Autologous Melanocyte Transplantation Research Articles

Dermabrasion Versus Microneedling in Transplantation of Autologous Noncultured Melanocyte-Keratinocyte Cell Suspension in Patients With Vitiligo.

Autologous noncultured melanocyte-keratinocyte transplantation is one of the procedures used to treat stable vitiligo with varying reported results. Recipient site preparation is one of the variables that could affect repigmentation outcomes. To assess the effectiveness of transplanting autologous melanocyte-keratinocyte suspension in patients with stable vitiligo and to compare recipient site preparation using dermabrasion versus microneedling. From March 2020 to September 2022, this randomized comparative study included 40 patients with 40 stable vitiligo lesions managed by suspension transplants of melanocytes. Patients were divided into 2 groups: group A, where the recipient site was prepared using dermabrasion, and group B, which was done by microneedling. The assessment was performed 3 months after the treatment based on the degree of repigmentation (excellent, ≥90%; good, 50%-89%; fair, 20%-49%; and poor response, <20%). Both modalities resulted in effective repigmentation, but the dermabrasion group showed a statistically significant improvement and a satisfactory repigmentation rate. Autologous melanocyte transplantation is a safe and effective treatment method for stable vitiligo lesions that have not responded to other therapies. When compared with microneedling, dermabrasion produced better outcomes for recipient site preparation.

One-Year Follow-Up of Vitiligo Patients Treated with Autologous Non-Cultured Melanocytes

Background: Vitiligo is a long-term multifactorial polygenic disorder, characterized by the patchy loss of pigments in the skin. Several treatments including therapeutic creams and oral drugs are used to treat vitiligo with varying degrees of success. Some medical treatments can reduce the severity of the disease, but it is difficult to cure the disorder. Autologous non-cultured melanocyte transplantation is an effective method of vitiligo treatment. The utilization of appropriate cell suspension is a safe and efficient strategy to cure such hypopigmentation disorder. Objectives: The aim of this study was to follow up patients suffering from generalized and stable vitiligo who were treated with transplanted cells. Patients with patches in four different parts (forehead, eyelids, trunks, and hands) were selected because skin thickness varied among different body parts. We compared melanin repigmentation in these areas. Methods: We recruited 39 patients with generalized and stable vitiligo who had patches on their forehead, eyelids, trunks, and hands. Partial grafts were taken from the gluteal regions of all patients. Epidermal cells including non-cultured melanocyte and keratinocyte suspensions were enzymatically isolated and found to be of > 98% viability. Cells were injected intraepidemally. After a 12-month follow-up, repigmentation was observed. Results: The mean repigmentation score continued to improve up to 12 months post-transplantation. The obtained results confirmed that the cellular suspension that consisted of a mixture of epidermal cells improved to restore the normal color of the repigmentation rate. The number of received cells per cm2 positively influenced the repigmentation score. Patches located on the face, neck, and trunk areas showed significantly higher responses to treatment. The pigmentation score was classified as “poor” (1% - 25%), “moderate” (26% - 50%), and “good” (51% - 75%). This study is a research and clinical study with a brief report registered with the Ethics Committee of Avicenna Research Institute, clinical trials (letter number: 93/22/01/89), Tehran, Iran. Conclusions: The application of autologous non-cultured melanocyte-keratinocyte cell suspension could restore the patchy skin color to a near-normal level and the majority of the patients were satisfied with the results.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Efficacy of Cyclosporine After Autologous Noncultured Melanocyte Transplantation in Localized Stable Vitiligo-A Pilot, Open Label, Comparative Study.

Understanding the pathogenesis of vitiligo has lead to innovation of new drugs and new uses of the existing drugs to enhance treatment outcome. The aim of this observational pilot study was to assess the role of cyclosporine (CsA) to tackle the commonest aesthetic problem "perilesional halo" after autologous noncultured melanocyte-keratinocyte cell transplant (NCMKT) for localized, stable vitiligo. Of the total 50 enrolled patients who underwent NCMKT for stable/resistant vitiligo, aged 12 to 68 years (mean 29.92 years), 18 were male and 32 were female. Group I (n = 25) patients did not receive any postoperative treatment. Group II (n = 25) patients received CsA postoperatively at 3 mg·kg·d for 3 weeks followed by 1.5 mg·kg·d for 6 weeks. In Group I, results were as follows: 28% (n = 7) achieved >75% repigmentation, 16% (n = 4) achieved 50% to 75% repigmentation, 52% (n = 13) achieved 25% to 50% repigmentation, and 4% (n = 1) achieved <25% repigmentation. In Group II, 100% (n = 25) achieved >75% (median 90.7%) repigmentation post-NCMKT at the end of 6 months. This new drug regimen using CsA resulted in rapid and uniform repigmentation without leaving any perilesional halo in Group II patients after NCMKT.

