US 20150.086513A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0086513 A1 Savkovic et al. (43) Pub. Date: Mar. 26, 2015

(54) METHOD FOR DERIVING MELANOCYTES (30) Foreign Application Priority Data FROM THE HAIR FOLLCLE OUTER ROOT

SHEATH AND PREPARATION FOR A. 36. 3. E. ------E.6 GRAFTNG l9. U. 414 ) ...... Publication Classification (71) Applicant: UNIVERSITAT LEIPZIG, Leipzig (DE) (51) Int. Cl. A6L27/38 (2006.01) (72) Inventors: Vuk Savkovic, Leipzig (DE); Christina CI2N5/071 (2006.01) Dieckmann, Leipzig (DE); (52) U.S. Cl. Jan-Christoph Simon, Leipzig (DE); CPC ...... A61L 27/3834 (2013.01); A61L 27/3895 Michaela Schulz-Siegmund, Leipzig (2013.01); C12N5/0626 (2013.01); C12N (DE); Michael Hacker, Leipzig (DE) 2506/03 (2013.01) USPC ...... 424/93.7:435/366 (57) ABSTRACT (73) Assignee: NIVERSITAT LEIPZIG, Leipzig The present invention relates to the field of biology and medi (DE) cine, and more specifically, to the field of stem-cell biology, involving producing or generating melanocytes from stem (21) Appl. No.: 14/354,545 cells and precursors derived from human hair root. Addition (22) PCT Filed: Oct. 29, 2012 ally, the present invention relates to the materials and method e a? 19 for producing autografts, homografts or allografts compris (86). PCT No.: PCT/EP2012/07 1418 ing melanocytes in general, as well as the materials and meth ods for producing autografts, homografts and allografts com S371 (c)(1), prising melanocytes for the treatment of diseases related to (2) Date: Apr. 25, 2014 depigmentation of the skin and for the treatment of Scars. Patent Application Publication Mar. 26, 2015 Sheet 1 of 17 US 2015/0O86513 A1

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Figs. 12A-H

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Fig. 13 Comparative changes in the Outer Root Sheath surface

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Figs. 14A-F

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Fig. 16 Comparative growth in the adherent culture, cell number per hair foilicle

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Fig. 17

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NHE HE US 2015/0O86513 A1 Mar. 26, 2015

METHOD FORDERVING MELANOCYTES inventors furthermore, found that Such melanocytes are very FROM THE HAIR FOLLCLE OUTERROOT suitable for treatment of depigmentation and are able to show SHEATH AND PREPARATION FOR unexpectedly high melanin production and a high enzymatic GRAFTING efficacy in three-dimensional structures. 0008. There are methods known how to obtain and culti CROSS-REFERENCE TO RELATED vate melanocytes. One approach is the differentiation and APPLICATIONS cultivation of melanocytes from stem cells from hair follicles 0001. This application is a U.S. National Stage filing (WO-A2 2009/049734). For such purpose, epilated hairs are under 35 U.S.C. S371 of International Patent Application No. treated enzymatically to release the stem cells from the hair PCT/EP2012/071418 filed on Oct. 29, 2012 which claims the follicle. After differentiation and cultivation, application to benefit of EP Application No. 11186944.2 filed Oct. 27, 2011 the affected areas was suggested. However, the inventors and EP Application No. 12182385.0 filed Aug. 30, 2012, found that by the method disclosed in WO-A2 2009/049734 which are incorporated herein in their entirety for all pur also cell types that are surplus to requirements of the treat poses. ment, such as fibroblasts and keratinocytes, are cultivated which show adverse effects on the efficacy of the treatment. FIELD With known methods the superfluous cell types are also being cultivated, which leads to a loss of efficacy of the depigmen 0002 The present invention relates to the field of biology tation therapy or makes it more complicated at its best. Hence, and medicine, and more specifically, to the field of stem-cell there is a need for improved methods to generate and obtain biology, involving producing or generating melanocytes from melanocytes. Moreover, there is a need to provide such cells stem-cells and precursors derived from human hair root. with high purity. Additionally, the present invention relates to a method for 0009 Dieckmann et al. (2010); Experimental Dermatol producing autografts, homografts or allografts comprising ogy: 19(6):543-545 discloses a non-invasive method for melanocytes. obtaining these adult stem cells from outer root sheath (ORS) of a plucked hair follicle. However, Dieckmann (loc. cit.) BACKGROUND does not disclose (1) that the bulb of an epilated human hair is 0003. Depigmentation is the lightening of the skin, or loss removed and the remaining part of the epilated hair be used; of pigment. Depigmentation of the skin can be caused by a and (2) that the epilated hair are treated with collagenase. In number of local and systemic conditions. The pigment loss addition, the cultivation in accordance with the herein pro can be partial, for instance injury to the skin, or complete, vided method can comprise a step of selecting and/or isolat Such as in vitiligo, temporary or permanent. ing melanocytes comprising Geneticin treatment. Such a step 0004 Depigmentation of the skin is a disease landmark in is not disclosed in Dieckmann (loc. cit.). people affected by vitiligo, which produces differing areas of 0010. As shown in Example 2, the method of the present light and dark skin. Next to the cosmetic disadvantages, skin invention allows for a much more pronounced growth and areas with low pigmentation are prone to Sun burns or even proliferation of melanocytes as compared to the method of more adverse effects, which, in the worst case, result in skin Dieckmann (loc. cit.). FIG. 15 shows that the herein provided cancer. Hence, Substantial efforts are currently undertaken to method provides an exponential increase in the cell number provide an easy and reliable method for protecting these over several passages, whereas the cell number even depigmented areas by providing pigmentation. One approach decreases if the Dieckmann (loc. cit.) method is used. As is to apply melanocytes on the depigmented skin areas. Mel demonstrated in Example 2, the Dieckmann (loc. cit.) method anocytes are melanin-producing cells, inter alia, located in did not allow for the production of more than 710,000 mel the bottom layer (the stratum basale) of the skin epidermis. anocytes in total whereas the method of the present invention Melanin is the pigment that primarily determines the color of allowed the production of about 80,875,000 melanocytes in the skin and enables its primary protection from Sun radiation. total after 6 passages under otherwise comparable conditions. 0005 Existing medical treatments for vitiligo are partially In other words, the present invention provides for a more than helpful and are either palliative or invasive. Therefore, a high 100-fold higher production of melanocytes as compared to health interest and market potential exist for a causative, the Dieckmann method (loc. cit.). If the corresponding num autologous, non-invasive treatment. bers of generated melanocytes per epilated human hair (or 0006. The developmental potential of the hair follicle likewise per follicle) are calculated, the Dieckmann (loc. cit.) outer root sheath (ORS), which contains pluripotent adult method did not allow for the production of more than about stem cells, transiently amplifying cells, precursors and dif 7,000 (more exactly 7,717 melanocytes) per epilated human ferentiated cells, is known. These cells are capable of giving hair (or per follicle), whereas the method of the present inven rise to melanocytes (among other cell types). Several meth tion allowed the production of about 2,700,000 melanocytes odological upgrades have been addressing duration, yield and per epilated human hair (or per follicle) after 6 passages under culture purity since the first long-term cultivation of human otherwise comparable conditions. In other words, the present hair follicle melanocytes (HM) in 1995 (Tobin DJ, Colen SR, invention provides for a more than 100-fold higher produc Bystryn J. C. J. Invest Dermatol 1995: 104: 86-89). tion of melanocytes as compared to the Dieckmann method 0007. However, there is a need for methods to isolate and (loc. cit.). The method of the present invention can advanta differentiate melanocyte-yielding cells in order to retrieve geously comprise adherent culture of melanocytes. pure melanocytes for further processing and application on 0011. A first difference of the herein provided method the skin. Furthermore, there is a need to provide a method to compared to the Dieckmann (loc. cit.) method is the removal increase the number of yielded melanocytes after a short of the bulb of an epilated human hair. It is believed that the period of culture. The inventors now provide means and bulb contains and carries over major amounts of fibroblasts. methods as disclosed herein below solving that problem. The By removing the bulb, the epilated hair to be used herein US 2015/0O86513 A1 Mar. 26, 2015

contains/yields less fibroblasts. Thus, there are less fibro allografts together with keratinocytes. The grafts provided blasts at the very start of the herein provided method as are stabilized by biocompatible (scaffold) carriers. compared to Dieckmann (loc. cit.). It is believed that this 0016. The present invention now allows to obtain melano allows a better growth of melanocytes, because the cell cul cytes non-invasively, through differentiation from stem cells ture is less contaminated with fibroblasts and there is there from the outer root sheath of epilated human hairs in high fore less competition for nutrients and space from the very quantity and purity, in a small time frame of 4 weeks and less. beginning on. 0012. A second difference of the herein provided method SUMMARY as compared to Dieckmann (loc. cit.) is the incubation of the 0017. The present invention provides for an enhanced epilated hair with a collagen degrading agent (like Collage method for generating melanocytes from stem cells and/or nase). Said incubation can take 10 minutes. The collagen melanocyte precursors. Hence, the present invention relates degrading agent loosens the extracellular matrix of the epil to a method for generating melanocytes from stem cells com ated hair. It is believed that said loosening facilitates the leave prising the steps of: or migration of stem cells from the extracellular matrix. Thus, 0018 (i) removing the bulb of an epilated human hair; the incubation with a collagen degrading agent can separate 0.019 (iii) (ii) incubating the remaining part of the epi stem cells from the outer root sheath. As demonstrated in lated hair with a collagen degrading agent to separate Example 2, FIGS. 13 and 14, the Outer Root Sheath surface/ stem cells from the outer root sheath; and cultivating the cell number is indeed increased as compared to Dieckmann separated stem cells in a medium that induces differen (loc. cit.), if the method of the present invention is employed. tiation and stimulates growth of stem cells, melanocyte Indeed, the Outer Root Sheath surface/cell number was precursors and melanocytes, wherein the medium com almost twice as high (factor 1.84) as compared to Dieckmann prises one or more growth factor for differentiation into (loc. cit.). melanotic melanocytes, until only the differentiated 0013. It is believed that due to that the reduction of fibro melanotic melanocytes remain in the culture. blasts and easier/quicker/increased leave of stem cells from 0020. The inventors unexpectedly discovered that con the extracellular matrix have the advantageous and Surprising tamination with fibroblasts and other cell types can be greatly effect that the present method allows for a pronounced reduced if the bulb of the hair is removed. The method accord increase in the growth and development of melanocytes in ing to the present invention produces an increase in the yield accordance with the present invention, as demonstrated in of pure melanocytes in a shorter timeframe. The aforemen Example 2. Dieckmann (loc. cit.) provided no hint to the tioned materials and methods provide for a way to increase removal of the bulb or collagenase treatment, let alone any the yield of melanocytes with high purity in a shorter time advantages conferred thereby. To the contrary, Dieckmann frame, especially when compared to, for example, the meth (loc. cit.) even taught away from enzymatic treatment, ods disclosed in Diekmann et al. (2010). because it was potentially unfavourable for the cells. 0021. The melanocytes generated by the method accord 0014. As explained below, the Geneticin treatment is a ing to the present invention are Surprisingly well Suited for further advantageous aspect of the herein provided method. autografts, homografts or allografts. Therefore, the present As shown in FIG. 15/16 the use of Geniticin in the method of invention also relates to a method for producing an autograft, Dieckmann (loc. cit.) does, in contrast to the herein provided homograft or an allograft comprising melanocytes, said method, not allow for a Substantial growth or selection/isola method comprising the steps of providing a suspension com tion of melanocytes. The Geneticin treatment in accordance prising melanocytes obtainable by a method for generating with the present method is advantageous because it targets melanocytes from stem cells according to the present inven primarily cells with a rapid metabolism and proliferation, tion; providing a biocompatible carrier, and cultivating the such as fibroblasts and keratinocytes, whereas it hardly melanocytes on said biocompatible carrier. affects slowly-dividing melanocytes. Thus, the Geneticin 0022. The present invention furthermore, relates to an treatment allows for an enrichment of melanocytes in the cell autograft, homograft or allograft obtainable by a method for culture and a pronounced increase in melanocyte number. producing an autograft, homograft or an allograft according Geneticin treatment in the Dieckmann (loc. cit.) method does to the present invention. not show any such desirable effects. Therefore, it is believed 0023 The invention further relates to melanocytes obtain that the cell culture according to Dieckmann (loc. cit.) prior to able by a method for generating melanocytes from stem cells Geneticin treatment contains, in contrast to the method of the and/or melanocyte precursors according to the present inven present invention, Substantial amounts of fibroblasts and keratinocytes and only a minor amount of melanocytes. Thus, tion. it is believed that the amount of melanocytes after Geneticin BRIEF DESCRIPTION OF THE DRAWINGS treatment of the Dieckmann (loc. cit.) cell culture is not sufficient for efficient selection/isolation and growth of mel 0024. The following drawings form part of the present anocytes. specification and are included to further demonstrate certain 0015. As explained above, the inventors now developed an embodiments. Some embodiments may be better understood improved non-invasive method for obtaining adult stem cells by reference to one or more of these drawings alone or in from outer root sheath (ORS) of a plucked hair follicle and combination with the detailed description of specific embodi differentiation into a pure culture of functional melanocytes. ments presented. The melanocytes prepared by the method according to the 0025 FIGS. 1A-1D are exemplary images of the cultiva present invention as set out herein can be readily used for tion, selection and expansion of ORS cells from the ORS of treatment of Vitiligo as Suspension, liquid or in the form of an the hair root into a pure culture of melanocytes within four aerosol, as single culture or combined with keratinocytes. The weeks. A: Mid part of the epilated, preparated temporal hair melanocytes are further provided in autografts, homografts or root set on the Transwell microporous membrane (medium US 2015/0O86513 A1 Mar. 26, 2015

air-interface) on the first day of cultivation. B: Outgrown ORS forming melanosomes. NKI/beteb labelling of melanosomes with first cells migrating out of ORS. C. Multiplied cells are in NHEM and HM are depicted. A: NHEMs on 3 um PCL forming aggregations apart from the ORS. D: Pure culture of scaffold, B: NHEMs on 10 pum PCL scaffold C: HM on 3 um ORS melanocytes after four weeks of cultivation. PCL scaffold D: HM on 10 um PCL scaffold. 0026 FIGS. 2A-2I are exemplary images of the improve 0034 FIGS. 10A-10D are graphical representations of the ments of the present invention in developing a pure culture of relative melanin content and L-DOPA-tautomerase activity ORS melanocytes. A: Unprepared, untreated hair. ORS is of NHEM and HM on PCL Scaffolds and adherent Surfaces. compact (arrow) B: Unprepared, Collagenase V-treated hair Black bars represent the NHEM and grey bars the HM. A: with visible migrated cells outside ORS at mid-part and also Melanin content in epidermal melanocytes on PCL scaffolds outside the proximal (bulb) part (arrows). C: Prepared hair compared to an adherent surface. B: Melanin content in ORS with the bulb part cut off (dash-line), untreated with Collage melanocytes on PCL Scaffolds compared to an adherent Sur nase V. showing compact ORS (arrow). D: Prepared hair with face. C: L-DOPA activity in epidermal melanocytes on PCL the bulb part cut off (dash-line), treated with Collagenase V. scaffolds compared to an adherent surface. D: L-DOPA activ showing outgrown ORS with migrated cells outside of ORS ity in ORS melanocytes on PCL scaffolds compared to an (arrow). E: Outgrown, nearly confluent propagated cells out adherent Surface. side of ORS on day 14 of culture. F: Primary culture of ORS 0035 FIGS. 11A-11B are ememplary images of outer root cell pool developed from non-collagenised hairs on day 20. sheath melanocytes (HMORS) embedded in the keratinocyte G: Primary culture of ORS cell pool developed from collage epidermal equivalent, which remain in the lower layer of the nised hairs on day 20. H: pure culture of selected melanocytes graft, equivalent to epidermal stratum basale, and express on day 26. I: Differentiated melanocytes in the pure culture at melanocyte markers. A: Epidermal melanocytes, normal day 30. light; B: Epidermal melanocytes, fluorescence. 0027 FIGS. 3A-3F are exemplary images of the immuno 0036 FIGS. 12A-12H are exemplary images of immuno cytochemical characterization of the ORS culture. A: cytochemical characterization of the various melanocyte cul NKI\beteb is expressed in all epidermal melanocytes tures. A: NKI-beteb expression in normal human epidermal (NHEM); B: Tyrosinase is expressed in all epidermal mel melanocytes (NHEM); B: Tyrosinase in epidermal melano anocytes (NHEM); C: CD 90 is expressed in all non-melano cytes;, C: CD-90 expression in pure dermal fibroblast cul cyte cells, prevalently fibroblasts; D: NKI\beteb is expressed ture, D: Lack of CD-90 expression in HM culture; E: HM in in almost all ORS melanocytes (HM); E: Tyrosinase is culture without L-DOPA addition: F: IBMX-mediated expressed in almost all HM; F: absence of CD 90 expression changes in HM morphology and increased melanin content, in pure HM-culture. without L-DOPA; G: Melanin content upon L-DOPA sub 0028 FIG. 4 is a graphical representation of the relative strate addition in HM; H: Increase in melanin content upon purity of an ORS melanocyte culture. The grey bars are show L-DOPA addition in IBMX-pre-treated HM. ing the percentage of NKI/beteb-positive melanocytes in cul 0037 FIG. 13 is a grahpical representation of the com ture. Coll--/- corresponds to Collagenase V treatment, HW+ parative changes of the Outer Root Sheath surface. Both the stands for presence of the bulb., HW-stands for absence of the current method (0) and the method of Dieckmann et al. () bulb, G+/- stands for Geneticintreatment, DLM for DermaL are depicted. ife culture media applied. The black bar represents epidermal 0038 FIGS. 14A-14F are exemplary images of compara melanocyte control (differentiated NHEM), grown in a sepa tive ORS surface changes. A. Current method, day 0: B: rate culture, showing 99% of NKI/beteb-expressing cells. Dieckmann method, day 0: C. Current method, day 7; D: 0029 FIG. 5 is a graphical representation of the relative Dieckmann method, day 7: E: Current method, day 14: F: fibroblast contamination in an ORS melanocyte culture. The Dieckmann method, day 14. grey bars are showing the percentage of CD-90-positive cells 0039 FIG. 15 is a graphical representation of the com in culture. Coll--/- corresponds to Collagenase V treatment, parative cell growth in the adherent culture growth curves of HW+ stands for presence of the bulb., HW-stands for absence ORS cells cultivated according to: (A) the current method, of the bulb (The bulb has been severed from the rest of the (O) Dieckmann method, and (*) Dieckmann method with root), G+/- stands for Geneticin treatment, DLM for Der Geneticin Supplement during the first 6 passages of adherent maLife culture media applied. The white bar represents fibro culture. blast control, grown in a separate culture, showing 100% of 0040 FIG. 16 is a graphical representation of the com CD-90-expressing cells. parative growth in the adherent culture, per hair follicle. 0030 FIGS. 6A-6B represent exemplary images of a Poli Growth curves of ORS cells per sampled hair follicle culti caprolactone (PCL) scaffold structure. A: REM capture of the vated according to: (A) the current method, (O) Dieckmann 8 to 10 um fibre. B: REM capture of the 2-3 um fibre. method, (*) Dieckmann method with Geneticin supplement 0031 FIGS. 7A-7D are exemplary images of laser-scan during the first 6 passages of adherent culture. The graph is ning microscope capture of HM and NHEM on PCL Scaf displayed logarithmically. folds. A: HM expressing Tyrosinase; B: NHEM expressing 0041 FIG. 17 is a graphical representation of the different NKI\beteb; C: Side view of the ORS melanocyte-populated sizes of cell soma and dendrite length in NHEM and HM. A. PCL-Scaffold; D: Side view of the epidermal melanocyte Relative cell size of NHEM and HM measured by Forward populated PCL-Scaffold. Scatter Plot (X-axis); B: Cell size of NHEM and HM in um: 0032 FIG. 8 is a graphical representation of melanocyte C: Dendrite length of NHEM and HM in p.m. marker expression on PCL Scaffolds and on a polystyrene adherent surface. The ratio of the number of nuclei against DEFINITIONS marker-positive cells is expressed as percentage. 0042 Antibiotics' in context with the present invention 0033 FIGS. 9A-9D are exemplary images of both epider refer to agents capable of inhibiting growth of microorgan mal melanocytes (NHEM) and ORS melanocytes (HM) isms and/or inducing the death of microorganisms, such as US 2015/0O86513 A1 Mar. 26, 2015

