Pigment cell transplantation for treatment of vitiligo: A progress report

Therapv Pigment cell transplantation for treatment of vitiligo: A progress report Werner Lantz, MD,* Mats 1. Olsson, PhD ,** Gisela Moellrnann, PhD, and Aaron B. Lerner, MD, PhD New Haven, Connecticut Background: Because available treatments for vitiligo generally provide unsatisfactory results, the search for viable therapeutic alternatives continues. Objective: Our purpose was to evaluate several transplantation procedures withcultured autologous melanocytes for their practicality in treating patients with vitiligo. Methods: Twenty-seven patients with stable or active vitiligo were treated after superficial dermabrasion with application ofsuspensions of autologous cultured melanocytes, melanocyte-keratinocyte mixtures, or epidermal sheets established in vitro. Results: Regardless of disease activity, use of each method resulted in repigmentation to a similar degree andwithout scarring in all patients. Melanocyte suspensions offer several advantages: They are easily prepared, can be applied in a controlled manner, permit coverage of large areas, and produce a homogeneous skin color that affords the best cosmetic restoration. Theultrastructure of transplant sites resembled that of uninvolved skin, with one exception: the me1anocytes were located slightly higher than in uninvolved skin. Conclusion: Application ofcultured autologous melanocytes to lightly abraded skin is an advantageous addition to the treatments available for patients with vitiligo. (J

AM ACAD DERMATOL

1994;30:591-7.)

Options for the repigmentation of skin of patients with vitiligo have increased slowly but steadily. Nonsurgical modalities include phototherapy (with or without orally or topically administered psoralens) and topically applied steroids of various potencies. Surgical approaches include transplantation of blister tops or minigrafts from normally pigmented areas.!" injection of cultured or uncultured autologous melanocytes into blisters raised by suction, friction, or freezing.e? application of autologous epidermal sheets established in vitro or collagen-backed cultured melanocytes onto skindenuded

Supported by the United States Publ ic Health Service (grant Nos.ROI AR39869 and P30 AR1942), the Eleanor Naylor Dana Cha ritable Trust, and The National Vitiligo Foundat ion.

by dermabrasion'S? or application of liquid nitrogen.10-12 We wanted to use highly expanded cultures of autologous pigmentcellsto repigment largesurfaces ofskin. To thisend we neededculture conditions that supported the proliferationof adult human melanocytes and a highly efficient method of preparing recipient sites for reimplantation of the cultured cells. Both an effective preparatory method (superficial dermabrasion) and highly stimulatory defined media that contain natural melanocyte growth factors such as basic fibroblast growth factor (bFGF) in place ofphorbol ester l3 are now available and have been put to optimal use in repigmentingareas of vitiligo.14• 15 We describe the progress made in this continued effort and compare several ways of replacing melanocytes into lightly dermabraded patches of depigmented skinin patients with vitiligo.

Reprint requests: Aaron B. Lerner, MD, PhD, Department of Dermatology, Yale University School of Medicine, P.O. Box 208059, New Haven, CT 06520-8059.

Study population

From the Department of Dermatology, Yale University School of Medicine.

*Dr. Lentz is currently at the Department of Dermatology, Restock University Faculty of Medicine, 18055 Rostock, F.R.G. **Dr. Olsson is currently at the Department of Dermato\ogy, Uppsala University, Uppsala, Sweden. Ccpyright w 1994 by the American Academy of Dermatology, Inc,

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MATERIAL AND METHODS

The 27 patients, 10 women and 17 men, were chosen at random and ranged in age from 18 to 61 years. The median agewas 41 years. Themedian ageofthe female patients was 7'12 years lower than the median age of the male patients. One patient was black (No.1, Table I),

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Table I. Summary of autologous pigment cell transplantations Patient No.