Efficacy of Autologous Melanocyte Transplantation on Amniotic Membrane in Patients With Stable Leukoderma: A Randomized Clinical Trial.

Efficacy of Autologous Melanocyte Transplantation on Amniotic Membrane in Patients With Stable Leukoderma: A Randomized Clinical Trial.

Role of Recipient-site Preparation Techniques and Post-operative Wound Dressing in the Surgical Management of Vitiligo.

Vitiligo is an acquired skin disorder characterized by the destruction of melanocytes resulting in achromic macules and patches involving the affected skin. Multiple methods of treatments have emerged to manage vitiligo, including medical and surgical techniques. Among the surgical techniques described in the management of vitiligo are minipunch grafting, split-thickness skin grafting, hair follicle transplantation, suction blister grafting, and cultured and non-cultured autologous melanocyte transplantation. However, prior to grafting optimal recipient-site preparation is needed for graft survival and successful repigmentation outcomes. Similarly, post-operative care of the recipient site is vital to yielding a viable graft irrespective of the transplantation technique employed. This article reviews the multiple methods employed to prepare the recipient site in vitiligo surgeries and the post-surgical conditions which optimize graft viability. A pubmed search was conducted utilizing the key words listed below.

Potential of regenerative medicine for treatment of vitiligo patients

The article presents a review of publishes sources on the efficacy of methods such as tissue engineering and cellular transplantation of autologous melanocytes for treatment of vitiligo patients. The article describes general principles of treatment and particular features of current melanocyte transplantation methods.

Autologous melanocyte transplantation for the treatment of vitiligo in patients with abnormal thyroid function:a clinical observation

Objective To evaluate the therapeutic efficacy of autologous melanocyte transplantation for the treatment of vitiligo in patients with abnormal thyroid function.Methods A total of 60 patients with vitiligo were enrolled in this study,including 30 with abnormal thyroid function and 30 without.Epidermal sheets were obtained by suction blister biopsy from the normal skin of all the patients followed by melanocyte isolation and culture.After 2-5 passages of subculture,the melanocytes were transplanted onto vitiliginous lesions,which were abraded previously by ultra-pulsed CO2 laser,in the corresponding patients.All the patients were followed for 6-12 months.Results Of the 30 patients with abnormal thyroid function,7 patients achieved more than 90％ repigmentation,9 patients 50％-89％ repigmentation,53.3％ more than 50％ repigmentation,with the average repigmentation rate being 47％ within 6 months after the transplantation.Meanwhile,13 out of the 30 patients without abnormal thyroid function showed more than 90％ repigmentation,11 showed 50％-89％ repigmentation,with the average repigmentation rate being 75％.Both the cure rate and response rate were significantly higher in the patients without abnormal thyroid function than in those with (cure rate,43.3％ vs.23.3％,P＜ 0.05; response rate,80％ vs.53.3％,P＜ 0.05).Significant differences were also found in the response rate for lesions on the face or neck and for those sized more than 20 cm2 between the two groups of patients (both P ＜ 0.05).The lesions transplanted with epidermal melanocytes from the waist exhibited the lowest cure rate and response rate.Conclusion Clinical or subclinical thyroid dysfunction may have a negative impact on the efficacy of autologous melanocyte transplantation in vitiligo. Key words: Vitiligo; Melanocytes; Autologous transplantation; Thyroid

A Randomized Controlled Study of the Effects of Different Modalities of Narrow-Band Ultraviolet B Therapy on the Outcome of Cultured Autologous Melanocytes Transplantation in Treating Vitiligo