bacteria or fungi, and/or as an additional selective agent for entiated cells, predominantly fibroblast. In a preferred quickly-dividing cells. Suited antibiotics may be chosen by embodiment also the distal part of the hair shaft, also called the skilled person. However, preferred antibiotics are selected the bulge is cut off if not lost during epilation. In a preferred from the group consisting of Penicillin, Amphotericin B, embodiment only the mid-part of the epilated hair root is Gentamycine and Streptomycin or combinations thereof. A used. The mid-part of an epilated hair in context with the preferred combination of antibiotics is a Penicillin, Strepto present invention is preferably the morphological unit mycin, Amphotericin B and Geneticin. between the bulb (proximal part) and the sebaceous gland 0043 “Hypoxic condition” refers to conditions with oxy channel. This part contains a pool of heterogeneously differ gen content below the normal atmospheric oxygen content. entiated cells, from pluripotent Neural-Crest-Like-Cells, Hence in one embodiment step (ii) of the method for gener over an array of partially differentiated precursors (epithelial, ating melanocytes from stem cells and/or melanocytes pre neuronal, adipocytic, chondrocytic, osteocytic to the fully cursors is performed under conditions with an O content of differentiated cells, that are in majority epithelial precursors 20% or less, preferably 10% or less, even more preferably and serve for regeneration of epidermis. In context with the with an O. content of between 1% and 5%. In a preferred present invention the terms epilated hair and epilated hair embodiment the cultivating steps of the method for producing follicle are used interchangeably. an autograft, homograft or allograft is performed under con 0051. The skilled artisan is able to adapt the amount of ditions with an O. content of 5%, and a CO content of 5%. epilated hairs used in context with the present invention in 0044) “Biocompatible carriers' in context of the present order to adjust the total amount of generated melanocytes. In invention are carriers that can be transplanted to a human or one embodiment the bulbs of 1 to 500 epilated hairs are animal body and provide a structure and biomechanical Sup removed in step (i) of the method for generating melanocytes port that allows the cells to adhere to the surface. Further from stem cells, preferably the bulbs of 10 to 80 epilated hairs more, the skilled artisan will understand that the carriers shall are removed in step (i) of the method for generating melano be non-toxic, compatible with biological functions of the cytes from stem cells, more preferably the bulbs of 30 to 60 receiver and they are either tissue compatible and/or are bio epilated hairs are removed in step (i) of the method for gen logically degradable in situ. erating melanocytes from stem cells. 0045 “Hypoxic condition” refers to conditions with oxy 0052. In a preferred embodiment of the method for gener gen content below the normal atmospheric oxygen content. ating melanocytes from stem cells, the method comprises a Hence in one embodiment step (iv) of the method for produc further step between step (ii) and (iii) of washing the remain ing an autograft, homograft or allograft is performed under ing part of the epilated hair with a washing solution after conditions with an O. content of 20% or less, preferably 10% incubation with the collagen degrading agent. or less, even more preferably with an O. content of between 0053 Suitable washing solutions are well known in the art 1% and 5%. In a preferred embodiment embodiment culti and can be chosen by those of skills in the art. In a preferred Vating steps of the method for producing an autograft, embodiment the washing solution is Dulbecco's Modified homograft or allograft is performed under conditions with an Eagle Medium (DMEM). In a further preferred embodiment O content of 5%, and a CO content of 5%. the washing Solution comprises an antibiotic, preferably 0046 “Normoxic condition” refers to conditions with selected from the group consisting of Gentamycin, Amphot oxygen content at the normal atmospheric oxygen content. ericin B, Penicillin, Streptomycin, Neomycin, Carbenicillin, Hence, in one embodiment the cultivation step(s) of the meth Penicillin G, Ampicillin, Polymyxin-B, tetracycline, Ciprof ods for producing an autograft, homograft or allograft is loxacin, Lincomycin, Spectinomycin, Cabenicilin, Thios performed under conditions with an O content of approxi trepton, Ceftacidin, Apramycin, Vancomycin, Tobramycin, mately 20%. Rifampycin, and Hygromycin, preferably the washing solu tion comprises an antibiotic, preferably selected from the DESCRIPTION group consisting of Gentamycin, Amphotericin B. Penicillin, 0047. In one embodiment the present invention relates to a Streptomycin. method for generating melanocytes from stem cells compris 0054 The skilled artisan is able to determine suited salts ing the steps of removing the bulb of an epilated human hair; and/or concentrations of antibiotics for the washing solution. 0048 i) incubating the remaining part of the epilated However, preferred antibiotics and preferred concentrations hair with a collagen degrading agent to release the stem in brackets are given in the following: Penicillin, e.g. (50 cells from the outer root sheath; U/ml to 150 U/ml, preferably 100 U/ml), Streptomycin-Sul 0049 ii) cultivating the separated stem cells from step fate (50 pg/ml to 20 mg/ml, preferably 100 ug/ml), Gentamy (ii) in a differentiation medium that induces differentia cin-Sulfate (5 g/ml to 3000 ug/ml, preferably 50 ug/ml), tion and stimulates growth of stem cells, melanocyte Amphotericin B (0.25 ug/ml to 30 ug/ml, preferably 2.5 precursors and melanocytes, wherein the medium com ug/ml), (25 g/ml to 600 ug/ml, preferably 50 g/ml), (50 prises one or more growth factors for differentiation into pg/ml to 10000 ug/ml, preferably 100 ug/ml), (25 ug/ml to melanotic melanocytes; until only the differentiated 3000 ug/ml, preferably 50 ug/ml), Ampicillintrihydrat (50 melanotic melanocytes remain in the culture. U/ml to 200U/ml, preferably 100 U/ml), -Carbenicillin, (50 0050. The inventors unexpectedly found that the yield and U/ml to 200 U/ml, preferably 100 U/ml) (50 U/ml to 200 purity of melanocytes using the method according to the U/ml, preferably 100U/mL), Polymyxin-B-Sulfate (25ug/ml present invention can be greatly increased if the proximal part to 3000 ug/ml, preferably 100 ug/ml), (2 g/ml to 80 ug/ml, of the follicle/epilated human hair is cut off, i.e. the bulb. preferably 10 ug/ml), 7-Hydroxy-Tetracyclin (each 2 to 25 Hence, the method according to the present invention com ug/ml, preferably 5 lug/ml), (5 to 35 ug/ml, preferably 10 prises removing the bulb of an epilated hair. The “bulb of an ug/ml), Erythromycin (50 ug/ml to 300 g/ml, preferably 100 epilated human hair in context of the present invention is the ug/ml), (2 ug/ml to 300 g/ml, preferably 10 g/ml), (2 ug/ml proximal part of the hair root, which contains mostly differ to 30 ug/ml, preferably 5ug/ml), Ciprofloxacin (1 lug/ml to 10 US 2015/0O86513 A1 Mar. 26, 2015

ug/ml), Lincomycin (50 g/ml), Spectinomycin) (5 g/ml to selected from the group consisting of Dermalife medium 50 ug/ml, preferably 10 ug/ml), Carbenicilin (100 U/ml), (DLM) base of low bovine serum content (Life Line Cell Thiostrepton (25 g/ml), Ceftacidin-Hydrate (100 lug/ml), Technology), Dulbeccos Modified Eagle Medium base Apramycin (2.5ug/ml to 25 g/ml), Vancomycin-Hydrochlo (DMEM, Sigma-Aldrich), F12 Ham's nutrient mixture ride (100 lug/ml), Tobramycin (4 g/ml to 100 g/ml, prefer (Sigma-Aldrich), and PC-1 medium base (Lonza). In a pre ably 80 g/ml), Rifampycin (400 g/ml), Hygromycin (200 ferred embodiment the differentiation medium comprises g/ml). DermaLife medium (DLM) base as a nutrient base, more 0055. In a preferred embodiment the washing solution preferably DLM is the only nutrient base. It is particularly comprises Gentamycin, preferably at a concentration of 10 preferred that the collagen degrading agent is Collagenase V: ug/ml to 100 g/ml, more preferably at a concentration of 25 and that the differentiation medium comprises DermaLife ug/ml to 75 g/ml, even more preferably at a concentration of medium (DLM) base as a nutrient base, more preferably 50 ug/ml. DLM is the only nutrient base comprised in the differentiation 0056. In a further preferred embodiment the washing solu medium. The method according to the present invention tion comprises Amphotericin B, preferably at a concentration unexpectedly allows the use of medium with a low or no of 1 Jug/ml to 20 ug/ml, more preferably at a concentration of serum content. Hence, it is preferred that the differentiation 5ug/ml to 15ug/ml, even more preferably at a concentration medium has a low serum content, preferably less than 2.5%, of 10 ug/ml. more preferably less than 2%, even more preferably less than 0057. In a preferred embodiment the washing solution 1%, yet more preferred 0.5% or less. It is even more preferred comprises two antibiotics, preferably Gentamycin and that the serum content of the differentiation medium is 0.5%. Amphotericin B. In a further embodiment the differentiation medium is serum 0058. In yet a further preferred embodiment the washing free. solution comprises DMEM, Gentamycin at a concentration 0061 Further nutrients and additives may be applied. In a of 50 lug/ml and Amphotericin B at a concentration of 10 preferred embodiment the differentiation medium further ug/ml. In a most preferred embodiment the washing Solution comprises one or more compounds selected from the group consists of DMEM, Gentamycin at a concentration of 50 consisting off-adrenergic receptor ligands. Such as epineph ug/ml and Amphotericin B at a concentration of 10 ug/ml. rine and derivatives thereof, Ca", L-glutamine, insulin, Vita 0059 Collagen degrading agents are known by the skilled min C, calf serum, Epidermal Growth Factor (EGF), basic person. Collagen degrading agents in context of the present Fibroblast Growth factor and its variants bFGF/FGF2, Endot invention are all agents which digest collagen, a polymer helin-1, C.-Melanocyte Stimulating Hormone (C-MSH), consisting of protein, into Smaller Subunits. The inventors Hydrocortisone, StemCell Factor (SCF), Nerve Growth Fac interestingly found that by the treatment with collagen tor beta (NGF-?3), Hepatocyte Growth Factor (HGF), StiMel degrading agents, cells of the Outer Root Sheath (ORS) are factor cocktail (Life Line Cell Technology), and antibiotics. more easily separated from the rest of the hair root sheath In a preferred embodiment the differentiation medium com cells during their cultivation. Digestion of collagen weakens prises DermLife medium (DLM) base as a nutrient base, and the extracellular matrix and releases the cells, including stem one or more compounds selected from the group consisting of cells, precursors and differentiated cells. In a preferred B-adrenergic receptor ligands, such as epinephrine and embodiment of the present invention the collagen degrading derivatives thereof, Ca", L-glutamine, insulin, Vitamin C, agent degrades a collagen selected from the group consisting calfserum, Epidermal Growth Factor (EGF), basic Fibroblast of collagen I, collagen IV and collagen V. In a preferred Growth factor and its variants bFGF/FGF2, Endothelin-1, embodiment the collagen degrading agent is an enzyme, pref C.-Melanocyte Stimulating Hormone (C-MSH), Hydrocorti erably a collagenase. In a further preferred embodiment the sone, Stem Cell Factor (SCF), Nerve Growth Factor beta collagen degrading agent is a collagenase selected from the (NGF-?3), Hepatocyte Growth Factor (HGF), StiMel factor group consisting of collagenase I, IV and V. He will recognize cocktail (Life Line Cell Technology), and antibiotics, prefer that collagen I, IV and V cover the cell surfaces, bases of cell ably DLM is the only nutrient base comprised in the differ basement membrane, the skin and the hair, respectively. The entiation medium and the differentiation medium comprises collagenases are preferably able to degrade more than one one or more compounds selected from the group consisting of type of collagen. In a particularly preferred embodiment the B-adrenergic receptor ligands, such as epinephrine and collagen degrading agent is Collagenase V. In an even more derivatives thereof, Ca", L-glutamine, insulin, Vitamin C, preferred embodiment the collagen degrading agent is Colla calfserum, Epidermal Growth Factor (EGF), basic Fibroblast genase V at a concentration of 5 mg/ml. Growth factor and its variants bFGF/FGF2, Endothelin-1, 0060. The skilled person is aware of suited compositions C.-Melanocyte Stimulating Hormone (C-MSH), Hydrocorti for the differentiation medium that induce differentiation and sone, Stem Cell Factor (SCF), Nerve Growth Factor beta stimulate growth of stem cells, melanocyte precursors and (NGF-?3), Hepatocyte Growth Factor (HGF), StiMel factor melanocytes, wherein the medium comprises one or more cocktail (Life Line Cell Technology), and antibiotics. Fur growth factors for differentiation into functional melanotic thermore, in a preferred embodiment the collagen degrading melanocytes. Standard differentiation media known in the art agent is Collagenase V; and the differentiation medium com can be used in the herein provided method. Exemplary dif prises DermLife medium (DLM) base as a nutrient base and ferentiation media are described in Szabad et al. Arch. Derm. one or more compounds selected from the group consisting of Res. (2007)299:191-200, Kauseretal. Endocrinology (2005) B-adrenergic receptor ligands, such as epinephrine and 146 (2):532-543, http://lifelinecelltech.com/docs/SPM Der derivatives thereof, Ca", L-glutamine, insulin, Vitamin C, maLife M. Preferably, the differentiation medium to be used calfserum, Epidermal Growth Factor (EGF), basic Fibroblast herein does not contain a tumor promoter (like Phorbol Growth factor and its variants bFGF/FGF2, Endothelin-1, Myristate Acetate (PMA)). In a preferred embodiment the C.-Melanocyte Stimulating Hormone (C-MSH), Hydrocorti differentiation medium comprises a nutrient base, preferably sone, Stem Cell Factor (SCF), Nerve Growth Factor beta US 2015/0O86513 A1 Mar. 26, 2015

