Safety and Efficacy of Autologous Melanocyte/Keratinocyte Transplantation in Patients with Refractory Stable Vitiligo

Background: Vitiligo is a common depigmentation skin disease associated with significant psychosocial morbidity and profound effect on the quality of life. The treatment of vitiligo is still a major challenge in the field of dermatology. Currently, topical steroids, calcineurin inhibitors, ultraviolet phototherapy, surgery, and cultured and non-cultured epidermal melanocyte transplantation are used for the treatment of vitiligo. However, the effectiveness of these treatment modalities is limited by the lack of response, long-term treatment periods, high cost, and inevitable adverse effects. Objectives: In this study, we aimed to evaluate the efficacy of intraepidermal injection of autologous non-cultured melanocytes and keratinocytes as an alternative therapy for the refractory and stable (RS) vitiligo. Methods: The treatment procedure was performed on thirty-nine RS vitiligo patients. The autologous skin grafts obtained from the buttock area and epidermis were separated from dermis using dispase. Single-cell autologous melanocytes and keratinocytes were prepared from the epidermis by trypsin/ethylene diamine tetra acetic acid and injected at the concentration of 100–400 × 103 cells/cm2, intra-epidermally to the selected vitiligo lesions. Vitiligo re-pigmentation was monitored employing photography. Photographs were taken prior to and 2, 4, and 6 months after the cell transplantation. Improvement of the skin depigmentation was classified as follows: <25% as minimal response, 26–50% as moderate response, 51–75% as good response, and finally 76–100% as excellent response. Results: Cell infusion appeared to be safe as none of the patients exhibited any adverse effects. At the end of the sixth month follow-up period, of the treated patients, 12.8% demonstrated an excellent response, 36% exhibited a good response, and 51.2% showed a moderate to minimal response to the administered therapy. Obtained significant p value for Wilcoxon test over the checkpoints at 2nd, 4th, and 6th month (p = 0.03, 0.04, and 0.039, respectively) post-cell transplantation confirmed notable growing trend in the re-pigmentation. Conclusion: Our findings provide a strong support for the therapeutic efficacy of autologous non-cultured melanocytes and keratinocytes in patients with RS vitiligo.


Introduction
With a relatively low prevalence rate, vitiligo is an acquired pigmentary disorder of the skin, clinically characterized by the presence of circumscribed depigmented macules resulted from impaired function of melanocytes [1].Amongst multiple theories proposed for unraveling the complex pathogenicity of vitiligo, theories based on the pivotal role of autoimmune and oxidative stress are the most accredited theories as they are in agreement with different clinical and experimental findings [2].Although no specific therapeutic guideline has been established for vitiligo, yet, certain approaches exist which can slow down the progress of the disease and promote pigmentation of depigmented areas.These approaches are mainly based on three strategies including modification of immune responses, suppressing melanocytes stress, and promoting the regeneration of melanocytes [1].Currently, medical and surgical interventions are employed to treat vitiligo [3][4][5].Medical treatments mainly consist of topical or systemic administration of corticosteroids, topical application of calcineurin inhibitors, and ultraviolet (UV) phototherapy (UV-B or psoralen plus UV-A [PUVA]) [3,4].Studies have shown that JAK inhibitors, that target the JAK/STAT pathway, are effective in the treatment of vitiligo [5].Recently, FDA approved Ruxolitinib (Topical) for the treatment of vitiligo patients [6].On the other hand, surgical interventions include autologous suction blister grafting, autologous melanocyte transplantation, and autologous non-cultured epidermal cellular grafting [7].
In a fraction of patients with stable vitiligo, surgical interventions are usually preferred over the medical treatments.This is mainly because these patients respond to medical treatments very poorly and thus, the outcome results are not satisfactory [8].As a recent surgical intervention, transplantation of healthy melanocytes in the depigmented or hypopigmented areas has been considered as a promising approach in the treatment of stable vitiligo [9].In this established treatment modality, the autologous skin is first treated with trypsin and ethylene diamine tetra acetic acid (EDTA) to separate the cells from the epidermal sheath [8,10].Thereafter, the obtained cell suspension is cultured in HU16 medium for 3 days and then treated with Geneticin to deplete keratinocytes and fibroblasts and obtain pure melanocytes [8,10].Finally, the melanocyte suspension is transplanted to vitiligo affected areas [8,10].
Most recently, melanocyte-keratinocyte transplantation (MKTP) or epidermal cell suspension has been introduced as a procedure which can be performed either in cultured or non-cultured forms [3,11].For this latter cell transplantation, cells derived from an autologous dermoepidermal junctions are first trypsinized and suspended in the patient's plasma.Thereafter, the obtained final cell suspension containing keratinocytes and melanocytes is transplanted into the recipients' lesion sites [7].As compared to the melanocyte-cultured procedure, MKTP method is more practical and cost-effective [12,13].Furthermore, the latter procedure is preferable as it provides more successful therapeutic outcomes with higher satisfaction rate particularly, in the treatment of small localized depigmented areas [8].Upon considering the simplicity and effectiveness of MKTP treatment method for vitiligo, this study was designed to evaluate the safety and efficacy of this method on re-pigmentation of patches in patients with refractory and stable vitiligo.

