC-CM alone Kouhkheil et al., 2019).

Clinical Studies
Only one clinical study evaluated the effects of MSC-CM on wound healing (Table 4). This research assessed the use of MSC-CM derived from human amniotic membrane for treating chronic plantar ulcers in leprosy. The mean age of the patients was 52.12 ± 1.33 years, the mean ulcer duration was 1.41 ± 0.36 years, and the mean ulcer size at baseline was 2.64 ± 0.5 cm 2 with a depth lower than 0.5 cm. Sixty-six patients were divided into groups to receive MSC-CM, MSC-CM+vitamin C, or MSC-CM+vitamin E topically applied every 3 days for 8 weeks. All groups improved wound healing, with MSC-CM+vitamin E being the most effective treatment. Wound size was reduced by 1.7 ± 1.05 vs. 2.01 ± 1.19 vs. 2.84 ± 1.67 cm 2 ; and depth was decreased by 0.35 ± 0.14 vs. 0.25 ± 0.11 vs. 0.27 ± 0.15 cm in MSC-CM, MSC-CM+vitamin C, and MSC-CM+vitamin E groups, respectively. No adverse events were reported (Prakoeswa et al., 2018).

Preclinical Studies
Nine studies evaluated the potential of MSC-CM in the treatment of hypertrophic scars or avoiding scar formation (Wu et al., 2015;Zhang et al., 2015;Du et al., 2016a,b;Li et al., 2016;Choi et al., 2017;Liu et al., 2018;Arjunan et al., 2020;Hu et al., 2020; Table 5). To obtain CM, MSCs were mainly isolated from human tissues: adipose tissue Choi et al., 2017;Liu et al., 2018) (33.33%, 3/9), umbilical cord blood (Arjunan et al., 2020), placenta (Du et al., 2016a), and bone marrow (Wu et al., 2015). Murine bone marrow (Hu et al., 2020), murine placenta (Du et al., 2016b), and rabbit adipose tissue (Zhang et al., 2015) were also used. CM was collected from MSCs between passages 3 and 13 at 70-90% confluence. The most common model was murine (88.89%, 8/9), although a rabbit model was also used (Zhang et al., 2015). CM was used mainly by subcutaneous injection (66.66%, 6/9) (Wu et al., 2015;Du et al., 2016a,b;Li et al., 2016;Liu et al., 2018;Hu et al., 2020). Intralesional (Zhang et al., 2015;Arjunan et al., 2020) and intravenous (Choi et al., 2017) injections were also used. MSC-CM was tested in 97 animals for treating hypertrophic scar and keloids or preventing scar formation. The mean follow-up time was 23 days. Scars were assessed mainly by macroscopic appearance, histology, immunohistochemistry, and qRT-PCR. Mesenchymal stromal cell-CM had better results than nontreated groups or other treatments groups (PBS, DMEM, and unconditioned medium) in terms of tumor volumes, tumor weight, collagen fiber deposition, and skin fibrosis. CM derived from MSCs cultured in hypoxic conditions showed greater effects on scar formation than cultured in normoxic conditions (Du et al., 2016a,b). Moreover, the MSC-CM-treated group exhibited significantly fewer proliferating cells than did MSC-CM with TGF-β3 blocked, showing that TGF-β3 may be responsible for parts of the antifibrotic effect of the MSC-CM (Wu et al., 2015). The combination of MSC-CM and botulinum toxin type A (botox) had a greater effect when treating human hypertrophic scars than did botox alone, MSC-CM alone, or DMEM. Scar weight was reduced by 21, 11, or 10% more compared with DMEM, MSC-CM, or botox, respectively (Hu et al., 2020). Although both MSCs and MSC-CMs reduced hypertrophic scars, MSC-CM might be more effective than MSCs (Zhang et al., 2015). The Scar Elevation Index was reduced in both MSC-and MSC-CM-treated groups compared with internal controls (44.04 and 32.48%, p < 0.01, respectively), and it was significantly lower in the MSC-treated group than in the MSC-CM-treated group (p < 0.01) (Zhang et al., 2015).