(NGF-?3), Hepatocyte Growth Factor (HGF), StiMel factor preferred the differentiation medium comprises Epidermal cocktail (Life Line Cell Technology), and antibiotics, prefer Growth Factor EGF at a concentration of 2.5 ng/ml. ably DLM is the only nutrient base comprised in the differ 0071. In another preferred embodiment the differentiation entiation medium and the differentiation medium further medium comprises Endothelin, preferably at a concentration comprises one or more compounds selected from the group of 0.2 ng/ml to 20 ng/ml, more preferably at a concentration consisting off-adrenergic receptor ligands, such as epineph of 1 ng/ml to 10 ng/ml, even more preferred the differentia rine and derivatives thereof, Ca", L-glutamine, insulin, Vita tion medium comprises Endothelin at a concentration of 5 min C, calf serum, Epidermal Growth Factor (EGF), basic ng/ml. Fibroblast Growth factor and its variants bFGF/FGF2, Endot helin-1, C.-Melanocyte Stimulating Hormone (C-MSH), (0072. In a further preferred embodiment the differentia Hydrocortisone, StemCell Factor (SCF), Nerve Growth Fac tion medium comprises StiMel factor cocktail (Life Line tor beta (NGF-?3), Hepatocyte Growth Factor (HGF), StiMel Scientific Inc.), preferably at a concentration of 0.1% to 5%, factor cocktail (Life Line Cell Technology), and antibiotics. more preferably at a concentration of 0.3 uM to 2.5uM, yet 0062. The further nutrients and additives may be applied more preferably at a concentration of 0.5% to 1.5%, even in suited concentrations well known in the art. However, more preferred the differentiation medium comprises StiMel preferred concentrations are given below: factor cocktail at a concentration of 1%. 0063. In a preferred embodiment the differentiation 0073. In a further embodiment the differentiation medium medium comprises L-Glutamine, preferably at a concentra does not comprise cholera toxin. In another embodiment the tion of 3 mM to 10 mM, more preferably 4 mM to 8 mM, yet differentiation medium does not comprise tumor promoters, more preferably 5 mM to 7 mM, even more preferred the such as phorbol-12-myristate 13-acetate (PMA, TPA). In a differentiation medium comprises L-Glutamine at a concen preferred embodiment the differentiation medium does nei tration of 6 mM. ther comprise cholera toxin nor a tumor promoter, Such as 0064. In a further preferred embodiment the differentia phorbol-12-myristate 13-acetate (PMA, TPA). tion medium comprises insulin, preferably at a concentration 0074. In a preferred embodiment the differentiation of 1 Jug/ml to 10 ug/ml, more preferably at a concentration of medium comprises Penicillin, preferably at a concentration 2 ug/ml to 8 g/ml, yet more preferably at a concentration of of 1.000 units/ml to 20.000 units/ml, more preferably at a 4 g/ml to 6 ug/ml, even more preferred the differentiation concentration of 5.000 units/ml to 15.000 units/ml, yet more medium comprises insulin at a concentration of 5ug/ml. preferred the differentiation medium comprises Penicillinata 0065. In a further preferred embodiment the differentia concentration of 10.000 units/ml. tion medium comprises Vitamin C, preferably at a concentra (0075. In a preferred embodiment the differentiation tion of 10 g/ml to 100 ug/ml, more preferably at a concen medium comprises Amphotericin B, preferably at a concen tration of 20 lug/ml to 80 ug/ml, yet more preferably at a tration of 1 ug/ml to 20 ug/ml, more preferably at a concen concentration of 40 ug/ml to 60 g/ml, even more preferred tration of 5ug/ml to 10 ug/ml, yet more preferred the differ the differentiation medium comprises Vitamin Cat a concen entiation medium comprises Amphotericin B at a tration of 50 ug/ml. concentration of 10 ug/ml. 0066. In another preferred embodiment the differentiation 0076. In a very preferred embodiment the differentiation medium comprises Epinephrin, preferably at a concentration medium comprises Gentamycin, preferably at a concentra of 0.1 uM to 5 LM, more preferably at a concentration of 0.3 tion of 10 g/ml to 100 ug/ml, more preferably at a concen uM to 2.5 LM, yet more preferably at a concentration of 0.5 tration of 25 ug/ml to 75 lug/ml, even more preferably at a uM to 1.5 uM, even more preferred the differentiation concentration of 50 ug/ml. medium comprises Epinephrin at a concentration of 1 M. 0067. In another preferred embodiment the differentiation (0077. In another preferred embodiment the differentiation medium comprises Calcium chloride, preferably at a concen medium comprises Penicillin, Stretpomycin and Amphoteri tration of 0.1 mM to 0.4 mM, more preferably at a concen cin. In a most preferred embodiment the differentiation tration of 1.5 mM to 2.5 mM, even more preferred the differ medium comprises Penicillin at a concentration of 10,000 entiation medium comprises Calcium chloride at a units/ml, Streptomycin at a concentration of 10,000 ug/ml, concentration of 0.2 mM. and Amphotericin B at a concentration of 25 ug/ml. 0068. In another preferred embodiment the differentiation 0078. In a particularly preferred embodiment the differen medium comprises Bovine Pituitary Extract, preferably at a tiation medium is DLM and comprises Penicillin, Stretpomy concentration of 10 g/ml to 40 ug/ml, more preferably at a cin and Amphotericin. In a most preferred embodiment the concentration of 15 ug/ml to 35 g/ml, even more preferred differentiation medium comprises Penicillin at a concentra tion of 10,000 units/ml, Streptomycin at a concentration of the differentiation medium comprises Bovine Pituitary 10,000 g/ml, and Amphotericin B at a concentration of 25 Extract at a concentration of 25 ug/ml. g/ml. 0069. In another preferred embodiment the differentiation medium comprises Basic Fibroblast Growth factor bFGF/ 0079. In another particularly preferred embodiment the FGF2, preferably at a concentration of 1 ng/ml to 20 ng/ml, differentiation medium base is Dulbecco's Modified Eagle more preferably at a concentration of 5 ng/ml to 15 ng/ml. Medium DMEM with a known formulation disclosed in Dul even more preferred the differentiation medium comprises becco, Rand Freeman, G. (1959) Virology 8:396), even more Basic Fibroblast Growth factor bRGF/FGF2 at a concentra preferably DMEM 5030, produced by Sigma(R) in GMP tion of 10 ng/ml. grade quality. 0070. In another preferred embodiment the differentiation 0080. During differentiation the medium may be medium comprises Epidermal Growth Factor EGF, prefer exchanged as desired. The skilled person knows when and ably at a concentration of 0.2 ng/ml to 20 ng/ml, more pref how to change the medium. In a preferred embodiment the erably at a concentration of 1.5 ng/ml to 5 ng/ml, even more medium is changed during differentiation every 2 days. US 2015/0O86513 A1 Mar. 26, 2015

0081. The skilled person may choose suited combinations human serum, L-Glutamine (6 mM), Ca" (0.2 mM), Bovine and concentrations for the differentiation medium. However, Pituitary Extract (25 g/ml), Insulin (5 ug/ml), Vitamin C Some preferred combinations and concentrations are given (0.2%), Epinephrine (0.2%), EGF (1 ng/ml), bFGF/FGF2 (10 below. ng/ml), Epidermal Growth Factor EGF (2.5 ng/ml), Endothe 0082 In a preferred embodiment DermaLife differentia lin-1 (20 nM), Gentamycin (50 ug/ml). tion medium purchased from Life Line Cell Technology is I0089. In an even more preferred embodiment the differen used as the differention medium, which comprises DLM tiation medium comprises DMEM medium base, 5% human medium base of low bovine serum content (0.5%), serum, L-Glutamine (6 mM), Ca" (0.2 mM), Bovine Pitu L-Glutamine (6 mM), Insulin (5 ug/ml), Vitamin C (50 itary Extract (25ug/ml), Insulin (5ug/ml), Vitamin C (0.2%), ug/ml), Epinephrin (1 uM), StiMel factor cocktail (1%) and Epinephrine (0.2%), EGF (1 ng/ml), bFGF/FGF2 (10 ng/ml), Antimicrobial supplement (Penicillin 10,000 units/ml, Strep Epidermal Growth Factor EGF (2.5 ng/ml), Endothelin-1 (20 tomycin 10,000 ug/ml, and Amphotericin B 25 g/ml). In nM), Gentamycin (50 ug/ml). another embodiment the differentiation medium comprises 0090 Growth factors and cultivation agents suited for the DLM medium base of low bovine serum content (0.5%), method according to the present invention are known by those L-Glutamine (6 mM), Insulin (5 ug/ml), Vitamin C (50 skilled in the art and can be adapted according to the needs. ug/ml), Epinephrin (1 uM), StiMel factor cocktail (1%) (mix Preferably these growth factors and cultivation agents trigger ture of growth factors, LifeLine Technology) and Gentamy the differentiation of stem cells to melanocytes, or at least do cine (50 ug/ml). not interfere with the melanocyte differentiation process and I0083. In a further preferred embodiment DermaLife dif favorize the growth and up-scaling of melanocytes. However, ferentiation medium purchased from Life Line Cell Technol in one embodiment of the method for generating melanocytes ogy is used as the differentiation medium, which comprises according to the present invention the growth factors and DLM medium base of low bovine serum content (0.5%), cultivation agents are selected from the group consisting of L-Glutamine (6 mM), Insulin (5 ug/ml), Vitamin C (50 ethanolamine, phosphoethanolamine, hydrocortisone, basic ug/ml), Epinephrin (1 uM), StiMel factor cocktail (1%) and fibroblast growth factor (bfGF), dibutyryl cyclic adenosine Gentamycine (50 ug/ml). In another embodiment the differ monophosphate (dbcAMP), melanocyte growth factor entiation medium comprises DLM medium base of low (MeCF), Kaposi's sarcoma derived FGF-like factor (hst/K- bovine serum content (0.5%), L-Glutamine (6 mM), Insulin FGF), hepatocyte growth factor (HGF), stem cell growth (5 g/ml), Vitamin C (50 ug/ml), Epinephrin (1 uM), and factor (SCF), endothelin-1, alpha-melanocyte stimulating StiMel factor cocktail (1%) (mixture of growth factors, Life hormone (C-MSH) and mixtures of native factors from the Line Technology). medium conditioned by cultured human keratinocytes, or 0084. In yet another preferred embodiment the differen from bovine pituitary extract (BPE), or bovine brain extract tiation medium comprises DLM basal medium, L-Glutamine (Wilkins et al. 1985). (6 mM), Ca" (0.2 mM), Insulin (5 ug/ml), Vitamin C (50 0091. In a preferred embodiment the differentiation step ug/ml), Epinephrin (1 uM), and/or EGF (1 ng/ml), and/or (ii) of the method for generating melanocytes from stem cells bFGF/FGF2 (10 ng/ml), and/or Endothelin-1 (20 nM), and/or and/or melanocytes precursors is performed under hypoxic C-MSH (5 nM), and/or Hydrocortison (0.5 lug/ml), and/or conditions or normoxic conditions. More preferably, the dif SCF (10 ng/ml), and/or NGF-?3 (20 ng/ml), and/or HGF (10 ferentiation step is performed under hypoxic conditions. ng/ml), and/or Gentamycin (50 ug/ml). 0092. Furthermore, in a preferred embodiment the differ 0085. In yet another preferred embodiment the differen entiation step (ii) is performed at a medium-air interface. The tiation medium comprises serum-free PC-1 medium base skilled artisan knows how to perform differentiation at a and/or DMEM and/or F12, 1-10% human serum, L-Glutamin medium-air interface. Preferably the remaining part of the (6 mM), Ca" (0.2 mM), Insulin (5ug/ml), Vitamin C (0.2%), epilated hair follicle of step (i) is placed on mesh, preferably Epinephrin (0.2%), EGF (1 ng/ml), and/or bFGF/FGF2 (10 a nylon mesh, wherein the nylon mesh is in contact with the ng/ml), Endothelin-1 (20 nM), and/or C-MSH (5 nM), differentiation medium. Preferred nylon meshes have a pore Hydrocortison (0.5ug/ml), SCF (10 ng/ml), and/or NGF-?320 size of 1 Lum or less, preferably 0.5um or less, more preferred ng/ml), and HGF (10 ng/ml), Gentamycin (fc. 50 g/ml). the nylon mesh has a pore size of 0.4 um. In a most preferred I0086. In yet another preferred embodiment the differen embodiment the medium-air interface is generated by placing tiation medium comprises DMEM basal medium, the epilated hair follicle of step (i) on a 24 mm Transwell(R) L-Glutamine (6 mM), Ca" (0.2 mM), Insulin (5 g/ml), nylon-meshe with 0.4 um Pore Polyester Membrane Insert Vitamin C (50 ug/ml), Epinephrin (1 uM), and/or EGF (1 (Corning) and grown in medium-air-interface conditions. In a ng/ml), and/or bFGF/FGF2 (10 ng/ml), and/or Endothelin-1 preferred embodiment of the medium-air interface the (20 nM), and/or C-MSH (5 nM), and/or Hydrocortisone (0.5 remaining part of the epilated hair follicle of step (i) are ug/ml), and/or SCF (10 ng/ml), and/or NGF-B (20 ng/ml), incubated under hypoxic conditions, preferably they are he and/or HGF (10 ng/ml), and/or Gentamycin (50 ug/ml). exposed to a hypoxic gas mixture. The hypoxic gas mixture 0087. In yet another preferred embodiment the differen preferably has a partial O gas pressure of 5%, more prefer tiation medium comprises DMEM medium base, 1-10% ably the hypoxic gas mixture further has a partial CO gas human serum, L-Glutamine (6 mM), Ca" (0.2 mM), Insulin pressure of 5%, most preferred the hypoxic gas mixture has a (5ug/ml), Vitamin C (0.2%), Epinephrine (0.2%), EGF (1 O gas pressure of 5%, a partial CO gas pressure of 5%, and ng/ml), and/or bFGF/FGF2 (10 ng/ml), Endothelin-1 (20 a N partial gas pressure of 90%. nM), and/or C-MSH (5 nM), and/or Hydrocortisone (0.5 0093. The inventors unexpectedly found that the cultiva ug/ml), and/or SCF (10 ng/ml), and/or NGF-?320 ng/ml), and tion step (iii) of the method for generating melanocytes HGF (10 ng/ml), Gentamycin (fc. 50 g/ml). according to the present invention may be performed until all 0088. In yet another preferred embodiment the differen other cells disappear as the medium is selective for the gen tiation medium comprises DMEM medium base, 1-10% eration of melanocytes from the stem cells. The other cell US 2015/0O86513 A1 Mar. 26, 2015 types are interestingly selected away and only the melano melanocytes, wherein the medium comprises one or cytes remain. Hence, in one embodiment of the present inven more growth factors for differentiation into melanotic tion step (iii) is performed until at least 85% of the cells are melanocytes, differentiated melanocytes, preferably at least 95%, more 0106 a. selecting the differentiated melanotic mel preferably 99%, even more preferably until all of the cells are anocytes in the culture, wherein the step of selecting differentiated melanocytes. comprises Geneticin treatment. 0094. The skilled artisan knows methods to determine 0107 The step of selection and/or isolation in the herein whether cells are differentiated melanocytes. For example the provided method may comprise isolating the melanocytes skilled artisan can determine whether cells express the mel using differential trypsinization and Subsequently Geneticin anocyte markers and not fibroblast markers. Known melano treatment. In other words, the step of selection and/or isola cytes markers are for example Tyrosinase, glycoprotein 100 tion in the herein provided method may comprise isolating the (gp100) variants, calcium-binding 5100 proteins, melanocytes using geneticin treatment along with differential Microphtalmia-associated Transcription Factor (MITF) as trypsinization. genes characteristically expressed in melanocytes; further on, 0.108 Different methods are known to select and isolate DOPA-tautomerase activity and Subsequent melanin content differentiated melanotic melanocytes from cultures. Methods are reliable markers of melanocyte function. Fibroblast mark of differential trypsinization or Geneticin treatment are ers are for example Cluster of Differentiation 90 cell surface known methods (In Vitro. 1984 May; 20(5):447-50, Exp Cell protein (CD-90), Cluster of Differentiation34 (CD34), Fibro Res. 1982 December; 142(2):309-15, Proc Natl Acad Sci blast Surface Antigen (SFA), Heat Shock Protein 47 (HSP47). USA. 1982 March; 79(6):2018-22). The skilled artisan is Fully differentiated melanocytes and fibroblasts also assume aware of these methods. However, the inventors unexpectedly characteristic morphology. found that the purity and amount of melanocytes obtained by 0095. However, in one embodiment of the method for the above-outlined method can be further enhanced by using generating melanocytes according to the present invention differential trypsinization and/or Geneticin treatment. Hence, the method comprises a further step (iv) selecting and isolat in one embodiment of the method for generating melanocytes ing the differentiated melanocytes in the culture. according to the present invention melanotic melanocytes are 0096. In accordance with the above, herein provided is a isolated in step (iv) using differential trypsinization and/or method for generating melanocytes from stem cells compris Geneticin treatment. The inventors now unexpectedly found ing the steps of that the selection and isolation of differentiated melanotic melanocytes is greatly enhanced if both methods are per 0097 (i) removing the bulb of an epilated human hair; formed, preferably the differential trypsinization is per 0.098 (ii) incubating the remaining part of the epilated formed at every passage of the cell culture, whereas the Gene hair with a collagen degrading agent to separate stem ticin treatment is applied once in the course of 24 to 48 hours, cells from the outer root sheath; preferably once in the course of 48 hours. In one embodiment 0099 (iii) cultivating the separated stem cells from step of the present invention the melanotic melanocytes are iso (ii) in a medium that induces differentiation and stimu lated using differential trypsinization combined with a single lates growth of stem cells, melanocyte precursors and Geneticin treatment. melanocytes, wherein the medium comprises one or 0109 Differential trypsinization is known by those skilled more growth factors for differentiation into melanotic in the art. In one embodiment of the differential trypsinization melanocytes, the cells are washed with a buffer and challenged with Trypsin 0100 (iv) isolating the differentiated melanotic mel and Ethylenediaminetetraacetic acid (EDTA). In a more pre anocytes in the culture, wherein melanocytes are iso ferred embodiment of the trypsinization the cells are washed lated in step (iv) using anatomic selection of hair root with HEPES buffer and challenged with 0.04% Trypsin/0. Subsections. Step (iv) may comprise, in the alternative or 03% EDTA for 4 minutes. The 4 minutes represent the time in addition to using anatomic selection of hair root Sub sufficient for melanocytes to withdraw their dendrites under sections, differential trypsinization. Step (iv) may, in the the stimulus of trypsin and detach from the polystyrene Sur alternative or in addition to using anatomic selection of face of the cell culture vessel. Fibroblasts and keratinocytes hair root Subsections, and/or in the alternative or in addi adhere to the surface with their large soma and therefore with tion to differential trypsinization, Geneticin treatment. more adherence force, and they are more robust to the stress 0101. As mentioned above, the step of incubating the stimuli altogether, therefore they need longer exposure to remaining part of the epilated hair with a collagen degrading trypsin than the melanocytes and still remain adherent after 4 agent to separate stem cells from the outer rootsheath accord minutes of trypsin digestion. The Supernatant with loose ing to the herein provided method can contain releasing/ trypsinized pure melanocytes is taken into the next passage of migrating/leaving of the stem cells from e.g. the extracellular the culture and cultivated further. matrix. 0110 Geneticin treatment is known by those skilled in the 0102 Provided is a method for generating melanocytes art. Geneticin is an antibiotic, a known inhibitor of the 80S from stem cells comprising the steps of ribosomes and thereby of protein synthesis. Exposure to Geneticin has most impact on the fast-dividing cells with (0103) 1. removing the bulb of an epilated human hair; large need for protein sysnthesis, in case of the ORS primary 0104 2. incubating the remaining part of the epilated culture keratinocytes and fibroblasts, which happen to be the hair with a collagen degrading agent to separate stem major contaminants of the targeted melanocyte culture. On cells from the outer root sheath; the other hand, the melanocytes are slow-dividing and there 0105 3. cultivating the separated stem cells from step fore quite immune to Geneticin stress, therefore, they all (ii) in a medium that induces differentiation and stimu survive the Geneticin treatment that takes up to 48 hours. lates growth of stem cells, melanocyte precursors and Upon this procedure, one can observe healthy adherent mel US 2015/0O86513 A1 Mar. 26, 2015 anocytes and apoptotic and necrotic rounded half-loose fibro also show important differences compared to other melano blasts and keratinocytes in the treated culture. The detached cyte cells, like NHEM. For example, the melanocytes obtain majority of the out-selected fibroblasts and keratinocytes are able by the herein provided method have different cell surface flushed away with HEPES buffer and those that remain markers as compared to NHEM. Due to these different sur attached do not survive and are hereby seceded within the face markers, the herein provided melanocytes can easily be next passage. In one embodiment Geneticin is applied for 12 distinguished from e.g. NHEM cells by routine methods, like hto 48 h, preferably for 24h to 48 h, more preferably for about FACS, as demonstrated in Example 3. 24h, yet more preferably for about 48 h. The skilled artisan 0115 Furthermore, the melanocytes obtainable by the will be able to decide on the time for which Geneticin is method of the invention have a smaller soma and shorter applied. This treatment blocks translation of the eukaryotic dendrites than other melanocytes, like adult skin melanocyte cells. The cells that divide quickly are much more sensitive to cells, such as NHEM; see Example 3 and FIG. 17. Also the Geneticin, since they have a very active protein production number of dendrites is slightly lower in the melanocytes (and hereby a very intensive translation). The inventors found obtainable by the method of the invention compared with that fibroblasts and keratinocytes divide quickly and they are other melanocytes, like adult skin melanocyte cells, such as selected away, whereas the melanocytes divide slowly and NHEM. Cell surface of NHEM is in average 23% larger than survive the selection. Hence, the skilled artisan can decide on that of melanocytes of the present invention (HM cells) and the time for which the cells are treated with Geneticin by their dendrites are 39% longer. Also, NHEM tend to have monitoring the amount of fibroblasts and keratinocytes and more dendrites than HM in average. establish an optimal time of treatment based on that. 0116 NHEM cells display an average surface of 377+135 0111. Furthermore, it has been demonstrated herein that um, whereas the HM display 290+74 um; hereby, NHEM the herein provided method allows the advantageous and soma is 23% larger in surface than HM soma (p=0.0451). improved generation of melanocytes. Following the medium Accordingly, the melanocytes obtainable by the method of air-interface cultivation of follicles as described herein (see the invention have an average Smaller Surface or Soma than e.g. method section of Example 1) the melanocytes can be other melanocytes, like adult skin melanocyte cells, such as further cultivated in adherent culture and regularly passaged. NHEM. The average surface or soma of the melanocytes Accordingly, the herein provided method can advantageously obtainable by the method of the invention is at least 5, 6, 7, 8, comprise adherent culture of melanocytes. The herein pro 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, preferably at least 20% vided method can further comprise passaging of melano Smaller than the average surface or Soma of other melano cytes. cytes, like adult skin melanocyte cells, such as NHEM. 0112 For example, the melanocytes can be passaged 1, 2, 0117 Dendrites of NHEM are in average 35.73+15 um 3, 4, 5, 6, or more times (and up to 15 times to increase the long, which is 39% longer than the 21.57+10 um long den melanocyte number or yield. “Passaging” means that a cer drites of the melanocytes obtainable by the method of the tain number of cells (e.g. sufficient to cover more than 10% of invention (HM cells) (p=0.045). Accordingly, the melano the cell vessel surface, preferably 100.000 cells per 25 cm cytes obtainable by the method of the invention have shorter vessel) are taken from a first medium-air-interface follicle dendrites on average than other melanocytes, like adult skin culture which has been produced in accordance with the melanocyte cells, such as NHEM. The average dendrite of the present invention. These cells are cultivated under adherent melanocytes obtainable by the method of the invention is at culture conditions described herein, like adherent culture in least 10, 15, 20, 25, 30, 31, 32, 33 or 34, preferably at least Example 1. When the cells proliferate and reach more than 35% shorter than the average dendrite of other melanocytes, 80% confluence on given surface, they are detached by like adult skin melanocyte cells, such as NHEM. trypsin and taken to a larger vessel Surface. This procedure? 0118. Furthermore, as shown in Example 3, NHEM have a cycle is called herein passaging. slightly higher average number of dendrites (3.13+1.19) than 0113. This procedure can be performed many times to HM (2.47+0.64), (p=0.065, non-significant). maintain a melanocyte population for a long period of time. 0119 Table 5 provided herein further below shows a com This procedure can also be used to produce melanocytes in prehensive Summary of markers characteristic for melano accordance with the present invention. cytes obtainable or obtained by the method of the present 0114. The present invention provides melanocytes obtain invention (a "+" indicates presence/expression of the able or obtained by the herein provided method for generating marker). The melanocytes obtainable or obtained by the melanocytes. As shown in the appended examples, the mel method of the present invention may express one or more of anocytes (also referred to herein as human hair follicle mel said markers of Table 5. In other words, the herein provided anocytes, or short “HM) obtained in accordance with the melanocytes may express one marker alone or in combination present invention have features which are characteristic of with any one of the other markers (i.e. in combination with melanocytes, like characteristic dendritic morphology, one or more of the other markers). expression of marker proteins (e.g. Tyrosinase, variants of I0120 Table 3 provides markers which are expressed/ gp100 protein, NKI-beteb, HMB45. Further, the herein pro present in the herein provided melanocytes and which are not vided melanocytes show reaction to isobutyl-methylxanthine expressed/present in NHEM cells. Also in this context, the (IBMX). They also show activity of Tyrosinase, CDT/TRP-2, herein provided melanocytes may express one marker alone and TRP-1, utilised in converting L-DOPA into melanin as or in combination with any one of the other markers (i.e. in well as spontaneous melanin production. The melanocytes combination with one or more of the other markers) of Table display melanin content and are characterized by the absence 3. As described herein, the presence of one a marker alone or of a CD90 signal/expression. Due to these characteristic fea in combination with other marker(s) can be determined/ tures, the herein provided melanocytes (HM) are a good sub analysed. stitute for normal human epidermal melanocytes (NHEM); I0121. As shown herein, the melanocytes obtainable in see Example 1 and 3. Yet, the herein provided melanocytes accordance with the invention express one or more well US 2015/0O86513 A1 Mar. 26, 2015

