Human placental mesenchymal stromal cell‐derived exosome‐enriched extracellular vesicles for chronic cutaneous graft‐versus‐host disease: A case report

1Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran 2Translational Type 1 Diabetes Research, Department of Clinical Research, Steno Diabetes Center Copenhagen, Gentofte, Denmark 3Department of Radiology, Interventional Regenerative Medicine and Imaging Laboratory, Stanford University School of Medicine, Palo Alto, California, USA 4Bone Marrow Transplantation Department, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran 5Department of Dermatology, School of Medicine, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran


| BACKG ROU N D
Allogeneic peripheral blood stem cells transplantation (PBSCT) is now among the treatments for different haematological diseases and malignancies such as acute myeloid leukaemia (AML). 1,2 Although this treatment is very advanced, graft-versus-host disease (GVHD) is still the most common and severe side effect. 3,4 Considering the time course, GVHD can be divided into two forms: acute and chronic. 4,5 The cumulative incidence of chronic GVHD (cGVHD) during the first year of transplantation is as high as 43.8 ± 10% among recipients of allogeneic marrow transplantation. 6 Moreover, studies have shown a 57% cumulative 5-year incidence of cGVHD in patients who have undergone haematopoietic stem cell transplants from their siblings. 7 Clinically, cGVHD involves mainly skin, lung, eye, liver and musculoskeletal system, 4,8 in which the cutaneous type is the most prevalent and earliest exhibiting type. According to the studies, the cutaneous cGVHD is associated with an inflammatory state in which anti-inflammatory agents are the most important treatment option. 5 Human mesenchymal stromal cells (hMSC) have been used to treat many inflammatory diseases/disorders in the clinic. A recent systematic review and meta-analysis showed that treatment with these cells is not associated with any severe or notable side effects. 9 Exosomes are natural extracellular vesicles released by different cell types and contain proteins, lipids and RNA. These vesicles have been known to participate in intercellular interactions and communications.
Depending on the origin and microenvironment of exosomes, they could play different roles, of which immune modulation is one of the most important ones. 10 Comparing to other sources such as bone marrow or adipose tissue, the placenta is a richer source of stem cells. Since in pregnancy, there is a 'tolerated allograft' in which the placenta is the immunoregulatory organ in this process, this tissue might be a better source in the allogeneic stem cell source. This issue has been shown in the xenotransplantation of this tissue, which resulted in low immunogenicity in immunocompetent animals. Furthermore, placenta cells lack MCH class II antigens, which are responsible for allograft rejection. These and many other reasons make the This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. placenta an immune privileged tissue to isolate mesenchymal stem/ stromal cells. 11 This study aims to report a case of cutaneous cGVHD who received human placental mesenchymal stromal cells (hPMSC)-derived exosome-enriched extracellular vesicles (EVs) as a treatment option.

| Cell isolation
hPMSCs derived from human placenta (single donor) tissue were isolated and identified by the method has been described by Pelekanos et al. 12 Also, the donor's blood sample was negative for viral infec-

| Exosome purification and characterization
hPMSCs passages 3-6 were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% Human AB serum (mycoplasma, CMV, HBV, HCV and HIV tests performed by PCR), 100 U/ml penicillin and 100 µg/ml streptomycin and incubated at 37°C in 5% CO 2 . Cells were grown to 70%-80% confluency, washed three times with PBS and incubated for 48 h in serum-free media (low-glucose DMEM/F12). In the presence of a cellular density of 80%, the hPMSCs-conditioned media were harvested every 48 h.
To sterilize the supernatants and remove larger EVs, the conditioned media were passed through 0.22 mm filter membranes and centrifuged at 700 g for 10 min at 4°C to remove cell debris. Another round of centrifugation was performed at 9000 g at 4°C for 30 min, and the supernatant was collected again. Exosomes were isolated by ExoEasy Maxi kit (76064; Qiagen) and resuspended in phosphatebuffered saline (PBS). Residual polyethylene glycol and soluble proteins that might have been coprecipitated were removed, pellets were washed with 0.9% NaCl and reprecipitated at 100,000 g at 4°C

| C A S E PR E S E NTATI O N
The  Table 1).
The exosomes-enriched EVs were isolated from placental-  Table 1. The patient was followed for 5 months, and the mentioned changes remained sustained.

| DISCUSS ION
This study aimed to evaluate the potential of treatment with hPMSC-derived exosomes-enriched EVs on a previously known case of cutaneous cGVHD. It was shown that the mentioned treatment could decrease the signs and symptoms caused by cutaneous cGVHD, specifically hyperpigmentations and ulcers caused by skin dryness. Also, the cutaneous inflammation decreased significantly and was more evident than other manifestations due to the antiinflammatory potential of the treatment.
As shown in Table 1, monocytes have been decreased from 18% to 5%, which is clinically significant. As mentioned, the patient was resistant to the many treatment options, including the corticosteroids. It has been shown that donor monocytes could be involved in the pathogenesis of GVHD. In patients diagnosed with GVHD, it has been shown that the intermediate CD14 ++ CD16 + monocytes could promote the induction of a subset of Th17 glucocorticoid resistance cells. 13 Thus, it seems that our intervention was able to reduce this effect in our patient who did not respond to corticosteroid therapy.

Human mesenchymal stromal cells have been used to treat
GVHD in patients undergoing haematopoietic stem cell transplantation. Bonig et al. have used these cells to treat cases with acute refractory GVHD (R-aGVHD). In their study, 92 patients diagnosed with R-aGVHD were enrolled in which two-thirds were therapyresistant patients, and one-third were only steroid refractory cases.
The authors declared that no toxicity was observed following the intervention (median of three doses). It was shown that the overall response rate was 82% in the first evaluation. 14 The only case of GVHD treatment by exosome therapy has been explained by Kordelas

| CON CLUS ION
Although many studies have used mesenchymal stem cells in clinics, data on using MSCs exosome therapy for GVHD are extremely limited. The experience of our team with hPMSC exosome-enriched EVs therapy on the described cutaneous cGVHD patient was clinically successful, making it a potential treatment for this pathology.
However, further complementary studies and trials are strongly suggested.

ACK N OWLED G M ENT
The authors are grateful for the kind permission from the patient for publishing the data.

CO N FLI C T O F I NTE R E S T
The authors declare no actual or potential conflict of interest related to this study. Writing -original draft (equal); Writing -review & editing (equal).

CO N S E NT
The patient signed a consent form freely and agreed for us to publish his records and receive the interventions after explaining the aims and methods of study according to his level of education and knowledge.