Clinical Studies
No clinical study specifically evaluated the effects of MSC-CM on hypertrophic scar formation. Nevertheless, the effect of MSC-CM has been tested on acne scars in combination with laser therapy, showing that combined therapy had a greater effect on reducing acne scars than laser alone (Zhou et

Preclinical Studies
Four studies evaluated the effects of MSC-CM for treating ischemic wounds or flaps (Mirabella et al., 2012;Cooper et al., 2018;Pu et al., 2019; Table 6). MSCs were isolated from human adipose tissue Cooper et al., 2018;Pu et al., 2019) or human amniotic fluid (Mirabella et al., 2012). CM was collected from MSCs between passages 3 and 8 at 70-90% confluence. All the models employed were murine.
Two studies tested MSC-CM in ischemia/reperfusion flap models by clamping the flap and then releasing it. Pu et al. evaluated the effects of MSC-CM applied to the subcutaneous layer between the flap and its bed and at the proximal, middle, and distal parts as compared with saline (n = 6/group) (Pu   et al., 2019). A porcine skin-derived gelatine membrane soaked in either MSC-CM or saline was also tested (n = 5/group) (Mirabella et al., 2012). Both studies observed that the MSC-CM treatment group enhanced skin flap recovery, attenuated the necrotic area, increased hair growth, and stimulated angiogenesis as compared with controls (Mirabella et al., 2012;Pu et al., 2019). MSC-CM were also applied topically on flap ischemic wounds in six mice. It was observed that MSC-CM accelerated the closure of flap ischemic wounds by 50% as compared with controls (Cooper et al., 2018). MSC-CM was also tested on skin allografts, intravenously injected, and compared with placebo and MSCs. MSCs and MSC-CMs increased skin allograft survival as compared with the control (23.9 ± 2.0, 19.6 ± 2.4, and 9.3 ± 1.4 days, respectively), without any differences between MSCs and MSC-CMs .
There are no studies at the clinical level.
The overexpression of a reprogramming factor in the CM, Nanog, improved hair induction even more than MSC-CM without Nanog . Moreover, it was observed that CM collected from human deciduous teeth accelerated hair growth more than CM derived from hair follicle cells. The transition from telogen to anagen stage for teeth-MSC-CM was between 8 and 12 days, while that at hair follicle-MSC-CM ranged from 12 to 15 days (Gunawardena et al., 2019a). CM enrichment with heparin binding-epidermal growth factor-like growth factor (HB-EGF) showed a greater effect in promoting hair growth compared with non-supplemented CM . Furthermore, CM collected from MSCs cultured in hypoxic conditions increased hair regeneration growth factors . No adverse events were reported when using MSC-CM in hair restoration.