Degree or repigmentation*

Age

Type/status

Transplanted cultured-ceU

Sex

(yr)

of ritiligo

preparation(s)

1

M M

59 61 26 45 27 22

A

2 3

M, M/E, Es M, M/E, Es MjEs,Es M, Es M,Es M M M M M M M M M, M/E, Es M, M/E, Es M,M/E M,M/E M, Es Es M M M M

Excellent Excellent Excellent Excellent Excellent Excellent Excellent Excellent Excellent Excellent Excellent Good Good Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate

M

Moderate Moderate Moderate Moderate

4 5 6

7 8

9 10 II

M M

F M M F F F M

12 13

M

14 15 16 17 18 19 20 21 22 23

M M F

24

F

M F M M M F M M

26

M F

27

F

25

44 60 22 19 45 28 46 37 42 36

42 41 48 51 25 18 28 47 39

33 59

I A I S

I S I

I I A I A I A I I I I I I I A A A A A

M M M

Moderate Moderate

A, Generalized, active; I, generalized, inaetive;S, segmental; M, melanocyte suspension; M(E, epidermal cell suspension aftercoculture; Es, epidermal sheet aftercoculture; M(Es, melanocytes, covered by epidermal sheet. * Excellent, Complete or nearly complete repigmentation of at least onerecipient site; good, greater than 50% repigmentation; moderate, lesser but definite takes.

another was an Indian (from India) (patient No.4), and a third was a dark-complected Puerto Rican (patient No. 7). The remainder of the patients were white with skin types I to II L Two of the patients had segmental vitiligo, 16 patients had generalized stable vitiligo, and II had generalized, moderately active vitiligo. Most had previously received nonsurgical treatments with limited or no success. None had received treatment within 2 months before or at any time after harvesting of the donor skin. These patients have been followed up postoperatively from 3 to 10 months.

Cell culture A shave biopsy specimen with a surface area of 5 to 7 ern? was taken from normally pigmented, unirradiated buttock skin after local anesthesia with 0.5% lidocaine without epinephrine and placed immediately into PC-ldefined medium (Ventrex Laboratories, Portland, Me.). A Goulian skin graft knife with a "0.008" shield (Edward

Week & Co., Research Triangle Park, N.C.) was used to obtain shaves shallow enough to prevent scarring or permanent hypopigmentation at the donor site. The wound was covered with one layer each of petrolatum-impregnated and dry gauze and secured with Tensoplast (Smith & Nephew, London, United Kingdom). Residual dermis was teased from the epidermis after flotation of the specimen on MEMjO.25% trypsin/O.l % EDTA-4 Na (Sigma Chemical Co., St. Louis, Mo.) for I hour at 37° C, dermal surface down. Melanocytes and keratinocytes were dissociated by vigorous vortexing and seeded in 75 em? flasks, either in a modified melanocyte medium 13 consisting of chemically defined PC-I medium (Ventrex Laboratories) supplemented with 5 ng/rnl bFGF (Promega Corp., Madison, Wis.), 0.5 mmol/L dibutyryl cyclic AMP (Sigma Chemical Co.), 50 U jml penicillin, and 0.1 mgjml streptomycin or in keratinocyte serum-free medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 2.5 ng/rnl bFGF to stimulate melanocyte growth (or, in some cases, endo-

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thelin-I from Boehringer, Ingelheim, Germany) and 5 JLg/mlgentamicin (Whittaker Bioproducts, WalkersvilIe, Md.). Cells in melanocyte medium were cultured for periods from 10 days to 5 weeks, depending on the desired ceII number. These cultures became 80% to 98% melanocytes without use of a phorbol ester or Geneticin to suppress keratinocytes or fibroblasts. Cultures in keratinocyte medium were used to establish stratified epidermal sheets that contained keratinocytes and melanocytes, suitable for direct transfer to graft sites. For this purpose the calcium concentration in the medium was raised by 1.5 X 10- 3 mol/L during the final 48 to 72 hours of incubation, 2 to 3 days after primary or secondary cultures had become confluent.

Preparing cultures for transplantation Melanocytes were released from the culture flasks by exposure to the above trypsin/EDTA solution for -3 minutes at 37° C, pooled, centrifuged into a pellet, washed and repelleted twice in phosphate-buffered saline solution, and resuspended inPC-I medium at I t02 X 1()6 cells/O.l ml, the concentration used for transplantation. Epidermal sheets were detached from the culture surface by changing the culture medium to a solution of dispase (Collaborative Research, Inc., Bedford, Mass.), 2000 caseinolytic U/ml Hanks' balanced salt solution (Sigma Chemical Co.), for periods of 15 to 20 minutes and then lifting the cellular sheet from the culture dish with Delnet (Frastec Wound Care Products, Bronx,

N.Y.).