Study Population and Ethical Considerations
A total of 38 subjects (16 male and 22 female) mean age 28.8 ± 11 (range 18-66) years old with stable vitiligo were admitted to the Dermatology Clinic of Helal Iran Pharmaceutical and Clinical Complex (Tehran, Iran) and enrolled in the current study according to the inclusion criteria.

Patients' Enrolment and Data Collection
A detailed questionnaire was completed by physician with interview for all subjects to record demographic data.Subjects were considered eligible for the study if all the following criteria were applicable: age over 18 years old with no active systemic or local infectious diseases (both donating and recipient area of the skin), stable vitiligo lesions for at least 6 months prior to the admission, no medications with immunosuppressive or cytotoxic agents and no treatments with UV phototherapy over the last 6 months, and no previous history of keloid formation.Positivity for viral hepatitis and HIV infections, pregnancy and/or lactation, thyroid dysfunction, any active inflammation, or tumor in the skin was considered as exclusion criteria.The duration of vitiligo varied from 2 to 30 years among included subjects which were divided into three distinct groups based on clinical diagnosis; 19 (50%) subjects diagnosed with generalized vitiligo, 18 (47.4%)with focal vitiligo, and 1 (2.6%) with universal vitiligo, all with a history of not responding to the routine medical treatments.The included patients were followed for the evaluation of re-pigmentation for a period of 6 months post-cell transplantation.Photographic documentation of the lesions of all subjects was performed at different intervals including prior to, and 2, 4, and 6 months after the intervention.The size of donated and transplanted areas and the improved re-pigmented area were assessed utilizing a square lattice grid on tracing paper.Re-pigmented skin surface was defined as less than 25% as minimal response, 26-50% as a mild response, 51-75% as a good response, and finally 76-100% as excellent response.Patients' demographic data are shown in Table 1.In the current study, we pooled both focal and segmental vitiligo and unfortunately, due to inaccessibility to the patients, we are not able to categorize them, separately.This can be considered as a limitation in this study.

Donor Site
Partial-thickness autologous skin grafts were obtained from the thigh-buttock region of every subject due to the low probability of scar formation after grafting, low cosmetic importance and being highly conserved with melanocytes.About one-tenth of the recipient area was irrigated with normal saline after cleansing with ethanol 70% and povidone iodine.Local epidermal anesthetizing was performed by local injection of 1.5-3 mL lidocaine 2%, producing a bumpy-lumpy appearance (Fig. 1a).Using a manual dermatome, a partial-thickness graft with the size of 2 × 2 cm 2 was obtained (Fig. 1b), placed on a normal saline moisturized using a sterile gauze and finally transferred to the laboratory in test tubes containing calcium and magnesium free HBSS plus 5% penicillin/ streptomycin.The donating area of the skin was then covered with non-adherent gauze and examined the next day for performing required managements.At the same day, 5 mL blood was collected from patients to produce autologous serum for cell suspension.