Clinical Studies
Four clinical studies evaluated the effects of MSC-CM on hair growth (Shin et al., 2015;Lee et al., 2020;Narita et al., 2020;Oh et al., 2020; Table 8). To obtain CM, cells were isolated from human adipose tissue (Shin et al., 2015;Lee et al., 2020;Narita et al., 2020) (75%) and human umbilical cord blood (Oh et al., 2020) (25%). All studies evaluated the effect of MSC-CM in androgenetic alopecia (AGA). MSC-CM was tested on 100 patients, both men and women, with an age range of 23-74 years. Two observational studies used intradermal injections of human AT-MSC-CM and observed that hair density increased by 17.3 hairs/cm 2 and hair thickness increased by 6.5 µm as compared with baseline (Shin et al., 2015;Narita et al., 2020). A clinical trial also evaluated the effects of human UC-MSC-CM enriched with TGF-1 and LiCl applied topically twice daily for 112 days. It was observed that MSC-CM increased hair density by 14.24%, hair thickness by 28.19%, and hair growth by 19.54% as compared with a placebo (Oh et al., 2020). Moreover, MSC-CM treatment was also evaluated in patients who underwent nonablative fractional laser treatment for AGA. It was found that concomitant treatment (MSC-CM+laser) had a greater effect than laser treatment only. The MSC-CM group had greater hair density (102.1 ± 4.09 vs. 89.3 ± 3.79/cm 2 ) and gross hair volume (2 ± 0.13 vs. 1.2 ± 0.19, evaluated by a 7-point global improvement score) than the placebo group after 12 weeks of treatment administration . No serious adverse events were reported in these studies.
Moreover, MSC-CM was also tested on acne scars (Zhou et al., 2016;Abdel-Maguid et al., 2019;El-Domyati et al., 2019;Park C. S. et al., 2019), suggesting that combined therapy (laser+MSC-CM) may increase the regenerative effects of fractional laser. Four studies evaluated the therapeutic potential in 71 patients with a mean follow-up of 82.5 days (range 60-90). After laser treatment, topical application of MSC-CM showed greater scar volume reduction than did with the control, by 23.5% on the MSC-CM side vs. 15.0% on the control side (Park C. S. et al., 2019). Furthermore, the MSC-CM-treated side improved subjective satisfaction and clinical assessment, showing better TEWL, SCH, elasticity, roughness, and collagen density than did the controls (laser alone or DMEM). Nevertheless, laser therapy+MSC-CM showed lower clinical improvement in acne scars than the combination of laser+platelet-rich plasma (Abdel-Maguid et al., 2019). Adverse events reported were erythema and edema, linked more to laser therapy than MSC-CM treatment.

Preclinical Studies
One study evaluated the effect of CM derived from murine adipose tissue for treating atopic dermatitis (AD) Table 11). Mice were followed up 33 days after AD lesions had developed and treated with subcutaneous injections every 3 days with saline, MSC, MSC-CM, or 2.5% cortisone lotion (n = 6/group). A higher AD severity index decrease was observed in groups treated with MSC, MSC-CM, or cortisone, as compared with the saline group. Moreover, the severity index (assessed by SCORAD) was lower in the MSC-CM group treated with MSCs or cortisone. Skin thickness decreases were similar between MSC-CM, MSC, and cortisone, and all greater than in the saline group. Likewise, mast cell infiltration was reduced in MSC-CM, MSC, and cortisone groups. Th2 expression, measured in optical densities, was lower in the MSC-CM groups than in the MSC, cortisone, or saline group (1.58 ± 1.84 vs. 3.79 ± 2.08 vs. 4.10 ± 3.32 vs. 19.19 ± 5.54). Ig E levels were slightly reduced in mice that were treated with MSC, MSC-CM, or cortisone. IL-4 was also reduced in the group treated with MSC-CM .

Clinical Studies
One study evaluated the effects of MSC-CM for treating AD , and one case report showed the outcomes in a patient with scalp psoriasis treated with MSC-CM (Seetharaman et al., 2019; Table 11).
Twenty-eight patients (15 males and 13 females; mean age 24.68 ± 4.32 years) were treated with human UC-MSC-CM in cream bases for 4 weeks. MSC-CM was applied on both eczematous lesions and non-involved skin twice a day. After treatment, SCH increased by 15.67 arbitrary units (AU), a relative unit of measurement to show the ratio of amount of some quantities to a predetermined reference measurement, on eczematous lesions and 14.49 AU on non-involved skin. Moreover, TEWL decreased by 15.09 g·m −2 ·h −1 AU on eczematous lesions and 3.08 g·m −2 ·h −1 on non-involved skin . In agreement with preclinical studies, these data showed that cosmetics that contained MSC-CM improved the skin barrier function and could be an effective treatment for AD patients.
One study also evaluated the clinical efficacy of MSC-CM derived from human adipose tissue for treating psoriasis scalp. A 38-year-old male was treated topically with MSC-CM on the lesions once a day. After 1-month follow-up, the number of psoriatic plaques was reduced and Psoriasis Scalp Severity Index (PSSI) changed from 28 to 0. Moreover, no relapse or adverse events were observed after 6 months' follow-up (Seetharaman et al., 2019).