known marker genes as exemplified in the Table 3 and 5 I0128 Quantification of the level of the gene product, in herein. Non-limiting examples of Such marker genes are particular RNA/mRNA can be performed by taking advan CD1a, CD28, CD77, CD79b, CD137 Ligand, CD140a, tage of Northern blotting techniques, hybridization on CD140b, CD282, HLA-A2, HLA-DQ, CD104, CD106, microarrays or DNA chips equipped with one or more probes CD142. or probe sets specific for mRNA transcripts or PCR tech 0122. It is preferred that the herein provided and described niques referred to above, like, for example, quantitative PCR melanocytes express at least one of the above genes alone or techniques, such as Real Time PCR. A skilled person is in combination with one or more of these or further marker capable of determining the amount of the component, in genes as exemplified in Table 3 and 5. particular said gene products, by taking advantage of a cor 0123. The nucleic acid or protein sequences of these relation, preferably a linear correlation, between the intensity marker genes to be used herein may be deduced from the ofa signal and the amount of the component to be determined. respective databases like NCBI. The tables also provide I0129. A person skilled in the art is easily in the position to accession numbers of the respective database. determine the expression level of the herein described marker 0.124. The denomination "+” (e.g. CD1 a+) indicates the genes based on his common general knowledge and the teach presence of the respective marker, i.e. indicates an expression ing provided herein. In particular, a skilled person can readily of the marker that may be reflected in a detectable expression decide whether a certain marker is or is not expressed in a level (protein, mRNA etc.) of this marker gene. Generally, a melanocyte obtainable by the herein provided method. cell is classified as “melanocyte obtainable or obtained by 0.130. An exemplary method to be used herein and used in the herein provided method if at least one of the herein the appended examples for determining expression of surface described marker genes is present at a detectable expression proteins, is based on combination of fluorophore-labelled level. Methods for detecting and evaluating expression levels antibody binding and analysis by flow cytometry. The method of Such genes are well known in the art and are also demon is based on running a single-cell Suspension through a capil strated in the appended examples. Accordingly the melano lary exposed to a laser beam. Difraction off the side of the cell cytes obtainable by the herein provided method can be mel walls (Forward Scatter, FSC) and through its intracellular anocytes (CD1a+, CD28+, CD77+, CD79b+, CD137 content (Side Scatter, SSC) can be quantified and interpreted Ligand--, CD140a+, CD140b-- CD282+, HLA-A2+, HLA as relative size (FSC readout) or complexity (SSC readout). DQ+, CD104+, CD106+, and/or CD142+). Both readouts can be plotted on X and y axis of a graph to 0.125 For example, the protein expression level can be define basic morphological parameters read out at the begin detected by taking advantage of FACS, immunoagglutina ning of the analysis (FIG. 17, a). Forward- and Side Scatter tion, immunoprecipitation (e.g. immunodiffusion, immu measurements are independent of the fluorescent signal. nelectrophoresis, immune fixation), western blotting tech Fluorescent signal itself is directly proportional to the number niques, (in situ) immunohistochemistry, (in situ) of attached fluorophores, in the case described in Example 3: immunocytochemistry, affinity chromatography, enzyme surface receptor markers labelled with fluorescing antibody immunoassays, and the like. These and other Suitable meth (FIG. 17, b). High-throughput measuring flow-cytometer ods of contacting proteins are well known in the art and are, devices such as BD FACS-Array, used in Example 3, allow for example, also described in Sambrook and Russell (2001), quick measurement of antibody panels without a risk of fluo Molecular Cloning: A Laboratory Manual, CSH Press, Cold rophore fadeout or loss. Spring Harbor, N.Y., USA; Burke et al. (1990), Methods in I0131 The principle and methodology of cell surface Yeast Genetics, A Laboratory Course Manual, Cold Spring marker analysis used herein is demonstrated and described in Harbor Laboratory Press, 1990.) Example 3 (see e.g. Methods section thereof) 0126. If the gene product is an mRNA, contacting and 0.132. The inventors unexpectedly found that the melano binding can be performed by taking advantage of Northern cytes derived by the methods for obtaining melanocytes blotting techniques or PCR techniques, like in-situ PCR. according to the present invention show extraordinarily good These and other suitable methods for binding (specific) properties as graft constituents. Hence, the present invention mRNA are well known in the art and are, for example, further relates to a method for producing an autograft, described in Sambrook and Russell (2001, loc. cit.). homograft or an allograft comprising melanocytes, said 0127 Quantification of the protein level can be performed method comprising the steps of by taking advantage of the techniques referred to above, in 0.133 (i) providing a suspension comprising melano particular Western blotting techniques. Generally, the skilled cytes obtainable by a method for generating melano person is aware of methods for the quantitation of polypep cytes from stem cells according to the present invention; tides. Amounts of purified polypeptide in solution can be 0134) (ii) providing a biocompatible carrier, and determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on 0.135 (iii) cultivating the melanocytes on said biocom specific binding, e.g. of antibodies. Specific detection and patible carrier. quantitation methods exploiting the specificity of antibodies 0.136 The Suspension comprising the melanocytes may comprise for example immunohistochemistry (in situ). West further comprise fibrin, thrombin, or both. ern blotting combines separation of a mixture of proteins by 0.137 The melanocytes may be further applied along the electrophoresis and specific detection with antibodies. Elec following routes: in the form of liquid Suspension, as an trophoresis may be multi-dimensional Such as 2D electro aerosol (spray), grown on a biocompatible carrier as a single phoresis. Usually, polypeptides are separated in 2D electro melanocyte culture or combined with keratinocytes to form phoresis by their apparent molecular weight along one an epidermal equivalent or as a cellular epidermal equivalent, dimension and by their isoelectric point along the other direc composed of a basal melanocyte layer and stratified kerati tion. nocytes. In a further embodiment the melanocytes may be US 2015/0O86513 A1 Mar. 26, 2015

combined with fibroblasts and keratinocytes to form a dermal for such method of scaffold production. In a preferred equivalent comprising melanocytes, keratinocytes and fibro embodiment of the invention the biocompatible carrier con blasts. sists of at least one of the materials selected from the group 0.138. In one embodiment the autograft, homograft or consisting of polycaprolactone (PCL), collagen, human col allograft to be produced by the method according to the lagen I, human collagen IV, human collagen V. shortened and present invention comprises keratinocytes. chemically cross-linked or otherwise modified collagen 0139 Methods for obtaining and/or stratifying kerati chains, fibrin, gelatine, and epidermis-equivalent kerati nocytes are known by those skilled in the art. However, the nocyte-based carriers. Biocompatible carriers comprising keratinocytes may be applied with the melanocytes in differ keratinocytes are known by those skilled in the art and are ent ways. For example, the keratinocytes may be comprised in commercially available. In one embodiment of the present the resulting autograft, homograft or allograft. Hence, in one invention the biocompatible carrier comprising keratinocytes embodiment of the method for producing an autograft, is an Epidex(R) keratinocyte-based epidermal sheath (Euro homograft or allograft according to the present invention the derm Biotech & Aesthetics, Germany). In a particularly pre method further comprises step (iv) stratifying keratinocytes ferred embodiment the biocompatible carrier consists of on the melanocytes cultivated on said biocompatible carrier; polycaprolactone (PCL). Methods to produce such biocom and step (V) cultivating said keratinocytes on the biocompat patible carrier are known by those skilled in the art. ible carrier comprising the melanocytes. Furthermore, the 0142. In one embodiment the melanocytes according to keratinocytes may be provided separately, e.g. as a Suspen the present invention are provided in a Suspension with col sion or spray, optionally on a further biocompatible carrier. lagen and fibrin. 0140. The inventors found that the melanocytes may be 0143. It may be desirable that the autograft, homograft or applied to the area to be treated in different ways. For allograft derivable by the methods according to the present example, a solid autograft, homograft or allograft may be invention is applied through different grafting application used. In this case the biocompatible carrier used in the method routes, such as Suspension, aerosol, Solid grafts as epidermal for producing the autograft, homograft or allograft serves as a equivalent (using a solid biocompatible carrier as a scaffold). scaffold and may be provided in the shape of the area to be The properties of the autograft, homograft or allograft for the treated. However, in other cases it may be advantageous that different routes may be adapted through the properties of the the autograft, homograft or allograft is provided in a non biocompatible carrier. Hence, the biocompatible carrier may predetermined shape. For such case the inventors found that be provided in different forms. The biocompatible carrier the provision as a Suspension or a spray shows great advan may be for example provided in a macroscopic form, such as tages. The Suspension or spray may also be provided without a solid biocompatible carrier as a scaffold, e.g. in a shape a biocompatible carrier. However, in one embodiment the fitting to the area to which the homo- or allograft shall be Suspension or spray comprises a biocompatible carrier as provided. Autograft, homograft or allograft can be provided outlined below, wherein the biocompatible carrier consists of as a Suspension and/or aerosol, i.e. it can be applied as such on microscopic particles (microscopic biocompatible carrier) the area to be treated or be used as a spray. Hence, in one polymeric network. Microscopic means that the particles or embodiment of the present invention the biocompatible car biocompatible carrier have a thickness in the range of rier is provided as a macroscopic matrix for the autograft, nanometre or micrometre. homograft or allograft to be produced. In another embodi 0141. In a preferred embodiment the biocompatible car ment the biocompatible carrier is provided as Suspension rier provides a three-dimensional structure consisting of a and/or spray. network of fibres. Such structure provides a scaffold for the 0144. The resulting autograft, homograft or allograft may cells which facilitates the cultivation, correct morphology also be prepared for stocking until used for treatment of the and functionality of the cells and furthermore increases the disease. Hence, the method for producing the autograft, adherence of the cells to the carrier and enhances the facili homograft or allograft according to the present invention may tated functionality of the graft. The inventors found that such further comprise the application of suited buffers and/or network of fibres serves as an efficient environment for the media. The skilled artisan will recognize that the resulting melanocytes and enhances their melanotic properties. Such autograft, homograft or allograft may be provided in different properties can be measured by the activity of Dopa-tau forms mostly depending on the form of the biocompatible tomerase and/or melanin production of the cells. The inven carrier used in the method, Supra. tors unexpectedly found that these properties are enhanced by 0145 The skilled person is able to adapt the period for the use of a biocompatible carrier according to the present cultivation of the melanocytes in the method for producing an invention. There is a variety of materials providing meshes, or autograft, homograft or allograft according to the present networks, whose fibres protrude in all directions, enabling the invention. The inventors found that the cultivation of the cells to interface the fibres. Furthermore, the fibres should melanocytes under the conditions that enable the formation of have a size? thickness that allows the cells to adhere to them. a stratum basale drastically enhances the treatment potential The inventors furthermore found that cells cultivated on such of the autograft, homograft or allograft by enabling a correct biocompatible carrier form a three-dimensional morphology morphology and cellular interactions, same as in epidermis. within these meshes, whereas on the flat surfaces in cell Hence, in one embodiment of the present invention the mel culture only two-dimensional networks of cells are formed. It anocytes are cultivated in the method for producing an will be acknowledged by the person skilled in the art that the autograft, homograft or allograft according to the present three-dimensional structure shall allow the melanocytes to invention in order to anatomically simulate formation of a migrate through the meshes formed by the network of the stratum basale. fibres and populate the entire scaffold. For example Loth, T. 0146 The skilled artisan knows how to determine whether (Electrospinning als Methode Zur Herstellung von fibrillaren the melanocytes form a stratum basale The stratum basale is Zellträgern aus bioabbaubaren Polymeren, 2009), provides a basal layer of the epidermis, where the melanocytes reside. US 2015/0O86513 A1 Mar. 26, 2015