Vitiligo is an acquired skin disorder with great social impact. It can be successfully treated using cultured autologous melanocytes transplantation. To evaluate the effect of different modalities of narrow-band ultraviolet B (NB-UVB) therapy on the outcome of cultured autologous melanocyte transplantation in treating vitiligo. Patients undergoing cultured autologous melanocyte transplantation were randomly assigned to four different study groups. Group 1 underwent 20 sessions of NB-UVB treatment before transplantation; Group 2 underwent 30 sessions of NB-UVB treatment after transplantation; Group 3 underwent 20 sessions of NB-UVB treatment before transplantation and 30 sessions after transplantation; Group 4 underwent only transplantation. Four hundred thirty-seven patients were enrolled. Group 3 responded best, more than 90% repigmentation was achieved in 81.3% of patients, and 94.8% patients experienced 50% or greater repigmentation. Statistical analysis showed that there was a highly significant difference between the four groups (χ(2) = 35.56, p < .001). Homogeneous skin color was obtained on the repigmentation areas, and no scarring or other serious side effects were observed. Cultured autologous melanocyte transplantation is an effective treatment for stable vitiligo. Combination of NB-UVB therapy with melanocyte transplantation can accelerate repigmentation of transplanted vitiliginous areas, especially if NB-UVB is given before and after transplantation.

Dermal Mesenchymal Stem Cells (DMSCs) Inhibit Skin-Homing CD8+ T Cell Activity, a Determining Factor of Vitiligo Patients’ Autologous Melanocytes Transplantation Efficiency

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients’ autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.

Generation of Melanocytes from Induced Pluripotent Stem Cells

Epidermal melanocytes play an important role in protecting skin from ultraviolet (UV) rays, and are implicated in a variety of skin diseases. Here, we developed an efficient method for differentiating induced pluripotent stem cells (iPSCs) into melanocytes. We first generated iPSCs from adult mouse tail-tip fibroblasts (TTFs) using retroviral vectors or virus-free piggyBac transposon vectors carrying murine Sox2, Oct3/4, cMyc and Klf4. The TTF-derived iPSC clones exhibited similar morphology and growth properties as mouse embryonic stem (ES) cells. The iPSCs expressed ES cell markers, displayed characteristic epigenetic changes and formed teratomas with all three germ layers. The iPSCs were used to generate embryoid bodies (EBs) and were then successfully differentiated into melanocytes by treatment with growth factors. The iPSC-derived melanocytes expressed characteristic melanocyte markers and produced melanin pigment. Electron microscopy showed that the melanocytes contained mature melanosomes. We manipulated the conditions used to differentiate iPSCs to melanocytes and discovered that Wnt3a is not required for mouse melanocyte differentiation. This report shows that melanocytes can be readily generated from iPSCs, providing a powerful resource for the in vitro study of melanocyte developmental biology and diseases. By inducing iPSCs without viruses, the possibility of integration mutagenesis is alleviated, providing iPSCs are more compatible for cell replacement therapies.

Autologous Noncultured Melanocyte Transplantation for Stable Vitiligo: Can Suspending Autologous Melanocytes in the Patients' Own Serum Improve Repigmentation and Patient Satisfaction?

Vitiligo is a cosmetically disfiguring acquired depigmenting disorder caused by the loss of functional melanocytes from the epidermis. Various approaches that have been used for the treatment of vitiligo can be classified as medical and surgical therapies. Noncultured autologous melanocyte transplantation is a new and effective surgical treatment for stable vitiligo. To compare the repigmentation results in stable vitiligo of transplantation of autologous noncultured melanocytes suspended in normal saline with that of those suspended in the patient's own serum. Twenty-five patients with 36 lesions of stable vitiligo were randomized into two groups for noncultured melanocyte transplantation. Patients in Group A received melanocytes suspended in normal saline, and those in Group B received melanocytes suspended in their own serum. Statistically significant difference in repigmentation results and reduction in Dermatology Life Quality Index (DLQI) score was observed between the two groups 16 weeks after surgery. Repigmentation results were excellent (>90%) and very good to excellent (>75%) in 44.4% and 66.7% of lesions, respectively, in Group A and 88.8% and 94.4% of lesions, respectively, in Group B. There was also a significant (p=.002) decline in DLQI score in both groups, with the mean reduction being significantly greater in Group B than Group A (p=.005). Results of noncultured melanocyte transplantation can be improved significantly more by suspending the melanocytes in the patients' autologous serum than in normal saline. This could be an important innovation in the surgical management of patients with stable vitiligo.