Fig. 1. Hands of 60-year-old white woman (Table I, patient 8) with extensive but stable vitiligo. A, Before treatment. B, Complete repigmentation to weeks after transplantation of free melanocytes. Ten months later no loss of pigment had occurred.

Preparing recipient sites With the cells or sheets ready at the patient's bedside and the recipient area lightly anesthetized with FluorEthyl Topical Skin Refrigerant (Gebauer Co., Cleveland, Ohio), vitiligo patches were superficially abraded at 25,000 revolutions per minute with an ASEPTICO AEU-17 dermabrader (Bersco Surgical Supply, Inc., Seattle, Wash.) fitted with regular diamond fraizes. The equipment contains tools appropriate for use on the largest practical areas down to the narrowest crevices.

Seeding recipient sites Pigment celIs were transplanted onto the denuded areas as melanocytes grown in melanocyte medium and applied free in suspension to a density of ~ 1500/mm2 (M, Table 0, or in suspension with keratinocytes after coculture of epidermal cells in the supplemented keratinocyte medium (M/E), or by being integrated into epidermal sheets (Es), or by being integrated into epidermal sheets that were applied immediately after application of free melanocytes, 500 cells/mm? (M/Es).

Cells in suspension were dropped onto the denuded surface from a 1 ml syringe without a needle and were spread with a spatula. The transplant was secured with Dacron netting (Millipore Corp. , Bedford, Mass.) and bandaged (description to follow). Epidermal sheets were cut to fit the vitiligo patches and were applied to the denuded skin, Delnet-side up. Regardless of the transplantation method used, the treated areas were covered with minimally overlapping 4 em 2 pieces of gauze previously soaked in medium (PC-I medium for transplants of suspended melanocytes and keratinocyte medium for all others), followed by an occlusive dressing (Bioclusive, Johnson & Johnson Medical, Inc ., Arlington, Tex.). To assure the best possible "take," the patient should remain as still as possible in bed for 8 to 10 hours, treated joints splinted and the patients fed and provided with a bedpan by others. After I week the occlusive dressing was changed to a dry dressing for an additional 5 to 7 days until reepithelialization was complete, as indicated by the sloughing of the Dacron or Delnet covers.

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Fig. 2. Abdomen and flank of 27 -year-old white woman (Table I, patient 5) with segmental vitiligo. A, Before treatment. B, Variegated area on left was photographed 6 weeks after transplantation of epidermal sheets prepared from epidermal-cell cocultures in the presence of melanocyte growth factors. Slightly hyperpigmented patch on right is pictured 5 weeks after transplantation of free melanocytes. The hyperpigmentation has since subsided without further loss of pigment.

Fig. 3. Flank of 59-year-old black man (Table I, patient 1) with active vitiligo. A, Before treatment. B, The three sites 6 weeks after transplantation of free melanocytes, epidermal sheets, or epidermal sheets over suspended melanocytes. Some repigmentation was evident as early as 6 days after transplantation. Photograph shows a slight hyperpigmentation, which had subsided 6 months later. There has been no further loss of pigment.

Electron microscopy

enriched epidermal sheets. In seven patients both methods were applied to patches on equivalent body sites (Table I, Figs. 2 and 3). Some patients were treated with mixed melanocyte-keratinocyte suspensions and others with melanocytes plus melanocyte-enriched epidermal sheets. The mixed-cell and sheet preparations were abandoned in favor of melanocyte suspensions relatively early in the course of this study because they were more cumbersome to prepare without providing better results and yielded less melanocyte amplification. Although melanocytes of patients who were older than 45 years appeared to have a somewhat slower rate of proliferation, at least 5 million cells were obtained from all patients regardless of age, race, or disease activity and irrespective of a past history of UV irradiation. In several patients more than 15