DISCUSSION
MSC-CM may be an alternative, safe, and easily delivered therapy for several skin conditions, but evidence in clinical studies is limited. They have been mainly tested in hair restoration (Shin et al., 2015;Lee et al., 2020;Narita et al., 2020;Oh et al., 2020) and skin rejuvenation (Abdel-Maguid et al., 2019;El-Domyati et al., 2019;, with promising results. Preclinical studies are mainly focused on wound healing (Shin et al., 2015;Lee et al., 2020;Narita et al., 2020;Oh et al., 2020), showing high rates in wound closure. MSC-CM may also be an effective treatment for decreasing hypertrophic scars (Arjunan et al., 2020;Hu et al., 2020), improving flap reperfusion (Mirabella et al., 2012;Cooper et al., 2018;Pu et al., 2019), and treating psoriasis (Seetharaman et al., 2019) and AD . Nevertheless, there was significant variability between studies in the cell source,     cell treatment, method of delivery, disease model used to assess efficacy, ways of assessment, and outcome evaluation. This review provides a useful summary regarding the use of MSC-CM for skin conditions and highlights the need to standardize manufacturing processes and characterize CM. The main findings of this review are summarized in Figure 2.
It is important to characterize the composition of CM, but there is a lack of evidence in this aspect. Some studies showed that CM contained high concentrations of growth factors and cytokines associated with angiogenesis and endothelial cell migration such as EGF, tissue inhibitors of metalloproteinase-1, or IGF-binding protein-7 (De Gregorio et al., 2020;. The properties of MSC-CM vary depending on the cell source (Sagaradze et al., 2019). More than 10 sources of MSCs were used to treat skin conditions. MSCs were mainly isolated from human adipose tissue, human bone marrow, and human umbilical cord blood. Another important aspect is the timing of the collection of CM from the cells and concentration steps carried out for obtaining CM (Im et al., 2019). Most studies used cells from passages 3 to 6 at 80-90% confluence. The secretome of cells may also vary depending on the age of the cells (Maguire, 2013). Furthermore, the same type of cells is reported to secrete different levels of paracrine factors depending on the culture condition  (hypoxic vs. normoxic) and the scaffold type (Li T. et al., 2019) (monolayer vs. 3D cultures). Hypoxic treatment is one of the most frequently used ways to improve CM, as hypoxic stress reduces oxygen and improves cellular functions (Du et al., 2017). CM collected from MSCs cultured under hypoxic conditions improved the cells' capacities of proliferation and self-renewal (Chen et al., 2014;Jun et al., 2014;Du et al., 2016aDu et al., , 2017. Determining the levels of paracrine factors secreted by MSCs at different passages with different conditions may also provide knowledge about the best stromal cell growth stage for obtaining a specific group of paracrine factors. Furthermore, MSC-CM should be considered both an alternative therapy and a synergic therapy for improving skin conditions, as MSC-CM combined with the conventional therapy has been proved to have better results than MSC-CM or the conventional therapy alone (Pouriran et al., 2016;Amini et al., 2018;Bagheri et al., 2018;Fridoni et al., 2019;Kouhkheil et al., 2019). In fact, some studies regarding wound healing in diabetic and infected wounds showed that PBM+MSC-CM application accelerated the process of wound healing, while it did not happen when using MSC-CM alone (Pouriran et al., 2016;Fridoni et al., 2019;Kouhkheil et al., 2019).
Several routes of administration have been also tested. Topical application and subcutaneous injection of CM were the most common routes of administration, and they are also the least invasive ways, appearing as promising routes for future research on MSC-CM.