Cell Isolation and Suspension
The isolation and preparation of skin melanocyteskeratinocytes were performed in a class B clean room and under a laminar flow hood.Skin grafts were transferred to 70% ethanol for 30 s to reduce the probability of contamination.Grafts were washed twice with HBSS and then cut into small pieces using a surgical scalpel blade.Skin fragments were then incubated at 4°C in Dispase II solution (Gibco).After 18 h of incubation, tissue was removed from Dispase II solution.Skin fragments were washed with HBSS, and epidermis was separated from dermis using forceps and scalpel blade.Obtained epidermis was incubated for 10 min at 37°C and then exposed to trypsin/EDTA solution (Gibco).Trypsin/EDTA was then inactivated through diluting with HBSS (Gibco).Finally, after centrifugation at 400 g for 10 min at room temperature and disposal of supernatant, obtained pellets were washed with HBSS and subjected for another centrifugation for 10 min at 400 g.Cell viability evaluation was performed for each sample using dye exclusion test.Characteristics of the depigmented patches and the volume of M/K suspension and number of cells have been shown in Table 2. Then cell number and volume were adjusted at 3-4 × 10 6 cell/mL in sodium chloride solution containing 10% autologous serum (obtained from the patients).Product was transferred in cold box (4-8°C) to the clinic, and the cell transplantation was done in less than 1 h (from the cell preparation process).In all cases, cell viability was more than 95%.

Cell Transplantation
The recipient site of the M/K cells was irrigated with normal saline after cleansing with 10% povidone iodine and 70% ethanol.After local anesthesia with 2% lidocaine, intraepidermal injection of the prepared cell suspension was performed carefully by an expert dermatologist using a 30 G needle with slow rotational movement.A portion of 0.05-0.1 mL of cell suspension was injected into each target area (1-4 × 10 5 cell/area) with approximately 0.5 cm intervals (Table 2).With a weak pressure to the injected areas, adjacent areas adjoined thus forming a uniformly swollen surface at recipient site.The target areas of transplantation were kept fully open, and no bandage were used for covering the recipient area.The number of dissociated cells injected to each target area was approximately 100-400 × 10 3 cells/cm 2 .Depending on the size of the patches, injection numbers ranged from 13 times for the largest and 1 for the smallest one.However, the size of treated area, in each patch, was restricted to the number of cells.Meaning that if the dimension of a patch was very large (e.g., 96 cm 2 ) we could not inject the cells to the whole lesion unless cell number was enough).Also, depending on the number and size of patches, the overall time required for completion of each subject's transplantation procedure varied between 5 and 10 min.In total face, forehead, neck, hands were among the treated area in which MKT was done on them.No dermabrasion or blister grafts required prior to the injection, no UV phototherapy was applied after the injections, and no bleeding or leakages were observed after injections.Subjects were discharged after surgical procedure and instructed to return for follow-ups at predetermined intervals.

Patients' Evaluation and Follow-Up
Patients were informed to contact the clinic in case of any problem in injection site(s) within 24 h or later.With the purpose of assessing inflammatory changes and possible side effects at the transplantation site, subjects were referred to dermatologist for their first examination 2 weeks after the transplantation and then examined bimonthly up to 6 months (i.e., at the end of 2nd, 4th, and 6th month posttransplantation) for assessing the status of repigmentation.During each examination, photographs were also taken from the site of transplantation to assess the status of repigmentation.Clinical response was evaluated by an expert dermatologist and was done based on the size of re-pigmentation in treated area.All treated area were photographed before and after

Statistical Analyses
Statistical analyses were performed using SPSS V.20.0,IBM, and descriptive indices including frequency, percentage, mean, and standard deviation were used to express data.The Friedman test was used to compare the status of pigmentation at preset intervals (months 2, 4, and 6).The Wilcoxon test was used for comparing the status of repigmentation two-by-two at the follow-up time points.A p value of less than 0.05 was considered as to be statistically significant.

Results
Re-pigmented skin surface in each follow-up checkpoint were evaluated as described in previous section (Table 3).Except in 3 (7.7%)cases, initiation of repigmentation was evident in transplantation sites at the end of the second month.At this point, 26 (69.2%) and 9 (23.1%) of subjects demonstrated a moderate and mild response, respectively.At the end of the fourth month, skin surface re-pigmentation was apparent in almost 97.4% of subjects ranging from a mild to excellent improvement.This trend of the skin re-pigmentation was more tangible at the end of the 6-month follow-up period since re-pigmentation in all depigmented patches of all subjects was evident.The process of re-pigmentation was also ongoing in cases with moderate or mild repigmentation.An excellent re-pigmentation was reported in 5 (12.8%) subjects and also good, moderate, and mild re-pigmentation were reported in 14 (36%), 16 (41%), and 4 (10.2%)subjects, respectively.Results of the Friedman test confirmed significant improvements at the end of each follow-up checkpoint (p value <0.05).Furthermore, comparison of follow-up checkpoints using Wilcoxon test demonstrated significant improvements during follow-up period (p value <0.05).A significant improvement in re-pigmentation was observed when the progress was compared between 2 versus 4 months