In four patients (Nos. 1, 2, 13, and 20, Table 1) a transplantation site was examined with the electron microscope approximately I lh, 5,4 1/2, and 31,,2 months after transplantation, respectively. In each case one 3 mm punch biopsy specimen was fixed in a buffered mixture of paraformaldehyde (2%) plus glutaraldehyde (2.5%) and then osmium tetroxide (1%) plus potassium ferrocyanide (1.5%), dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were counterstained with lead citrate and viewed in a Hitachi HU-I1BI electron microscope. RESULTS

In 27 patients more than 60 patches of different sizes were treated, most with melanocyte suspensions (Table I, Fig. 1), and nine with melanocyte-

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Fig. 4. Transmission electron micrograph from melanocyte transplant site on finger of patient No. 20, an olive-complected, readily tanning white patient with bright blue eyes. All nuclei in this micrograph belong to basal keratinocytes; beginning of papillary dermis is shown in lower left corner. Micrograph includes a melanocyte (M), three melanocytic dendrites (arrows), and incipient perimelanocytic degeneration of a keratinocyte (asterisk), which is typical in vitiligo.!? Age of transplant at time of biopsy was 3Y2 months. (X6237.)

million melanocytes were derived from a single biopsy specimen and used for transplantation, which was consistent with an estimated greater than 100fold expansion in pigment cell number through culturing. An end point of proliferative activity was reached at 5 to 6 weeks, when the cells appeared to have differentiated irreversibly. Nevertheless, grafting these cells did not diminish success per se. It remains to be seen whether repigmentation persists as long in these sites as in other graft sites. All patients but not necessarily all recipient sites had at least some repigmentation. Pigment started to appear as early as 6 days after transplantation in a dark-skinned patient (Fig. 3) and reached its final extent after 3 months. Whether melanocytes were transplanted in suspension or as part of epidermal sheets, the degree of repigmentation was dependent mainly on body site. Areas on the trunk, face, and back of the hand showed good to excellent results, followed by arms and legs, which had only moderate repigmentation. Elbows and fingers were initial problem areas and were more difficultto repigment than other areas. These differences were seen across

the entire patient population and in individual patients treated at multiple sites. I n the four patients in whom a transplantation site was examined with the electron microscope, the epidermis was pigmented uniformly and according to the individual patient's skin type (Fig. 4). Nevertheless, abnormal ultrastructural characteristics of "uninvolved" skin of patients with vitiligo'< 17 were evident at some of the healed and fully pigmented transplantation sites. The melanocytes, however, were located somewhat higher among the basal keratinocytes in the positions in which they occur in spontaneously or PUVA-mediated repigmenting skin, solar lentigines, and embryonic skin (G. Moe1lmann, K. Holbrook, unpublished observations). DISCUSSION

Whereas all the early techniques of placing autologouspigment cellsinto leukodermic sitesbrought encouraging results, l-12 in most studies, including those from this laboratory, the transplantations were cautiously restricted to patients with stable disease, and the homogeneous repigmentation of extensive

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surfaces continued to present problems. We have largely overcome these limitations. We have been able to cultivate melanocytes from small shave biopsy specimens in numbers sufficient to cover surfaces up to 180 cm2, even from patients who had progressive vitiligo at the time of harvesting or transplantation. As a method of preparing the recipient site, superficial abrasion is rapid, uncomplicated, allows the denuding of large areas and relatively inaccessible small lesions with fine control over the margins, and provides what appears to be a fertile growth surface for the transplanted cells. Both melanocyte suspensions and epidermal sheets provided sufficient numbers of pigment cells to the leukodermic areas, but in most cases the grafting of free melanocytes produced superior, often cosmetically perfect results. The major factor that determined repigmentation independent of the method of transplantation was not age or disease activity but the anatomic location of the recipient site. This was particularly evident when cells from the same biopsy specimen were used to treat different body areas in one session. Aside from the face and neck, which are hospitable to implanted pigment cells, the comparative success of repigmentation at different body sites seen in this study can be summarized as follows: trunk (11 sites) > legs/arms (I8 sites):::z hands/feet (21 sites) > fingers/elbows (5 sites). Fingers (especially the knuckles) and elbows were the most difficult areas to repigment, in part because of the relative uncertainty in controlling the depth of abrasion of these more heavily cornified areas and in part because of the high mobility of skin covering these joints. Resurfaced joints require strict immobilization, which was not assured in this series. The attention brought to these problem areas in later transplantations has provided satisfactory results. A minor side effect was hyperpigmentation of the transplantation site that faded in several months, ending in a good match with the patient's normal skin color. The overshoot may be caused by a carryover of hyperactivity of the transplanted cells from culture or an oversupply of growth factors and melanogenic peptides during wound healing such as bFGF, mast cell growth factor, and endothelin-l , all of which are melanogenic melanocyte growth factors elaborated by proliferating or basal keratinocytes. 18-21 The confirmation that vitiligo melanocytes have rather normal growth characteristics in culture/? and the finding that disease stabilization is not a