The epidermis is composed of 4-5 layers depending on the embodiment the neutral cultivation medium comprises one or region of skin being considered. Those layers in ascending more components selected from the group consisting of order are the basal/germinal layer (stratum basale? germinati medium base, preferably DLM base or DMEM base, fibro Vum), spinous layer (stratum spinosum), granular layer (Stra blast growth factor, N6-2'-dibutyryl adenosine-3'-5'-cyclic tum granulosum), clear/translucent layer (stratum lucidum), monophosphate (db AMP), L-glutamine, 0.5% to 20% serum, and cornified layer(stratum corneum). The keratinocytes rep preferably 0.5% of foetal serum or 10% or less human serum. resent the majority of the cells in the upper four layers. There 0151. In a preferred embodiment the cultivation step(s) of fore the keratinocytes in culture can be stratified in order to the method for producing an autograft, homograft or allograft mimick epidermal anatomy (so called epidermal equiva is performed under hypoxic conditions; or normoxic condi lents). The inventors found that the provision of the autograft, tions. homograft or allograft that corresponds to epidermal anatomy 0152. Furthermore, in one embodiment the cultivation preserves the functionality of melanocytes and provides a may be performed as a medium-air interface or as a medium potential for eventual treatment of depigmentation diseases. covered epidermal equivalent. 0147 The inventors furthermore found that the cultivation 0153. The method for producing an autograft, homograft of the keratinocytes until the formation of a stratum corneum or allograft in a preferred embodiment is performed under the drastically enhances the treatment results of the autograft, following conditions: O content of approximately 5%, CO homograft or allograft. Hence in one embodiment of the content of approximately 5%, and a temperature of 37°C. (as present invention the keratinocytes are cultivated in a revealed in Dieckmannet al. Exp Dermatol. 2010 June; 19(6): medium-air interface until formation of a stratum corneum 543-5). prior to stratification of the method for producing an 0154 The skilled artisan knows methods for performing autograft, homograft or allograft according to the present cultivation and/or incubation of cells. For example the cells invention. Stratum corneum is the upper stratum of the epi may be covered with the medium or the cells may be present dermis. It consists of flattened keratinized keratinocytes. It at the medium-air interface. In one embodiment of the present can be morphologically determined in the cross sections of invention the biocompatible carrier is located at the medium the stratified epidermal equivalents that the upper stratum is air interface, i.e. the biocompatible carrier is in contact with keratinized. Also, a possibility of staining keratinized cells to the medium at its lower side and in contact with the air at its corroborate the morphological analysis exists. The person upper side. In another embodiment of the invention the bio skilled in the arts, hence, is able to determine when the kera compatible carrier with cells is covered with the medium, i.e. tinocytes have formed a stratum corneum. However, in a not in contact with the air. preferred embodiment of the method for producing 0.155. In a further preferred embodiment the incubation autografts, homografts or allografts according to the present step of the method for producing an autograft, homograft or invention the keratinocytes are cultivated for 8 to 20 days, allograft is performed under normoxic conditions, wherein preferably for 10 to 15 days, even more preferably for 11 to 12 the biocompatible carrier is covered with the incubation days. In a preferred embodiment the keratinocytes are culti medium. In another embodiment, the incubation step of the vated at a medium-air interface. method for producing an autograft, homograft or allograft is 0148 Medium and conditions for cultivation of kerati performed under normoxic conditions, wherein the biocom nocytes are known by the skilled person. A Suited cultivation patible carrier is at the medium/air interface. In yet another methodology, e.g. medium and conditions, is disclosed in Int embodiment, the incubation step of the method for producing Wound J. 2009 June; 6(3):226-32. an autograft, homograft or allograft is performed under 014.9 The inventors unexpectedly found that a ratio of hypoxic conditions, wherein the biocompatible carrier is at keratinocytes to melanocytes of 10:1 increases the efficacy of the medium/air interface. In yet another embodiment, the the autograft, homograft or allograft, by maintaining melano incubation step of the method for producing an autograft, cyte functions and cellular interactions similarly to that in homograft or allograft is performed under hypoxic condi epidermis, and hereby prospects of the eventual treatment of tions, wherein the biocompatible carrier is covered with the diseases as outlined herein below. Hence, in one embodiment incubation medium. of the method for producing an autograft, homograft or 0156 The incubation step of the method for producing an allograft according to the present invention, the keratinocytes autograft, homograft or allograft is performed until the are stratified on top of the cultivated melanocytes in a ratio of desired properties are reached. These include the biome 10:1. chanical stability, i.e. the resulting biocompatible carrier cov 0150. The cultivation in step of the method for producing ered with keratinocytes and melanocytes shall provide a an autograft, homograft or allograft should be performed in rigidity that allows the application of the derived autograft, order to cultivate both, melanocytes and keratinocytes. In one homograft or allograft to the target region without the col embodiment the cultivation step is performed in a neutral lapsing of the autograft, homograft or allograft. Furthermore, cultivation medium. A "neutral medium' in context with the cross sections along with staining of melanin, tyrosinase, present invention relates to a medium that does not contain gp100 variants, should show presence of melanocytes at the Supplements that affect melanocytes or keratinocytes. Hence, lower phase of the graft, which is the anatomic equivalent of in one embodiment of the present invention the neutral culti the stratum basale. Melanocytes stretch their dendrites into Vation medium does not comprise a growth factor selected the upper phase, composed of keratinocytes (anatomically from the group consisting of B-adrenergic receptor ligands, equivalent to the upper strata—spinous layer (stratum spino such as epinephrine and derivatives thereof, and/or Ca" and/ Sum), granular layer (stratum granulosum), clear/translucent or Vitamin C and/or EGF and/or bRGF/FGF2 and/or Endot layer (stratum lucidum), and cornified layer (stratum cor helin-1, C-MSH, SCF and/or NGF-B and/or HGF and/or Gen neum). Each melanocyte interfaces with up to 40 kerati tamycin, epidermal growth factor, hydrocortisone, cholera nocytes, forming so-called epidermal units. Cell Soma and the toxin, adenine, triiodothyronine, and alpha-MSH. In a further dendrites of melanocytes should contain visible melano US 2015/0O86513 A1 Mar. 26, 2015 somes of type I-IV, especially at the points of interface with In another embodiment the autograft, homograft or allograft the keratinocytes. Melanosomes are delivery packages— is applied to the area to be treated as a solid autograft, vesicles that serve for secretion of melanine. Melanosomes homograft or allograft. In a preferred embodiment the contain melanine and also various isoforms of glycoprotein autograft, homograft or allograft is applied on the area to be 100 (gp100), therefore they may be observed by staining of treated after dermabrasion as outlined above. In a one gp100 proteins (for example, HMB45 and NKI-beteb vari embodiment of the present invention the autograft, homograft ants). or allograft comprises separated melanocytes obtainable by a 0157. In a preferred embodiment the incubation step of the method according to the present invention. method for producing an autograft, homograft or allograft is 0163 The application of the autograft, homograft or performed for 1 to 10 days, preferably 2 to 3 days. allograft may be performed in different ways. For example if 0158. The skilled artisan furthermore is aware of the fact a solid autograft, homograft or allograft is used, the autograft, that the incubation temperature has to be adapted to the needs homograft or allograft may be directly applied on the area to of the cells. All the incubations within the present invention be treated. However, as outlined above the autograft, are performed at a temperature of approximately 20° to 37 homograft or allograft in one embodiment of the present C., preferably all the incubation steps of the methods dis invention is a Suspension or spray. In such case the autograft, closed herein are performed at 37°C. homograft or allograft is applied as a Suspension onto the area 0159. The inventors found that an autograft, homograft or to be treated or sprayed thereon, respectively. allograft according to the present invention and/or melano 0164. In one embodiment melanocytes and keratinocytes cytes according to the present invention can be used for the are applied onto the area to be treated. If this is performed treatment of depigmentation of the skin. Hence, the present with Suspensions or sprays with separated melanocytes and invention also relates to an autograft, homograft or allograft keratinocytes it may be performed in different orders. For according to the present invention and/or melanocytes example a suspension or spray comprising melanocytes according to the present invention for use intreating a disease, according to the present invention may be mixed with a Sus preferably for use in treating Scars and/or for use in treating a pension comprising keratinocytes as outlined herein and then disease selected from the group of leukoderma, vitiligo, be applied together as a suspension or a spray. In another quadrichrome vitiligo, Vitiligo ponctué, syndromic Albinism, embodiment a Suspension comprising melanocytes accord Such as AleZZandrini syndrome, Hermansky-Pudlak Syn ing to the present invention is applied on the area to be treated drome, Chédiak-Higashi syndrome, Griscelli syndrome (Ele as a Suspension or as a spray and then a suspension compris jalde syndrome, Griscelli syndrome type 2 and Griscelli syn ing keratinocytes as outlined herein is applied on said area as drome type 3), Waardenburg Syndrome, Tietz syndrome, a suspension or a spray. The spray may further comprise Cross-McKusick-Breen syndrome, ABCD syndrome, Albi fibroblasts and keratinocytes as outlined above. nism-deafness syndrome and Vogt-Koyanagi-Harada Syn 0.165. In one embodiment of the present invention the area drome, oculocutaneous albinism, hypomelanosis (idiopathic to be treated is sealed after application of the respective sus guttate hypomelanosis, phylloid hypomelanosis, progressive pension or solid autografts, homografts or allografts. macular hypomelanosis), piebaldism, nevus depigmentosus, (0166 The area treated as outlined herein is in one embodi post-inflammatory hypopigmentation, pityriasis alba, Vaga ment left unstimulated after treatment to recover and gain bonds leukomelanoderma, Yemenite deaf-blind hypopig own pigmentation. In a further embodiment the area is stimu mentation syndrome, Wende-Bauckus syndrome, Woronoffs lated with UV light using suberythematogenous doses of 311 ring, amelanism, Leucism, and diseases associated with nm UVB 2-3 times weekly, as described previously (Derma depigmentation of the skin. tology. 2009; 218(4):342-3). 0160. In one embodiment the present invention relates to 0167. The autograft, homograft or allograft and/or mel an autograft, homograft or allograft derivable by a method for anocytes according to the present invention can also be used producing an autograft, homograft or allograft according to for aesthetic purposes, for example, in cosmetics. Accord the present invention for use in treatment of a disease. In a ingly, a cosmetic composition comprising the autograft, preferred embodiment, the present invention also relates to an homograft or allograft and/or melanocyte(s) according to the autograft, homograft or allograft according to the present present invention is provided. Likewise, the present invention invention and/or melanocytes according to the present inven relates to a method for the cosmetic treatment comprising tion for use in the treatment of scars and/or the treatment of a treating an individual or Subject with an autograft, homograft disease selected from the group of leukoderma, vitiligo, Syn or allograft and/or melanocytes as provided herein. dromic Albinism piebaldism, nevus depigmentosus, postin 0.168. The following non-limiting examples and figures flammatory hypopigmentation, and diseases associated with illustrate the invention: depigmentation of the skin. 0161 The autografts, homografts or allografts may be EXAMPLES applied directly onto the depigmented skin area or scars. 0169. The following examples are included to demon However, the area to be treated may in one embodiment be strate preferred embodiments of the invention. It should be pretreated. In a preferred embodiment the skin area to be appreciated by those of skill in the art that the techniques treated according to the present invention is pre-treated by disclosed in the examples which follow represent techniques dermabrasion of an epidermal layer corresponding to the discovered by the inventors to function well in the practice of thickness of the graft, preferably by usingerbium:YAG Laser. the invention, and thus can be considered to constitute pre 0162. In a preferred embodiment the autograft, homograft ferred modes for its practice. However, those of skill in the art or allograft derivable by a method for producing an autograft, should, in light of the present disclosure, appreciate that many homograft or allograft according to the present invention for changes can be made in the specific embodiments which are use in treating a disease the autograft, homograft or allograft disclosed and still obtain a like or similar result without is applied to the area to be treated as a suspension or a spray. departing from the spirit and scope of the invention. US 2015/0O86513 A1 Mar. 26, 2015 14