Discussion
Successful surgical approaches as treatment choices for vitiligo have been established for more than half of a century [14].These types of interventions are especially helpful in subjects with stable vitiligo with undesirable effectiveness of existing medications [15].In the cases of stable vitiligo with no response to usual treatments, whole tissue grafting methods including split thickness grafting, punch grafting, suction blister, and cellular grafting techniques are the main surgical approaches.Results of the study performed by Bao et al. [16] in the treatment of stable vitiligo in a mutual selfcontrol study confirmed previously stated [17] superiority of blister roof grafting with 91% improvement in repigmentation as compared to cultured melanocyte transplantation and non-cultured epidermal cell suspension transplantation (NCES) with 82% and 81% re-pigmentation respectively.However, the need for 1:1 donor site to recipient skin size ratio (DR ratio) undermines its superiority against cultured melanocyte transplantation and NCES with 1:20 and 1:5 DR ratio, respectively [16].Thus, in contrast to the whole tissue grafting methods, cellular grafting techniques can be used to treat large, depigmented patches with cells originated from small areas of the donor skin [18].Furthermore, intraepidermal route of administration restricts the scar formation and induction of Kobner phenomenon.
It has been shown that non-cultured melanocytes are able to induce re-pigmentation within 25-30 days [19].Consistent with this finding, we also observed that repigmentation initiated 4 weeks after the transplantation of non-cultured melanocytes/keratinocytes. Together these observations suggest that a time period of 21-28 days is required for transplanted melanocytes to establish themselves at the site of transplantation, initiate proliferation and reach to a certain number at which they become capable of producing required amounts of melanin for re-pigmentation.In this connection, it is of interest to elucidate the role keratinocytes and thereof factors in the initiation and establishment of repigmentation by the grafted melanocytes.
In our study, the maximum response was observed in subjects with focal vitiligo.Consistently, Mulekar et al. [19] reported that the maximum response (95% re-pigmentation) to the autologous M/K transplantation in subjects with segmental vitiligo.Together these findings suggest that noncultured M/K transplantation can be employed as the treatment of choice for patients with segmental vitiligo, a subtype of vitiligo that is refractory to the conventional medical treatment, [20].Obviously, further studies are required to validate this suggestion.
Finally, as transplantations were applied in diverse regions, extent of re-pigmentation was also varied depending on the site of transplantation.This is mainly due to the fact that based on the location of vitiligo; the response to different medications is different.For instance, based on literature, lesions presented on the head and neck are usually the most responsive area to all kind of treatment modalities.Vitiligo lesions on the lips or the tips of the fingers or toes (also mentioned as lip-tip vitiligo) usually fairly respond to the treatments and finally, vitiligo lesions of hands and feet (also referred as acral vitiligo) are the most resistant area to the medications [21][22][23].

Conclusion
In general, the modified MKTP method performed in this study has been introduced as an effective and welltolerated method for the treatment of stable vitiligo.As the process of re-pigmentation was ongoing at the end of the 6 months of follow-up period, better outcomes may be

Key Message
Autologous melanocyte/keratinocyte cell transplantation is safe and could effectively treat resistant vitiligo.

Statement of Ethics
The study protocol was reviewed and approved by the Ethics Committee in the Research of Avicenna Research Institute (Tehran, Iran) (reference number: 93/5984) in conformity with the Declaration of Helsinki.All participants were informed in detail about the procedure of treatment and signed written informed consents prior to the enrolment in the study.

Table 1 .
Demographic and clinical data of study subjects

Table 2 .
Characteristics of the depigmented patches and the volume of melanocyte-keratinocyte suspension and number of injected cells

Table 3 .
Evaluation of re-pigmentation rate after melanocyte-keratinocyte transplantation procedure at different check points of follow-up period (months 2, 4, and 6) Autologous Melanocyte Transplantation for Vitiligo Treatment predictable with further follow-up as seen in similar studies.Subjects with all types of focal, general, and universal stable vitiligo may benefit from this approach especially in cases where other medicinal interventions were shown to be ineffective.