Journal of the American Academy of Dermatology April 1994

prerequisite in determining success of transplantation suggest that vitiligo is a condition with cyclic time-limited activity confined primarily to progressively depigmenting areas that, after abrasion, will sustain a healthy, autologous population of transplanted melanocytes. Only long-term follow-up will disclose whether the repigmentation and the associated higher position of the transplanted melanocytes among the nourishing basal keratinocytes are permanent. From our combined experience with more than 40 patients, of whom 10have been followed up for more than 2 years,14, 15 we are confident that autologous pigment cell transplantation after culture into lightly dermabraded skin is a viablemodality in the therapy of vitiligo, is safe and efficient, and can be used even in patients experiencing active depigmentation in whom conventional therapeutic approaches have failed. REFERENCES I. Falabella R. Repigmentation of leukoderma by autologous epidermal grafting. 1 Dermatol Surg OnooI1984;10:13644. 2. Suvanprakorn P, Dee-Ananlap S, Pongsomboon C, et a1. Melanocyte autologous grafting for treatment of leukoderma. J AM ACAD DERMATOL 1985;13:968-74. 3. Koga N. Epidermal grafting using the top of suction blisters in the treatment of vitiligo. Arch Dermatol 1988; 124:1656-8. 4. Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol SurgOncoll986;12:5l421. 5. Lerner AB, Halaban R, Klaus SN, et al. Transplantation of human melanocytes. 1 Invest Dermatol 1987;89:21924. 6. Lerner AB, Halaban R, Leffell D. Melanocytes in culture from patients with disorders of hypopigmentation. In: Program abstracts of the Fourteenth International Pigment Cell Conference. Kobe, Japan: The International Pigment Cell Society, 1990:100. 7. Gauthier Y, Surleve-BazeiIle lE. Autologous grafting with noncultured melanocytes; a simplified method for treatment of depigmented lesions. J AM ACAD DERMATOL 1992;26:191-4. 8. Brysk MM, Newton RM, Rajamaran S, et a1. Repigrnentation of vitiliginous skin by cultured cells. Pigment Cell Res 1989;2:202-7. 9. Zachariae H, Zachariae C, Deleuran B, et a!. Autotransplantation in vitiligo: treatment with epidermal grafts and cultured melanocytes. Acta Derm Venereol (Stoekh) 1993; 73:46-8. 10. Plott R, Brysk MM, Newton RC, et a1. A surgical treatment for vitiligo: autologous cultured-epithelial grafts. J Dermatol Surg OnoolI989;15:1161-6. 11. Falabella R, Escobar C, Borrero L Transplantation of in vitro-cultured epidermis bearing melanocytes for repigrnenting vitiligo. J AM ACAD DERMATOl 1989;21 :257-64. 12. Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured

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epidermal autografts bearing melanocytes. 1 AM ACAD DERMATOL 1992;26:23~. 13. Halaban R, Rubin1S, FunasakaY, et a1. Met and hepatocyte growth factor/scatter factor signaltransduction in normalmelanoeytes andmelanoma cells. Oncogene 1992;7: 2195-206. 14. Olsson MJ, Juhlin L. Melanocyte transplantation in vitiligo. Lancet 1992;340:981. 15. Olsson MJ, Juhlin L. Repigmentation ofvitiligo by transplantation ofcultured autologous melanocytes. Acta Derm Venereol (Stockh) 1993;73:49-51. 16. Moellmann GE, Klein-Angerer S, Scollay DA, et a1. Extracellular granular material and degeneration of keratinocytes in the normally pigmented epidermis of patients with vitiligo. J Invest Dermatol 1982;79:321-30. 17. Bhawan J, Bhutani LK. Keratinocyte damagein vitiligo. J Cutan PathoI1983;10:207-12.