Autologous melanocyte–keratinocyte suspension in the treatment of vitiligo

In stable vitiligo, several techniques of autologous transplantation of melanocytes are used. Autologous melanocyte transplantation of non-cultured melanocytes is one of those techniques with variable reported outcomes. The objective of this study was to evaluate the response to autologous melanocyte-keratinocytes suspension transplantation in cases of stable vitiligo. A total of 25 cases of vitiligo were treated by autologous melanocyte-keratinocytes suspension transplantation. After 6-17 months, patients' response was evaluated according to the extent of pigmentation (excellent 90-100%, good 50-89%, fair 20-49% and poor response <20%). Of the 25 patients treated, 22 continued the follow-up period. Five (23%) patients showed excellent response, 7 (32%) good, 6 (27%) fair and 4(18%) showed poor response. Unlike transplantation of cultured melanocytes, which requires experience in culture technique, autologous melanocyte-keratinocytes suspension transplantation is an easy economic technique, which may be used in resistant areas of stable vitiligo.

Amniotic Membrane as a Scaffold for Melanocyte Transplantation in Patients with Stable Vitiligo

Vitiligo is an acquired skin disease that significantly impacts the quality of life of patients. Medical treatment of vitiligo includes the use of melanocyte transplant, but the results are variable. We have treated 4 patients with either focal or generalized stable vitiligo using a graft of autologous melanocytes' culture on a denuded amniotic membrane (AM). A culture biopsy was obtained in every patient and grown in melanocytes' media for 10–14 days after which cells were transferred to a denuded AM and transplanted into the achromic lesions. Patients were followed for up to 6 months using clinical assessment of achromic lesions. Treated areas ranged between 4 cm2 and 210.6 cm2. Response to treatment was excellent in all patients with 90–95% repigmentation success rate. Our results demonstrate that transplantation of autologous melanocytes cultured on AM is a new, simple, and effective treatment for stable vitiligo.

Transplantation of individualized cultured autologous melanocytes for the treatment of vitiligo.

Objective To evaluate the therapeutic effect of transplantation of autologous melanocytes cultured with individualized medium in vitiligo. Methods Donor skin was obtained by suction blisters from a normally pigmented area of the abdomen of 155 patients with vitiligo. The roof of the blisters was clipped and digested with trypsin, then the suspension of epidermal cells and melanocytes were cultured in Hu16 medium.The cell division time (DOT) and melanin content of cultured melanocytes were measured followed by the adjustment of concentration of fetal calf serum, cytokines and cAMP elevating agents based on the DOT,melanin content and morphology of melanocytes for the individualized culture of melanocytes. After 2 - 5 passages, melanocytes were harvested and inoculated into ultrapluse CO2 laser-denuded lesions. All patients were followed up for at least 6 months. Results One hundred and fifty-five vitiligo patients with 204 lesions were treated with transplantation of autologous melanocytes. Of the 155 patients, 119 received 1 session of transplantation, 36 received 2 to 4 session of transplantation. Cells were expanded by 50 - 80 times in vitro after individualized culture. Repigmentation was more than 50% in 84.8% of these lesions, more than 90% in 52.94% of the lesions. A homogeneous skin color was obtained in repigmented skin, and no scarring or other side effects were observed. No influence was noted on the outcome of transplantation for sex, age, course of disease or lesion size of patients. Segmental vitiligo showed better response than vitiligo vulgaris: the effective rate and cure rate were 93.62% and 65.96% respectively for segmental vitiligo, 82.16% and 49.04% respectively for vitiligo vulgaris. Lesions located on the arms and legs (not including elbows and knees) showed the best response, with a cure rate of 73.08%, whereas acral sites were the most difficult area to repigment, with a cure rate of just 25.93%. Conclusions Individualized culture can significantly increase the success rate of melanocyte culture and expanding times of melanocytes. Transplantation of cultured autologous melanocytes is an effective modality deserving clinical application in the treatment of stable vitiligo, with the advantage of treating large depigmented area with melanocytes from a small donor site. Key words: Vitiligo; Melanocyte; Cell culture techniques; Transplantation

Repigmentation by Outer-Root-Sheath-Derived Melanocytes: Proof of Concept in Vitiligo and Leucoderma

Background: Treatment of depigmented skin is an unmet medical need. Objective: Melanocytes or stem cells thereof residing in the outer root sheath (ORS) of hair follicles might be used to repigment skin. Methods: After de-epidermisation, autologous ORS cell solutions were applied to 5 patients with vitiligo and 1 with leucoderma. Results: Stable repigmentation in a variable percentage was documented in all the patients. Conclusion: Applying ORS-derived melanocytes is a promising technology to improve autologous melanocyte transplantation.