Example 1 Adherent Culture (0176 Cells were further cultivated in DLM medium until Cultivation, Expansion, Differentiation, Selection of they reached over 90% confluence. They were then Pure Melanocyte Culture trypsinized, harvested and taken into an advanced primary culture. The obtained outer root sheath cells were seeded on Experimental Design and Methods an adherent surface of the 10 cm wells culture dishes (Corn ing) in the third week of cultivation (day 18+2). The start of Hair Biopsy the culture at the adherent Surface was marked as start passage (p0). 0170 To begin the procedure of extracting the melano cytes fromhair roots, one needs to pluck 30 to 80 anagen hairs Differential Trypsinization from the temporal region of the scalp. The biopsy is painless, Scarless and requires less hairs than one spontaneously looses (0177. The cells were further differentially trypsinized on daily basis. Up to five hairs of the temporal area are held upon reaching 80% confluence at the uncoated adherent Sur close to the root with a disinfected forceps and pulled in the face. After 4 minutes of 0.04% Trypsin/0.03% EDTA incu direction of the hair growth. The hairs were stored in Phos bation at room temperature, detached melanocytes were col phate Buffer Saline (PBS, without Calcium addition until lected and passaged into the T25 cell culture flasks. The rest preparation. of the cells, keratinocytes and fibroblasts, remained adherent and they were not transferred into the next passage of the culture. Differential trypsinization was performed at every Selection of the ORS Mid Part, Preparation passage as routine measure of culture maintenance. 0171 The long part of the hair shaft was cut off and a short part of the shaft was left for handling purposes. The hair roots Geneticin Treatment were shortened from the proximal end by cutting the hair bulb 0.178 Growth of keratinocytes and fibroblasts in later off (FIG.1). As the bulb usually yields over 90% fibroblasts in stages of differentiation was foremost prevented by treating our experience, a significant part of cellular contaminants is the whole culture with Geneticin (50 ug/ml) for 48 h at day avoided by removing this portion. 22+2 of differentiation. The effect of Geneticin is to block 0172. The hairs were serially washed five times for one translation in all cells, hereby affecting primarily the quickly minute in separate 50 ug/ml Gentamycine (Ratiopharm) and dividing cells, e.g. keratinocytes and fibroblasts. The metabo 10 ug/ml Amphotericin (Amimed) in Dulbecco's Modified lism and division of melanocytes in the culture is slow, there Eagle Medium (DMEM) and subsequently washed with PBS. fore they remained unaffected by short treatment with Gene ticin (FIG. 2, F-H). Loosening of Extracellular Matrix (0179 The advanced primary culture of ORS cells was maintained in DermaLife Melanocyte medium (Life Line 0173 Extracellular matrix was loosened by digestion with Cell Technology, Walkersville, Md., USA), DLM medium solution of Collagenase V (Sigma-Aldrich) in Phosphate base of low bovine serum content (0.5%), L-Glutamine (6 Buffer Saline (PBS), 5 mg/ml, at 37° C. for 10 mM. mM), insulin (5ug/ml), Vitamin C (50 g/ml), epinephrine (1 uM), StiMel factor cocktail (1%) and gentamycine (50 Media ug/ml). The medium did not contain cholera toxin nor any tumor promoters such as phorbol 12-myristat 13-acetate 0.174. The hair follicles and ORS cells were cultivated in (PMA, TPA) DermaLife Melanocyte medium (Life Line Cell Technology, Walkersville, Md., USA), DLM medium base of low bovine Control Cells serum content (0.5%), L-Glutamine (6 mM), Insulin (5 ng/ml), Vitamin C (50 ng/ml), Epinephrine (1 nM), StiMel 0180 Epidermal melanocytes (normal human epidermal factor cocktail (1%) and Gentamycine (50 ng/ml), according melanocytes, NHEM, PromoCell) were cultivated in the Mel to the manufacturers specifications. The medium did not con anocyte M2 Basal Medium supplemented with M2 Supple tain cholera toxin nor any tumour promoters such as phorbol ment Mix (Promocell) upon the manufacturers guidelines, at 12-myristate 13-acetate (PMA, TPA). 37° C., under normoxic conditions. The cells were used as fully differentiated control cells. 0181 Fibroblasts used as non-melanotic control were cul Medium-Air Interface Cultivation Conditions tivated in DMEM, supplemented with 10% bovine serum, 2 0.175. The shortened hair roots were placed on 24 mm mM L-Glutamine, and 100 U/ml penicilline, as well as 0.1 Transwell(R) nylon-meshes with 0.4 um Pore Polyester Mem mg/ml streptomycine. brane Insert (Corning) and grown in medium-air-interface 0182 Production of the biocompatible carrier (PCL scaf conditions. Nutrition was supplemented by 1 ml DLM folds) medium (DermaLife), the amount sufficient to enable contact 0183 The biocompatible carrier (in this case a scaffold with the mesh and the cells from underneath, hereby supply consisting of PCL: referred to as PCL scaffolds) were pro ing the necessary nutrients. The upper parts of the follicles duced by the standard electrospinning procedure, as revealed were exposed to hypoxic gas mixture, 5% O, 5% CO, and in Loth, T., Electrospinning als. Methode Zur Herstellung von 90% N. The hair roots were allowed to expand for 2 weeks fibrillaren Zelltragern aus bioabbaubaren Polymeren. 2009. and were then covered with DLM medium. This period is 0.184 Polycaprolacton (PCL)-Fiber Meshes originate in usually sufficient to reach up to 40% confluence on the mesh electrospinning process that creates a network of fibers by (FIG. 1 e). Subjecting a jet of polycaprolactone solution to high Voltage. US 2015/0O86513 A1 Mar. 26, 2015

Polimer solution is prepared from the PCL powder solved in mann L. Kuphal S. Melanoblasts in culture as an in vitro Chloroform (CHC13): Methanol (MetOH) (7:1) agitated for 1 system to determine molecular changes in melanoma. Exp h. The solution was placed in a syringe with a blunt-end Dermatol. 2011 May; 20(5):435-40). needle and mounted onto a syringe pump (KD Scientific Inc., 0191 The melanocytes were characterized based on mor USA). DC current was connected to both the syringe needle phological criteria for melanocytes reported by Bosserhoffet and grounded collector glass plate, forming a closed circuit. al. The set of criteria used in this study in terms of melanotic This creates an electric field between the PCl polymer solu features, proliferation and attachment helped us discriminate tion and the glass collector plate and leads to the formation of the derived Human melanocytes HM cells, abbreviated from a continuous jet between a maintained droplet at the top of the Hair follicle-derived Melanocytes from melanocyte precur needle and the glass collector plate. As the jet strikes against sors. Obtained cells in the end culture were melanotic, pro the glass plate, it assumes the future network formation. Solu liferated at comparable pace as NHEM epidermal melano tion of chloroform and methanol evaporates quickly, and the cytes and weakly adhered to the surfaces of cell culture PCL fibre remains in the mesh. The mesh is withdrawn from vessel, unlike the amelanotic, quickly dividing and strongly the glass by 100% ethanol. Finally, the fibre mesh is dried out adherent precursors reported by Bosserhoff. using an exicator device coupled to a membrane Vacuum 0.192 Morphological criteria for melanocytes were com pump (VP 820; VWR International GmbH, Deutschland). bined with the expression of marker proteins: Tyrosinase 0185. By combining the parameters of electrospinning (FIG. 3e) and variants of gp100 protein (melanosome procedure, varying fibrethickness, fibre orientation and mesh marker) NKI-beteb (FIG. 3d) and HMB45 (FIG. 3?). Fur thickness can be obtained. In this application, two basic thermore, reaction to 3-isobutyl-1-methylxanthine (IBMX) parameter sets were used to form 100 um thick meshes net (FIG. 12.f. 12 h), activity of Tyrosinase, Dopachrome-tau worked from thin and thick fiber. Thin fibre mesh (2-3 um tomerase/Tyrosinase-related protein 2 (DCT/TRP-2) was diameter) were obtained by using 14% PCL at flow-rate 2 tested as well as the DHIC-oxidase/Tyrosinase-related pro ml/h, with the electrode 18.5 cm apart from the collector tein 1 activity (TRP-1) in the L-DOPA conversion test (FIG. electrode, using a 22 Gauge needle to generate the jet from a 12 e, 12 g). Moreover, melanin content was determined at 0.4-0.5 ml droplet. Thick fibre mesh (8-10 um diameter) 13.7 pg/cell in unstimulated HM cells and at 22.6 pg/cell after were obtained by using 20% PCL at flow-rate 10 ml/h, with converting L-DOPA into melanin. the electrode 30 cm apart from the collector electrode, using 0193 Expression of CD90 was used as a criterion for a 14 Gauge needle to generate the jet from a 0.4 to 0.5 ml undifferentiated cells, by rule fibroblasts (positive CD90 sig droplet. nal, FIG.12c), and as exclusion factor for differentiated mel 0186 The structure, fibre orientation and fibre thickness anocytes (absence of CD-90 signal, FIG.12d). were analyzed and quantified by the means of light micros 0194 mmunofluorescence copy (Nikon Eclipse TE 2000-S; Nikon Corporation/Instru (0195 Melanocytes ments, Deutschland), and electron microscopy (Raster elec 0196. The cells grown in chamber slides coated with tron microscope CS 44; CamScan: Obducat CamScan Ltd, gelatine were washed with PBS and fixed with ice-cold Para UK). formaldehyde (PFA, 4%) for 8 minutes, then washed with 0187. The scaffolds were circle-shaped with a sterile hol PBS again. The cells were blocked with 0.5 ml of 2% bovine low punch, with an 8 mm diameter so that they covered the 10 serum albumin and permeabilised with 0.1% Triton-PBS 7 mm wide bottom of the 96-well plate in calibrated condi Solution for 1 h. Solutions of primary mouse-generated anti tion. They were sterilized with 75% ethanol for 30 min, bodies against NKI\beteb Tyrosinase and HMB-45 (Abcam) washed with PBS five times. Before the cells were seeded were set at 2% in PBS with 2% BSA/0.1% Triton and incu onto the scaffolds, the material was calibrated in the DLM bated with the cells on scaffolds overnight at 4° C. After medium or PC-1 medium for 24 hours. washing with 2% BSA/0.1% Triton solution, biotinylated 0188 The cells were seeded onto the scaffolds, 10.000 anti-mouse secondary antibody was added to the cells and cells pro scaffold, suspended in 200 ul medium. incubated for 1 h, then washed. Solution of 2% BSA-PBS/0. 1% Triton/0.25% IGG was incubated with the cells for one Characterization of Cell Types hour, then washed. Finally, Streptavidine-coupled Alexa594(R) and Alexa 488R) fluorochrome 0.25% solution in 0189 The cells were characterized by their morphology, 2% BSA/0.1% Triton were incubated with the cells together expression of marker proteins Tyrosinase, variants of gp100 with /1000 DAPI nuclear dye for 1 h, then washed two times protein (melanosome marker)—NKI-beteb and HMB45, with 2% BSA/0.1% Triton Solution. Cells were fixed onto activity of DOPA-tautomerase, and reaction to IBMX and glass with Fluoromount(R) Sealing liquid and covered with a melanin content. glass slip. 0190. In agreement with the literature data several cell types were determined based on morphology: Small round 0197) The marker signals were analysed by means of fluo cells, small spindle-like cells, cells with characteristic fibro rescence microscopy (Nikon Eclipse Ti-S; Nikon Corpora blast morphology and cells with pebble-like keratinocyte tion, Deutschland). The cellular and nuclear signals were morphology, tri- or multidendritic cells with melanocyte mor counted by means of Image.J 1.42q NIH public software. phology (Tobin DJ, Colen SR, Bystryn J C.J Invest Dermatol Grafts 1995: 104: 86-89; Dieckmann C, Milkova L., Hunziker T, Emmendorffer A, Simon J C. Human melanocytes can be 0198 The cells on scaffolds were washed with PBS and isolated, propagated and expanded from plucked anagen hair fixed with ice-cold Para formaldehyde (PFA, 4%) for 8 min follicles. Exp Dermatol. 2010 June; 19(6):543-5; Zhu WY. utes, then washed with PBS again. The cells were blocked Zhang R Z. Ma HJ, Wang D G. Pigment Cell Res. 2004 with 0.5 ml of 2% bovine serum albumin 2% BSA-PBS and December; 17(6):668-73. Isolation and culture of amelanotic permeabilised with 0.1% Triton-PBS solution for 1 h. Solu melanocytes from human hair follicles; Bosserhoff A K. Ell tions of primary mouse-generated antibodies against US 2015/0O86513 A1 Mar. 26, 2015

NKI\beteb and Tyrosinase were set at 2% in PBS with 2% prepared (100: 50:25; 12.5: 6.25; 3.125ug/ml), measured for BSA/0.1% Triton and incubated with the cells on scaffolds extinction at 475 nm and used for concentration comparison. overnight at 4°C. After washing with 2% BSA/0.1% Triton Solution, biotinilised antimouse secondary antibody was Graft added to the cells and incubated for 1 h, thenwashed. Solution of 2% BSA-PBS/0.1% Triton/0.25% IGG was incubated with (0204. The cells together with PCL scaffolds were treated the cells for one hour, then washed. Finally, Streptavidine with 150 ul of 1M NaOH and washed manifold with a pipette. coupled Alexa594(R) (red) and Alexa 488(R) (green) fluoro Scaffold was subsequently withdrawn. The cell pellet was chrome 0.25% solution in 2% BSA/0.1% Triton were incu incubated at 37°C. for 6 hours. 150 ul of the cell lysate was bated with the cells together with /1000 DAPI nuclear dye, for transferred to a flat-bottom 96-well plate, filled up with 300 ul 1 h, then washed two times with 2% BSA/0.1% Triton solu lysate solution and the extinction at 475 nm was measured tion. Scaffolds with the cells were left to dry out for 2 min and with a plate reader in order to quantify the melanin content. fixed onto glass with Fluoromount(R) Sealing liquid and cov ered with a glass slip. 0205 Synthetic melanin was used as solution for the stan 0199 The marker signals were analysed by means of fluo dard curve. Melanin solution 100 ug/ml was incubated in 1M rescence microscopy (Nikon Eclipse Ti-S; Nikon Corpora NaOH for 5 hours at 60° C. and serial dilutions were prepared tion, Deutschland) and Laser-Scanning microscope (LSM (100: 50: 25; 12.5: 6.25; 3.125 ug/ml), measured for extinc 510 Meta, Carl Zeiss Jena GmbH, Deutschland). tion at 475 nm and used for concentration comparison. Incorporation into Epidermal Equivalents L-Dopa Tautomerase Activity 0206 Melanocytes and keratinocytes were cultivated 0200 Change of colour due to the build-up of melanin from ORS of the hair follicle, melanocytes as described in this upon L-DOPA tautomerization and Subsequent oxidation was application and keratinocytes as described in Cells Tissues visually registered. The cells, together with the scaffolds were Organs 2002: 172:79-85. Melanocytes were seeded on the 24 submitted to a five-fold freeze/thaw procedure on dry ice/37 mm Transwell(R) nylon-meshes with 0.4 um Pore Polyester C. water bath in order to disrupt the cell membranes. The Membrane Insert (Corning). Upon a 4 hadhesion, Suspension lysates were centrifuged at 4°C. at 14000 rpm for 30 min. The keratinocytes in 9-fold suffix were added and allowed to supernatant was used to test the Tyrosinase, TRP-1 and stratify over 11 days in the DermaLife Keratinocyte medium TRP-2 enzymes activity. The lysate (100 ul) was incubated (Life Line Cell Technology, Walkersville, Md., USA), or with 200 ul of L-DOPA solution (10 mg/ml) for 6 h. The DMEM/F12 (3:1) supplemented with 10% human serum, 20 extinction at 475 nm was measured by the means of Multi ng/ml epidermal growth factor, hydrocortisone, cholera toxin scan R. Spectrum (Thermo Fisher Scientific Germany Ltd. & 10 ng/ml, adenine and triiodothyronine. The stratified con Co. KG, Deutschland) Elisa plate reader in order to determine struct represents an epidermal equivalent which mimics epi the exact amount of the melanin produced. The presence of dermal anatomy. Melanocytes reside in stratum basale and melanin synthetized from L-DOPA substrate confirmed the the keratinocytes are the constituents of the upper layers. At activity of Tyrosinase, DOPA-tautomerase/Tyrosinase-re day 12, the upper keratinocyte stratum should be equivalent to lated Protein 2 (TRP-2) and DHIC-oxidase/Tyrosinase-re stratum corneum, morphologically identifiable by flattened lated Protein 1 (TRP-1). cells. 0207. The epidermal equivalents were cross-sectioned by IBMX Induction the means of cryotome into 200 um slices, fixed, dyed using 0201 The HM cells were incubated with 20 mg/ml the described immunfluorescence procedure and analysed by 3-isobutyl-1-methylxanthine (IBMX) for 7 days and then fluorescent microscope. Additionally, slices were dyed with analyzed for melanin content against untreated HMs, as Nile Blue in order to mark the cells and their subsections with described earlier on in this application. melanin content. Melanin Content Data Validation 0208 All experiments were performed using material of Cells four donors in five biological experiments, set up in three 0202 The cells were trypsinized, counted or adjusted to technical replicas. The data were validated for statistical sig total of 10000 cells and treated with 150 ul of 1M NaOH and nificance of the intertreatment differences by the means of washed several times with a pipette, then pelleted by centrifu two-tailed Student's t-test. gation at 14000 rpm for 30 min, 4°C. The cell pellet was incubated at 37° C. for 6 hours in 1M NaOH. The amount of Comparative Data 150 ul of the resulting cell lysate was transferred to a flat bottom 96-well plate, filled up with 1MNaOH lysing solution 0209 We have additionally compared the method of until 300 ul end volume and the extinction at 475 nm was Dieckmann et al. (Exp Dermatol. 2010 June; 19(6):543-5. measured by means of a plate reader in order to quantify the Epub 2010 Mar. 30) to the method according to the present melanin concentration and Subsequently its total content in Mention, using this similar experimental platform to cultivate the measured sample. Content of melanin was adjusted to HM melanocytes from hair samples. We have followed both amount per cell. cultivation and differentiation procedures synchroniously. At 0203 Synthetic melanin was used as solution for the stan the point of Geneticin selection, we have synchroniously dard curve. Melanin solution of 100 ug/ml was incubated in treated all the cells in order to examine the fibroblast and 1M NaOH for 5 hours at 60° C. and serial dilutions were keratinocyte content of the both preparations at that stage. US 2015/0O86513 A1 Mar. 26, 2015