Stiller et al. 18. Halaban R, Langdon R, BirchallN, et a1. Basic fibroblast growth factorfromhuman keratinocytes is a natural mitogen for melanocytes, J Cell Bioi 1988;107:1611-9. 19. Scott G, StolerM, Sarkar S, et al, Localization of basicfibroblastgrowth factormRNA in melanocytic lesions byin situ hybridization. J InvestDermatoI1991;96:318-22. 20. Longley BJ, Morganroth GS, Tyrrell L, et a1. Alteredmetabolism ofmastcellgrowthfactor (c-KIT ligand)in cutaneous mastocytosis. N Engl 1 Med 1993;328:1302-7. 21. Yohn11,Morelli 1G, WalchakSJ, et al. Cultured human keratinocytes synthesize and secreteendothelin-l. J Invest Dermatol 1993;100:23-6. 22. MedranoEE, Nordlund JJ. Successful culture ofadult humanmelanocytes obtained fromnormaland vitiligo donors. J InvestDennatol 1990;95:441-5.

A double-blind, placebo-controlled clinical trial to evaluate the safety and efficacy of thymopentin as an adjunctive treatment in atopic dermatitis Matthew J. Stiller, MD, Jerome L. Shupack, MD, Clare Kenny, RN, Lorrie Jondreau, RN, David E. Cohen, MD, MPH, and Nicholas A. Soter, MD New York, New York Baekground: Multiple immunologic abnormalities such as impaired T-cell function, elevated serum 19Blevel, and increased interleukin 4 production have been demonstrated in patients with atopic dermatitis. Objective: As part of a 12-week, multicenter, double-blind, placebo-controlled clinical trial, we evaluated the safety and efficacy of thymopentin (Timunox) as an adjunctive treatment in patients with severe atopic dermatitis. Methods: Thirty-nine patients at least 2 years old with severe atopic dermatitis on a minimum of 200/0 of their cutaneous surface area were randomly selected to receive either thriceweeklysubcutaneous injections of thymopentin, 50 mg, or placebo. Use of triamcinolone 0.1 % or hydrocortisone 1.00/0 cream and oral antihistamines were permitted during this trial. Results: After 12 weeks, thymopentin-treated patients had significantly greater improvement than those receiving placebo. No thymopentin-related adverse events occurred. Conclusion: Thymopentin may be a safe effective adjunct to therapy in patients with severe atopic dermatitis. (J AM ACAD DERMATOL 1994;30:597-602.) From the Ronald O. Perelman Department of Dermatology, New York University School of Medicine, and the Dennatopharmacology Section, Charles C. Harris Skin and Cancer Pavilion, New York University Medical Center. Supported by a grant from the Immunobiology Research Institute, Annandale, N.J. Presented in part at the Eastern Regional Society for Investigative Dermatology, New York, Oct. 9-10, 1992 and the annual meeting of the Society for Investigative Dermatology, Washington, D.C., Apri128May 1,1993. Reprint requests: Matthew J. Stiller, MD, The Ronald O. Perelman Department of Dermatology, 562 First Avenue, New York, NY 10016. Copyright @I 1994 by the American Academy of Dermatology, Inc. 0190-9622/94 S3.00 + 0

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Atopic dermatitis (AD) affects more than 5% of the population.!" Although its pathogenesis is uncertain, immunologic abnormalities are a prominent feature. These include defective T-cell function.v'' an elevated serum IgE level." as well as decreased interferon gammaf and increased interleukin 4 (IL-4)9 production by mononuclear cells. The synthetic inununomodulatory pentapeptide, thymopentin, (Timunox, TP-5) is composed of ammo acids 32-36 (Arg-Lys-Asp-Val-Tyr) of the 52 amino acid thymic hormone thymopoietin. 10-12 Thymopentin appears to have all the biologic and phar-

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