No adverse events were reported to be related to MSC-CM in either preclinical or clinical trials. This is one of the main advantages of using MSC-CM instead of CM, as adverse events related to cells such as hyperimmunogenicity or tumorigenicity can be avoided (Gunawardena et al., 2019b). Only three studies evaluated differences between MSCs and MSC-CM, showing similar results between them (Tamari et al., 2013;Zhang S. et al., 2020). Similar effectiveness was observed between UC-MSCs and UC-MSC-CM for accelerating wound healing rate. The wound area of UC-MSC and UC-MSC-CM was ≈15% reduced as compared with controls without differences in UC-MSC and UC-MSC-CM Zhang S. et al., 2020). Other research also showed similar epithelialization rate when using MSC or MSC-CM (Tamari et al., 2013). Moreover, it was observed that the effect of autologous AT-MSCs and AT-MSC-CM was similar for treating AD in mouse and higher than in the control group .
It is also important to differentiate MSC-CM from culture media only. MSC-CM is the one in which MSCs have been grown for a period of time and with some requirements, and the culture media only is the media in which we grow the MSC-CM but has not been exposed to the cells. The culture media were used as control in some studies to prove the effectiveness of MSC-CM in wound healing (Jun et al., 2014;Deng et al., 2019;Rong et al., 2019). Higher wound healing rates and faster healing were observed with MSC-CM treatment than DMEM. Moreover, it was observed that hypertrophic scar reduction was higher using MSC-CM than DMEM (Zhang et al., 2015;Liu et al., 2018;Hu et al., 2020). It was also found that the topical application of MSC-CM after laser treatment showed less erythema and less pigmentation than the application of DMEM (Zhou et al., 2013(Zhou et al., , 2016. This might mean that the effect observed of MSC-CM in these diseases is due to the secretome and is not being caused by the culture media alone. One of the limitations of MSC-CM is that, to translate its use to patients, it is necessary to know the exact composition of each CM and that its use validation should be conducted for every disease it is to be used for. Moreover, CM needs to be given more frequently than MSC, as the half-lives of cytokines and growth factors are mostly shorter than those of stromal cells, which may survive for rather long periods (Pawitan, 2014). Further research is needed to determine the components of MSC-CM that improves skin diseases. Moreover, regulatory requirements for manufacturing, standardization, and quality control of MSC-CM regarding MSC origin, isolation methods, and culture conditions are necessary to establish the safety and efficacy profiles of these products .
Although there are some reviews regarding MSC-CM (Pawitan, 2014;Mizukami and Yagihashi, 2016;Bogatcheva and Coleman, 2019), they include little information about cutaneous disease. Pawitan et al. included some studies where MSC-CM was used for skin repair and alopecia. Bogatcheva and Coleman only included two articles where MSC-CM was used for skin diseases, one on treating dermatitis and the other on burns. Mizukami and Yagihashi reviewed the status of preclinical studies on stromal cell therapy for diabetic polyneuropathy, but they focused on MSCs and did not include information on other diseases. As far as we know, here, we review the use of MSC-CM in all skin conditions reported in the literature for the first time.
In conclusion, MSC-CM is a promising therapy for skin conditions. Studies on animals showed important rates of wound closure after MSC-CM treatment and clinical studies showed good results for skin rejuvenation. Further studies are needed to corroborate safety and effectiveness and to standardize CM manufacturing.

AUTHOR CONTRIBUTIONS
TM-V and SA-S contributed to conceptualization, formal analysis, investigation, and writing -original draft preparation. TM-V, ÁS-S, and SA-S contributed to methodology. AM-L contributed to software. TM-V, AM-L, and SA-S contributed to validation and visualization. MQ-V contributed to resources. TM-V, ÁS-S, MS-D, and MQ-V contributed to data curation. TM-V, SA-S, ÁS-S, and AM-L contributed to writing -review and editing. All authors have read and agreed to the published version of the manuscript.