Results nin (FIG. 12e and g). IBMX treatment induced dendrite growth and melanin synthesis in all cells (FIG. 12 fandh). Cultivation, Expansion, Differentiation, Selection of Pure 0217 Conclusions Melanocyte Culture 0218. The present invention provides an improved method 0210. The procedure has yielded a higher expanded cul for generating melanocytes comprising the microdissection ture of 95-100% pure, almost fully differentiated human mel of the epilated hair, the treatment with Collagenase and treat anocytes, which reached the melanotic stage within 3 to 4 ment with antibiotics, the differentiation in a differentiation weeks post-biopsy, as early as p3 or p4 of the primary culture medium as defined above. Further improvements were (FIG. 1-5). In particular, the cultivation and differentiating reached by maintenance of the obtained culture purity by procedure from 60 hair follicles yielded 10 pure, differenti differential trypsinization. This method leads to a higher ated human melanocytes which reached melanotic stage yield, quicker differentiation of melanocytes and enhanced within 3-4 weeks postepilation, in the third to fifth passage of purity of HM culture when compared to the method disclosed adherent culture. in Dieckmann et al. (Dieckmann C. Milkova L., Hunziker T. 0211. In comparison to the results of Dieckman et al., the Emmendorffer A, Simon J C. Human melanocytes can be method presented here reached an output of 10° HM in half isolated, propagated and expanded from plucked anagen hair the time. Further on, the difference between methods was follicles. Exp Dermatol. 2010 June; 19(6):543-5. Epub 2010 obvious already at the point of harvesting the cells from the Mar. 30). The synergy of faster growth, higher yield, ample Transwell nylon meshes in order to take them into adherent differentiation and out-selection of competing cells reduced culture; the number of harvested cells exceeded that of the the time necessary to cultivate 10 HM by 50% compared to Dieckmann method (loc. cit.) by factor 2.46. Further on, the the method of Dieckmann (loc. cit.). portion offibroblasts and keratinocytes in the harvested mate 0219. In the method according to the present invention the rial was much higher according to the Dieckmann method proximal fibroblast-rich part of the follicle was excised. (loc. cit.), since most of them died during the Geneticin selec Fibroblasts divide quickly, competitively out-selecting the tion and an extremely low number of cells remained after the slower-dividing melanocytes. By reducing the initial fibro Geneticin treatment. In contrast to the outcome of Geneticin blast content in culture, the nutritional and spatial competi selection of cells cultivated according to Dieckmann et al tion is lowered. (loc. cit.), large number of cells cultivated as described herein 0220 Partial digestion of the follicle collagen by Collage survived this selection step and were able to give raise to 10° nase V loosened the extracellular matrix and accelerated cell HM in four weeks. migration from the ORS pool. 0212. The uppermost purity was reached by synchroni 0221 Single short-term treatment by Geneticinefficiently ously combining DLM medium, microdissection (severance removed keratinocytes and fibroblasts from the culture, leav of the proximal part of the follicle (bulb)), digestion by Col ing melanocytes unharmed. lagenase, selection by Geneticin and differential trypsiniza 0222 Differential trypsinization increased and main tion, yielding 96% melanocyte-marker-expressing cells tained HM purity. Melanocytes are weakly adherent and (FIG. 4), with negligible residual content (2.39-4.2%) of quickly retract dendrites upon short trypsinization, ending up CD-90-expressing cells (FIG. 5). in Suspension early on and leaving adherent fibroblasts and 0213 Combined microdissection, Collagenase and Gene keratinocytes behind. Negligible residually adherent melano ticin treatment increased the purity of HM culture by 87.65% cyte loss was compensated by high HMyield (10° cells within compared to cultures without treatments (FIG. 4) and lowered 3-4 weeks). fibroblast content in HM culture by 82.12% (FIG. 5). Gene 0223) The ORS cell pool was amplified from minimal, ticin treatment increased HM culture purity by 67.7% (FIG. non-invasively gained material into quickly, putatively devel 2d) and diminished fibroblast content by 68% (FIG. 5). oped melanocytes. 0214 Collagenase treatment increased the purity of Gene 0224 All of the above provides pure, defined and sterile ticin-treated HM cultures by 12.72% (FIG. 5). HM culture and makes the improved method preferable in 0215 Excision of the proximal follicle part (microdissec non-invasive HM cultivation for purposes of graft-based tion) increased HM content by 20% when combined with treatmentS. Subsequent Collagenase and geneticin treatment (FIG. 4), 0225. The cell expansion was accelerated and the cell lowering fibroblast presence by 14.15% (FIG. 5). All differ purity was increased by the means of the above described ences between treatments were statistically significant (p<0. combined treatments. The cultures obtained by a complete 05) to very highly significant (p<0.005) according to Stu combination of the treatments grew most quickly, reached the dent's t-test, except between DLM/-Col/-HF/+G (non highest purity yielding a 96% of melanocyte-marker-express digested shortened follicles grown in DLM and selected with ing cells (FIG. 2), hereby showing the lowest presence of geneticin) and other treatments. Culture purity of HM was contaminant cells with a negligible residual content of con maintained by cell passages using differential trypsinization, taminant CD-90-expressing cells (FIG. 5). exposing adherent cell culture to Trypsin/EDTA solution for 0226. Similar results were obtained with material from all 4 minutes at room temperature and using the detached mel probands and biological experiments interms of cell yield (at anocytes in the Supernatant for further cultivation (based on In least 1 million cells in 4 weeks) and purity. No significant Vitro. 1984 May; 20(5):447-50, Exp Cell Res. 1982 Decem differences between probands and experiments have occurred ber; 142(2):309-15). in terms of purity/contamination. As for the yield, individual 0216. After the third passage of adherent cultivation, HM differences were observed in the time necessary to obtain at cells fully displayed features of differentiated melanocytes. least 1 million of differentiated melanocytes. However in all The cells had a typically large tri-to-multidendritic Soma probands, biological experiments and replicas, a minimal (FIGS. 1d and 2h), expressed NKI-beteb, HMB45 and Tyro yield of one million differentiated melanocytes was always sinase (FIG.2a-c), converted L-DOPA and synthesized mela reached in four weeks or less. US 2015/0O86513 A1 Mar. 26, 2015

Characterization ml/ml, Basic Fibroblast Growth Factor (recombinant human) 1 ng/ml. Insulin (recombinant human) 5 mg/ml, Hydrocorti 0227 Characterization criteria were fulfilled at the third sone 0.5 mg/ml, and Phorbol Myristate Acetate (PMA) 10 passage or later. Before the p3, the cells were still negative for ng/ml. Cultivation conditions for the adherent culture accord certain characterization parameters for melanocytes. ing to Dieckmann (loc. cit.) involves medium Melanocyte 0228. The cells had a typically large soma and at least Growth Medium M2, supplemented with SupplementMix, three dendrites (FIG. 1 D), they expressed Tyrosinase, NKI produced by Promocell, (Heidelberg, Germany). Because the beteb and HMB45 (FIG. 3, 7, 9), successfully converted L-DOPA and synthesized melanin (FIG. 10). All of the cells SupplementMix of this medium contains PMA, which is a reacted to the IBMX test, being successfully induced into potent tumor promoter, the cells obtained by the method of melanin synthesis and dendrite growth. Dieckmann (loc. cit.) are disadvantageous for therapeutical Integration into PCL Fibre Mesh SC. 0229. The cells grown on PCL scaffolds were properly 0233. At the point of Geneticin selection, we have syn nested and assumed a typical morphology of melanocytes in chroniously treated all the cells cultivated according to the the three-dimensional tissue context. They extended den present invention in order to selectively discard the fibroblast drites in multiple directions and interfaced the fibre of the and keratinocyte content of the preparation at the stage of scaffolds (FIGS. 7A and 7B). The cells migrated into the adherent culture as described herein. Further on, in order to scaffolds and populated the entire depth of the network (FIG. clarify the role of the contaminant fibroblasts and kerati 7C). The diffuse pattern of Tyrosinase and the localized pat nocytes as competitors against differentiating melanocytes, tern of gp100 variants were also typical for melanocytes (FIG. we have treated a separately set up experiment of the Dieck 7. 9). The gp100 variants were localized in melanosome mann (loc. cit.) cultivation, at the point of the initial passage vesicles near the dendrite interfaces (FIG. 9). Equally high of the adherent culture, with Geneticin. percentage of cells, both in NHEM and HM, displayed gp100 0234. The cells were stained with Trypsin blue and variants and Tyrosinase on PCL scaffolds and on the polysty counted in Neubauer chamber at each passage. rene surface (FIG. 8). 0230. Moreover, L-DOPA activity and melanin content of Measurement of the Outer Root Sheath Surface Changes: the cells on scaffolds were manifold higher than the cells on flat adherent polystyrene surfaces—roughly 6-7-fold in epi 0235 Surface of the follicle ORS was measured by the dermal melanocytes and 2-fold in ORS melanocytes (FIG. means of Nikon NIS Elements BR (Version: 3.00) software. 10). The ORS, which is morphologically clearly distinguishable, Incorporation into Keratinocyte-Based Epidermal Equiva was selected from surface micrography of hair follicle by the lents means of an adjustable polygonal frame and the Surface val 0231. The epidermal NHEM were successfully incorpo ues were read out by software. The average values of ORS rated into keratinocyte-based epidermal equivalents. They Surface of four independently set up cultivation experiments remained oriented towards the lower layer which corresponds with three technical replicates were compared at day 0, day 7 to the stratum basale of human skin epidermis. The melano and day 14 (FIG. 13, 14). cytes maintained their characteristics and fully displayed the Comparison of the Dieckmann (Loc. Cit.) Method and Herein characterization parameters for melanocytes as described Provided Method with Geneticin Addition: herein (FIG. 11). 0236 Additionally, in order to withdraw the contaminant fibroblasts and keratinocytes as competitors against differen Example 2 tiating melanocytes, we have treated the Dieckmann (loc. cit.) adherent culture with 50 lug/ml Geneticin after the p0 as The Herein Provided Method is More Efficient in a corresponding selection measure used in the current Producing Melanocytes Compared to the Method of method. Dieckmann (Loc. Cit.) Results Materials and Methods 0232. The method of Dieckmann et al. (Exp Dermatol. Cultivation, Expansion, Differentiation, Selection of Pure 2010 June; 19(6):543-5. Epub 2010 Mar. 30) was compared Melanocyte Culture to the method according to the present invention, using this similar experimental platform to cultivate HM melanocytes 0237. The procedure has yielded a higher expanded cul from hair samples. We have followed both procedures syn ture of 95-100% pure, almost fully differentiated human mel chroniously. The procedure of preparation, cultivation, dif anocytes, which reached the melanotic stage within 3 to 4 ferentiation and characterization according to the method weeks 20 post-biopsy, as early as p3 or p4 of the primary described herein was conducted as described in Example 1. culture (FIG. 1-5). In particular, the cultivation and differen As described above, the herein provided method differs, inter tiating procedure from 60 hair follicles yielded 10° pure, alia from the method of Dieckmann by the use of an epilated differentiated human melanocytes which reached melanotic hair, whereby the bulb of the hair has been removed, and by stage within 3-4 weeks postepilation, in the second to fifth the incubation of the epilated hair with a collagen-degrading passage of adherent culture. In comparison to the results of agent. In addition, the method of the present invention can Dieckman et al., the method presented here reached an output comprise Geneticin treatment. The procedure of Dieckmann of 10° HM inhalf the time according to the published material et al. (loc. cit.) was performed according to the given refer and by far exceeded the yield of protocol by Dieckmann (loc. ence. The supplement for M2 as used in the method of Dieck cit.)already in very early passages of adherent culture accord mann (loc. cit.) contains Bovine Pituitary Extract 0.004 ing to our findings. US 2015/0O86513 A1 Mar. 26, 2015

ORS Surface Growth start with. The number of cells harvested from the follicles 0238. Difference in growth between methods was visible cultivated by the current method on day 0 of the adherent already on day 7 and day 14 of follicle cultivation on nylon culture exceeded that of Dieckmann (loc. cit.) method by meshes, with clearly displayed higher growth of the follicles factor 1.84 (FIG. 15). cultivated upon the method described herein in comrarison to the Dieckmann (loc. cit.) method (FIG. 14). The ORS surface Comparative Growth in the Adherent Culture of hair follicles digested with collagenase and cultivated 0243 The cells in the adherent culture were continuously according to the present invention, as described in Example 1, dividing in cultivation experiments according to the method increased from 42.480+17.451 um on day 0 to 87.735+45. described herein, reaching 1,000,000 cells at the passage 2 of 397 um on day 7 and 125.838+58.446 pum on day 14, the adherent culture, (half the time needed for the same num whereas the follicles cultivated according to the Dieckmann ber by Dieckmann et al. (loc. cit.)) and as much as 80,875,000 (loc. cit.) method increased from 43.302+9.226 um on day 0 by the passage 6. The cells cultivated according to the Dieck to 50.039+10.753 um on day 7 and 77.488+29.569 um on mann (loc. cit.) method never reached those numbers, as a day 14. The ratio of the Outer Root Sheath surface between matter of fact, their numbers persistently declined after the the Dieckmann (loc. cit.) method and the current method passage 2 of the adherent culture. hereby declined from 0.94 on day 0 to 0.69 on day 7 (p=0.01*) and 0.72 on day 14 (p=0.018) (FIG. 13), displaying faster Genetycine Treatment growth and migration of cells from collagenised follicles. At 0244 Clearly, the portion of fibroblasts and keratinocytes the point of harvesting the cells from the Transwell nylon in the material harvested from medium-air-interface follicle meshes in order to take them into adherent culture, the num culture was much higher in the preparation according to the ber of harvested cells exceeded that of the Dieckmann (loc. Dieckmann (loc. cit.) method, since most of them died during cit.) method by factor 1.84 when all the sampled hair follicles the Geneticin selection and an extremely low number of cells were taken into account. (FIG. 16). survived the Geneticin treatment, indicating that the Dieck mann (loc. cit.) method could not provide enough stem cells Comparative Growth in Adherent Culture and precursors to quickly cultivate melanocytes in the adher 0239. The cells in the adherent culture, cultivated accord ent culture (FIG. 15). In contrast to the outcome of Geneticin ing to the method described herein, continued to divide and selection of the cells cultivated according to Dieckmann et al. reached the numbers of 1,000,000 at the passage 2, adherent (loc. cit.), large number of cells cultivated as described herein culture day 11 and as much as 80,875,000 at passage 6. In survived this selection step and were able to give raise to very contrast to our method, the cells in the adherent culture cul high cell numbers (FIG. 15). tivated according to the Dieckmann et al. (loc. cit.) protocol 0245 Synergy of the higher start number of stem cells and reached 710.000 cells at passage 1 of the adherent culture, as precursors and more favourable adherent culture conditions the highest cell number and 650.000 cells by passage 2 at day given by the current method significantly advanced the yield 11 of the adherent culture. Unlike the cells cultivated upon the of HM (FIG. 15). method described herein, the cells cultivated according to the Dieckmann (loc. cit.) method decreased in numbers with Example 3 further passages (FIG. 15.16). The Melanocytes Obtainable by the Herein Provided Genetycine Treatment Method are Distinct from Known Melanocytes 0240 Additional treatment of adherent culture by Dieck 0246. Even though melanocytes obtained according to the mann (loc. cit.) with 50 ug/ml Geneticin with as a correspond present invention (HM) correspond morphologically and ing selection measure used in our current method resulted functionally to adult skin melanocytes, they are a distinct and with extremely low numbers of survivor cells (<2% conflu novel cell entity, as shown by the expression of cell surface ence) (FIG. 15,16). markers (Tables 1-5) and by quantitative morphological 0241 The cells cultivated according to the current method parameters (FIG. 17). reached higher numbers than the cells cultivated according to Dieckmann method (loc. cit.). Material and Methods Conclusions Morphological Characteristics ORS Surface Changes 0247 Surface of cells and dendrite length were measured by the means of Nikon NIS Elements BR (Version: 3.00) 0242 Collagenase effect on loosening the extracellular software. Total of 5 cells per experiment in 3 cultivation matrix through collagen proteolysis had obviously enabled experiments, as described in the example 1, were evaluated. easier migration of the cells out of ORS. This effect is visible The surface of the cells was selected by the means of an by the enlarged surface of the ORS and by a more intensive adjustable polygonal frame and the Surface values were read migration of cells from ORS to the nylon mesh (FIG. 14). The out by the software. The dendrite length was measured by a initial growth of ORS in follicles digested with Collagenase V multipoint line length measuring tool. as per the current method exceeded that of non-digested fol licles according to the Dieckmann (loc. cit.) method on all the days of adherent culture following day 0. That gave the Surface Marker Analysis by Lyoplate, FACS Measurements method according to the invention the advantage of harvest 0248 Expression of surface markers was analysed by the ing more cells from the medium-air-interface follicle culture means of BD Lyoplate Human Cell Surface Marker Screen and having more cells for the set-up of the adherent culture to ing Panel. This panel of 242 antibodies against human cell US 2015/0O86513 A1 Mar. 26, 2015 20 surface markers was used to label Normal Human Epidermal 0253. The markers characteristic for NHEM and HM Melanocytes (NHEM) and ORS-derived melanocytes (HM). respectively, have been assigned a NP number from the NCBI The cells were cultivated and characterized as described in protein database (www.ncbi.nlm.nih.gov/protein). The use of the methods section of the Example 1, harvested in passages Such protein databases are known and therefore the corre 10 to 11 and resuspended in Stain Buffer provided by the sponding amino acid and nucleic acid sequences of the mark manufacturer. Aliquots of 200.000 cells/well were seeded in ers to be used herein are easily accessible to a person skilled 96-well plates. Cells in each well were labelled with a sepa in the art. rate mouse-generated or rat-generated primary antibody for a single Surface marker and Subsequently labelled with a cor Conclusions responding secondary antibody against mouse or rat, respec tively, labelled with Alexa 647 fluorophore. Between the Morphological Parameters 40-minute incubation steps, the cells were washed with Stain Buffer and centrifuged at 300 g for 4 minutes. After the 0254 HM and NHEM share a common morphology, both staining, labelled cells were fixed for 10 minutes with 4% have a large soma with protruded dendrites. HM display a paraformaldehide, washed and measured by the means of smaller cell surface, shorter dendrites in a somewhat lower Becton Dickinson FACS Array system. The measurements number than HM. were analysed using BD DIVA Software. Negative controls Surface Markers (unlabelled cells) were used to establish the threshold of the fluorescence signal. Fluorescence intensity of 2x10 was set 0255. The melanocytes obtained in accordance with the as a threshold of marker expression Fluorescence intensity present invention and NHEM differ in surface marker expres above the threshold in at least one proband was marked as sion (10.74% of the 242 examined markers) and overall positive expression (+). marker expression (34.7% of all expressed markers). The difference in marker expression reflects a difference in cellu Results lar identity between HM and NHEM. 0256 Expression of markers typical for other lineages Morphological Characteristics complies with the nature of HM and NHEM. Expression of CD57, neuronal adhesion molecule, goes along with the com 0249 NHEM cells display an average surface of 377+135 mon developmental origin of melanocytes and neural cells. um, whereas the HM display 290+74 um; hereby, NHEM CD40a, CD40b are receptors for mitogens in mesenchymal soma is 23% larger in surface than HM soma (p=0.0451). cells, which might render HM as susceptible to mesenchymal Dendrites of NHEM are in average 35.73+15um long, which signalling cues; nevertheless, it is not within goals of this is 39% longer than the 21.57+10 Lim long dendrites of the HM application to place HM in mesenchymal niche. cells (p=0.045). NHEM have a slightly higher average num 0257 CD40a, CD40b, CD104 and CD106 are involved ber of dendrites (3.13+1.19) than HM (2.47+0.64), (p=0.065, cell growth and adhesion and expressed in HM. Along with non-significant) (FIG. 17). the absence CD117 expression, this reflects the stem cell origin of HM and points towards residual naivity of the HM Surface Markers cells, placing them in advanced partially melanotic melano blast stage, the last before the full adult melanotic melanocyte 0250 Out of 242 examined cell surface markers, 229 were Stage. expressed in HM, out of which 13 markers exclusively in HM (0258 CD221 (Insulin-like Growth Factor 1 tyrosine (5.37% of the total number of cell markers). 229 markers kinase receptor) is expressed in most human cells. CD221 were expressed in NHEM, 13 markers exclusively in NHEM mediates Insulin-like growth factor IGF-1 and IGF-2 signal (5.37%). Both NHEM and HM expressed 84 cell surface ling that affects cell growth, a very important feature for the markers out of 242 of all examined markers. Differences in cell culture. Absence of CD221 expression in HM deprives the cell surface marker expression between NHEM and HM the HM cells from IGF-1 and IGF-2 signalling through this comprise 10.74% of all examined markers and 34.7% of the receptor and renders the IGF-1 and IGF-2 factors to a com expressed markers (Table 1-5). petitive binding of Insulin receptors. This does not interfere 0251 Most of the markers that are differentially expressed with our cultivation system, since the insulin Supplement in between HM and NHEM are involved in regulation of immu the medium eliminates this competitive binding with its 100 nity (CD195 CD108 CD40 CD36 CD15s CD282 CD173, fold higher affinity to insulin receptors and allows normal cell CD274 in HM, CD1, CD28, CD79b, CD137, CD282, HLA growth. A2(A2), HLA-DQ in NHEM) and do not play a major role in 0259. The melanocytes obtained herein differin expressed development. Certain markers are generally functional or markers and in qualitative morphologic traits which can be expressed in other lineages than melanocytic (CD40a, CD40b quantified as described herein above. Due to these differ receptors for mesenchymal cell mitogens in HM, neuronally ences, the herein provided and obtained melanocytes are a expressed adhesion molecule CD57 in NHEM). Markers nor novel cellular entity mally expressed in melanocytes, CD117 and CD221, were 0260 Series of similarities at different levels of character expressed in NHEM and not in HM. ization show that ORS-differentiated HM are morphologi 0252 Tables 2 and 3 show surface markers that are differ cally and functionally a peer to the epidermal skin-derived entially expressed in NHEM cells and melanocytes obtain NHEM. Those features qualitatively and quantitatively show able by the herein provided method (HM). Tables 4 and 5 that the HM are a good substitute for NHEM, which means a show a comprehensive list of Surface markers expressed in great deal given that they are obtained non-invasively. NHEM cells and melanocytes obtainable by the herein pro 0261 ORS melanocytes express the same intracellular vided method (HM). markers (Tyrosinase, gp100 variants), Sustain an active enzy US 2015/0O86513 A1 Mar. 26, 2015 21 matic set for melanogenesis (Tyrosinase, TRP-1 And TRP-2) TABLE 3 that converts L-DOPA substrate into melanin, displaying ability to produce melanin from the supplemented substrate Table of surface markers expressed in melanocytes obtainable by as well as spontaneously. Moreover, HM are able to migrate the present method (also referred to herein as “HM') and populate novel niches. As we have shown, the HM are morphologically quite similar to the NHEM, apart from Marker Name Reference smaller soma and shorter dendrites, as determined by For CD1 a T-cell surface glycoprotein CD1a NP 001754.2 ward Scatter in FACS analysis as well as by quantitative CD28 Cluster of Differentiation 28 NP OO123OOO6.1 morphological parameters such as cell Surface and dendrite CD77 Ganglioside GA3 NLM Registry length. Also, the cell Surface marker expression differs Number 71965-57-6 between NHEM and HM. Due to these differences the herein CD79b immunoglobulin-associated beta NP OOO617.1 provided melanocytes (HM) are a new cellular entity com CD137 a) TNFRSF9 tumor necrosis factor NP 001552.2 pared to epidermal NHEM. Accordingly, the melanocytes Ligand receptor Superfamily, member 9 obtainable by the herein provided method are novel. TABLE 1 Table of differences between epidermal melanocytes (NHEM) and ORS-derived melanocytes HM) obtained in accordance with the present invention in Surface marker expression. complete difference in expression partial difference in expression % of markers