Cultured Melanocytes: From Skin Biopsy to Transplantation

Restoration of cutaneous pigmentation has been achieved in stable vitiligo by autologous melanocyte transplantation. This study was aimed to develop a methodology to deliver melanocytes to vitiliginous area following their processing and culture in a centralized facility. Here we report a methodology to culture melanocytes on carrier films, transport the cells, and graft them on vitiliginous areas. The salient features of this study include: 1) development of polylactic acid (PLA) films that support melanocyte attachment, growth, and delivery; 2) establish transport conditions for skin biopsies from hospitals; 3) establish transport conditions for cultured cells from cell processing center to hospitals. Results suggest that PLA films could serve as carriers for melanocytes during transport. "Upside-down" application of the graft results in the migration of cells from the films into the dermabraded area. The transport conditions ensure cell viability for 96 h. This system could help clinicians, who do not have access to cell culture facilities, transplant cultured melanocytes in a cost-effective manner.

Repigmentation of skin and hairs in stable vitiligo by transplantation of autologous melanocytes in fibrin suspension

Repigmentation of skin and hairs in stable vitiligo by transplantation of autologous melanocytes in fibrin suspension

Human adult epidermal melanocytes cultured without chemical mitogens express the EGF receptor and respond to EGF

We describe a novel chemical mitogen-free in vitro culture technique for obtaining pure melanocyte cultures using normal human adult epidermis as a source. The culture medium consists equal parts of the commercially available Keratinocyte Basal and AIM-V media (both from Gibco), as basal medium, which is supplemented with fetal bovine serum, bovine pituitary extract and recombinant human epidermal growth factor (EGF). Melanocytes harvested from human adult skin proliferate extensively and can be passaged serially up to 10-15 times using this medium. We have verified the identity of the cultured cells by tyrosinase mRNA expression and TRP-1 protein staining. Moreover, we showed that autologous human serum alone, without additional supplements is able to provide sufficient growth support for the cultured cells in the basal medium, making this culture technique suitable for autologous melanocyte transplantation. In this culture system normal human adult melanocytes expressed both EGF receptor (EGFR) mRNA and protein and EGF showed a dose dependent mitogenic effect on the cells. EGF itself had no significant influence on EGFR mRNA expression.

THE EFFECT OF SERUM CONCENTRATION ON THE SPHEROID FORMING ACTIVITY AND CELL GROWTH OF HUMAN MELANOCYTES ON CHITOSAN SURFACE

We have developed a chitosan-based melanocyte patch to improve the drawbacks encountered in autologous melanocyte transplantation for vitiligo treatment by use of melanocyte suspension. Melanocytes spontaneously grow into three dimensional spheroids on chitosan-coated surface and melanocyte spheroids show a better survival in growth factor and serum-deprived condition. Hence, preculturing melanocytes into spheroids on chitosan surface can provide melanocytes a survival advantage in stringent conditions. Up to date, the mechanism of the melanocyte spheroid formation is not clear. It has been shown that certain factors in serum are crucial for multicellular spheroid formation in cancer cell lines. In this work, we investigate the effect of serum concentration on the melanocyte spheroid formation. The trend for melanocyte spheroid formation is gradually decreased when serum concentration is serially increased from 0% to 20%. Our result shows that melanocyte spheroid formation is not inhibited in the absence of serum and serum deprivation accelerates melanocyte spheroid formation. However, melanocyte growth is greatly reduced when serum is completely removed. Our results suggest that melanocyte spheroid formation on chitosan is not dependent on serum factors. However, the presence of serum contributes to the cell growth on chitosan-coated surface. This result is important in the future development of serum free culture system for spheroidal melanocyte patch.