10.74 7.44 not not stronger stronger expression expression expressed expressed expression expression varies between varies between in NHEM in HM in NHEM in HM HM-NHEM and HM-HM HM probands % of markers

5.37 5.37 1.24 O.83 3.72 1.65 Nomenclature CD1a CD15s CD49a CD10 CD27 CD66 (a,c,d,e) CD28 CD36 CD49d CD13 CD90 CD209 CD77 CD40 Disialoganglioside GD2 CD97 CD271 CD79b CD48 CD99R CD273 CD137 Ligand CD57 CD2OO CD140a CD108 CD337 CD14Ob CD112 HLA-DR CD282 CD117 HLA-DR, DP, DQ HLA-A2 CD163 CD49f HLA-DQ CD195 CD104 CD221 CD106 CD274 CD142 CD2O1

TABLE 2 TABLE 3-continued Table of surface markers expressed in NHEM: Table of surface markers expressed in melanocytes obtainable by the present method (also referred to herein as “HM') Marker Name Reference CD15s sialyl Lewis X Nat Chem Biol Mark8Ke N8t Ref{{{Ce 2008; 4: 751 CD140a Alpha-type platelet-derived growth NPC O06197.1 CD36 glycoprotein IV (gpIV) NP OOOO63.2 factor receptor CD40 TNF receptor superfamily member 5 NP OO1241.1 C CD48 SLAMF2 NP OO1242959.1 CD140b platelet-derived growth factor NP 002600.1 CD57 HNK-1 (human natural killer-1) carbo- NP 061114.2 receptor, beta polypeptide hydrate PDGFRB CD108 Semaphorin 7A NP 0011395.01.1 CD282 Toll-like receptor 2 NP OO3255.2 CD112 Poliovirus receptor-related 2 (herpesvirus NP 001036189.1 HLA-A2 human leukocyte antigen serotype NP 001229687. entry mediator B, nectin-2) within HLA-AA serotype GenBank: AAA766.08.2 CD117 c-kit ligand, maststem cell growth factor NP 000213.1 group GI:33 1871.48 receptor Kit HLA-DQ human leukocyte antigen-DQ NP 00123O890.1, CD163 Scavenger receptor for the hemoglobin- NP OO4235.4 NP 002113.2 haptoglobin complex in b d CD195 chemokine (C-C motif) receptor 5 NP 000570.1 CD104 integrin beta-4 NP 000204.3 CD221 Insulin-like Growth Factor 1 (IGF-1) NP OOO866.1 CD106 vascular cell adhesion molecule 1 NP 001069.1 Receptor (VCAM-1) CD274 Programmed cell death 1 ligand 1 NP OO1254635.1 CD142 platelet tissue factor, factor III, NP 001171567.1 CD2O1 primary receptor for protein C activation NP O06395.2 thrombokinase US 2015/0O86513 A1 Mar. 26, 2015 22

TABLE 4 Markers characteristic for NHEM melanocytes. NHEM Name Reference C D15S + sialyl Lewis X Nat Chem Biol 2008; 4: 751 D36 glycoprotein IV (gpIV) NP OOOO63.2 D40 TNF receptor superfamily member 5 NP OO1241.1 D48 SLAMF2 NP OO1242.959.1 D57 HNK-1 (human natural killer-1) carbohydrate NP 061114.2 D108 Semaphorin 7A NP 0011395 O1.1 D112 Poliovirus receptor-related 2 (herpesvirus entry mediator NP 001036189.1 B, nectin-2) D117 c-kit ligand, maststem cell growth factor receptor Kit NP OOO213.1 D163 : scavenger receptor for the hemoglobin-haptoglobin NP OO4235.4 complex D195 chemokine (C-C motif) receptor 5 NP 000570.1 D221 Insulin-like Growth Factor 1 (IGF-1) Receptor NP OOO866.1 D274 Programmed cell death 1 ligand 1 NP OO1254635.1 D2O1 primary receptor for protein C activation NP OO6395.2 D9 D10 D13 D26 D27 D29 D34 D44 D4SRB D4SRO D46 D47 D49a D49b D49c. D49d D49e DS1,61 DS4 D55 DS6 D58 D59 D61 D63 D66 (a, c, d, e) D71 D73 D81 D90 D91 D95 D97 D98 D99 D99R 05 O7a O7b 09 19 20a 30 41 46 47 51 52 53 64 65 66 71 83 84 96 D 2O O US 2015/0O86513 A1 Mar. 26, 2015 23

TABLE 4-continued Markers characteristic for NHEM melanocytes. NHEM Name Reference

D209 D227 D271 D273 D321 (F11 cptr) D337 D340 (Her2) 2micro em. Prog. Cell LA , B, C LA-DR LA-DR, DP,

US 2015/0O86513 A1 Mar. 26, 2015 25

TABLE 4-continued Markers characteristic for NHEM melanocytes. NHEM Name Reference y8TCR Invariant NKT MICAAB NKB1 SSEA-1 SSEA-4 TRA-1-60

gG1 gG2a gG2b gG3 D12Ob D132 D210 D212 D267 D294 D326 Cutaneous Lymph. Antigen Integrin B7 SSEA-3 rigN rigG1 rlgG2a rigG2b

TABLE 5 Markers characteristic for melanocytes obtainable by the method of the present invention (HM). HM Name Reference CD1a. + T-cell surface glycoprotein CD1a NP OO1754.2 CD28 + Cluster of Differentiation 28 NP OO123OOO6.1 CD77 + Ganglioside GA3 NLM Registry Number 71965-57-6 CD79b + immunoglobulin-associated beta NP OOO617.1 CD137 Ligand + a) TNFRSF9 tumor necrosis factor NP OO1552.2 receptor Superfamily, member 9 CD140a + Alpha-type platelet-derived growth NP OO6197.1 factor receptor CD14Ob + platelet-derived growth factor receptor, NP 002600.1 beta polypeptide PDGFRB CD282 + Toll-like receptor 2 NP OO3255.2 HLA-A2 + human leukocyte antigen serotype NP OO1229687.1 within HLA-A 'A' serotype group GenBank: AAA766.08.2 GI:33 1871.48 HLA-DQ + human leukocyte antigen-DQ NP 00123O890.1, NP 002113.2 CD104 + integrin beta-4 NP 000204.3 CD106 + vascular cell adhesion molecule 1 NP 001069.1 (VCAM-1) CD142 + platelet tissue factor, factor III, NP 001171567.1 thrombokinase CD9 -- CD10 -- CD13 -- CD26 -- CD27 -- CD29 -- CD34 -- CD44 -- CD4SRB -- CD4SRO --

US 2015/0O86513 A1 Mar. 26, 2015 27

TABLE 5-continued Markers characteristic for melanocytes obtainable by the method of the present invention (HM). HM Name Reference

C p ) 21a. 21b 22 23 24 26 27 28b 34 35

US 2015/0O86513 A1 Mar. 26, 2015 29

TABLE 5-continued Markers characteristic for melanocytes obtainable by the method of the present invention (HM). HM Name Reference Integrin B7 SSEA-3 rigN

0262 FIGS. 1: Cultivation, selection and expansion of ture, showing 99% of NKI/beteb-expressing cells. All of the ORS cells from the ORS of the hair root into a pure culture of differences are statistically significant (p<0.05 to very highly melanocytes within four weeks. A: Mid part of the epilated, significant (p<0.005), except the differences between DLM/- preparated temporal hair root set on the Transwell Col/-HW/+G (non-digested shortened follicles treated with microporous membrane (medium-air-interface) on the first Geneticin and differentiated in DLM medium) and the rest of day of cultivation. The compact Outer Root Sheath (ORS) is the categories. visible. B: Outgrown ORS with first cells migrating out of 0266 FIG. 5: Fibroblast contamination in the ORS mel ORS.C. Multiplied cells are forming aggregations apart from anocyte culture. The grey bars are showing the percentage of the ORS. D: Pure culture of ORS melanocytes after four CD-90-positive cells in culture (prevalently fibroblasts). The weeks of cultivation. The cells display a large Soma and triple table displays the procedures of expansion, differentiation to multiple dendrites (tripolar to stellar morphology). and selection of melanocytes in culture, showing a clear trend 0263 FIGS. 2: Combination of methodological improve of a decrease of fibroblasts in culture that reaches 2% of ments in developing a pure culture of ORS melanocytes. A: CD-90-expressing cells with the optimal combination of the Unprepared, untreated hair. ORS is compact (arrow) B: procedures. Coll--/- corresponds to Collagenase V treatment, Unprepared, Collagenase V-treated hair with visible migrated HW+ stands for presence of the bulb., HW-stands for absence cells outside ORS at mid-part and also outside the proximal of the bulb (The bulb has been severed from the rest of the (bulb) part (arrows). C: Prepared hair with the bulb part cut off root), G+/- stands for Geneticin treatment, DLM for Der (dash-line), untreated with Collagenase V. showing compact maLife culture media applied. The white bar represents fibro ORS (arrow). D: Prepared hair with the bulb part cut off blast control, grown in a separate culture, showing 100% of (dash-line), treated with Collagenase V. showing outgrown CD-90-expressing cells. All of the differences are statistically ORS with migrated cells outside of ORS (arrow). Scale bars significant (p<0.05) to very highly significant (p<0.005), correspond to 500 um. E: Outgrown, nearly confluent propa except the differences between DLM/-Col/-HW/+G and the gated cells outside of ORS on day 14 of culture. F: Primary rest of the categories. culture of ORS cell pool developed from non-collagenised 0267 FIG. 6: Policaprolactone (PCL) scaffold structure. hairs on day 20. Several cell types can be seen. G: Primary A: REM capture of the 8 to 10 um fibre, 800-fold magnifica culture of ORS cell pool developed from collagenised hairs tion. B: REM capture of the 2-3 um fibre, 800 fold magnifi on day 20. The cultivated cells display a more typical mor cation. The scale bars correspond to 25um. phology. H: pure culture of selected melanocytes on day 26. I: 0268 FIG. 7: Laser-Scanning Microscope capture of HM Differentiated melanocytes in the pure culture at day 30. and NHEM on PCL Scaffolds. PCL-fibre (signal from Scale bars correspond to 100 nm. reflected red light). NHEM melanocytes expressing Tyrosi 0264 FIG. 3: Immunocytochemical characterization of nase (green fluorescence) and the nuclei (DAPIUV-fluores the ORS culture. A: NKI\beteb is expressed in all epidermal cence) are visible. A: HM expressing Tyrosinase; B: NHEM melanocytes (NHEM); B: Tyrosinase is expressed in all epi expressing NKi\beteb; C: Side view (cross-section 3-D dermal melanocytes (NHEM); C: CD 90 is expressed in all model) of the ORS melanocyte-populated PCL-Scaffold: non-melanocyte cells, prevalently fibroblasts; D: NKI\beteb HM have been labelled with Tyrosinase antibody (green sig is expressed in almost all ORS melanocytes (HM); E: Tyro nal). D: Side view (cross-section 3-D model) of the epidermal sinase is expressed in almost all HM; F: absence of CD 90 melanocyte-populated PCL-Scaffold. expression in pure HM-culture. Bars correspond to 50 lum. 0269. The cross-sections show that the cells are populat 0265 FIG. 4: Purity of the ORS melanocyte culture. The ing the full depth of PCL scaffold. Scale bars correspond to grey bars are showing the percentage of NKI/beteb-positive 200 nm. The arrows are pointing at typical cell Soma and melanocytes in culture. The table displays the procedures of extended dendrites interfaced with mesh fibre. expansion, differentiation and selection in culture, showing a 0270 FIG. 8: Melanocyte marker expression on PCL scaf clear trend of an increase of melanocytes in culture that folds and on polystyrene adherent surface. The ratio of the reaches 96% of NKI-beteb-expressing melanocytes with number of nuclei against marker-positive cells is expressed as proper combination of the cultivating procedures. Coll--/- percentage. The Tyrosinase and NKI/beteb marker expres corresponds to Collagenase V treatment, HW+ stands for sion of epidermal (dark brown) and ORS melanocytes (beige) presence of the bulb., HW-stands for absence of the bulb (The is equal to that of epidermal melanocytes on polystyrene bulb has been severed from the rest of the follicle), G+/- adherent surface (black), showing that both HM and NHEM stands for Geneticin treatment, DLM for DermaLife culture are maintaining their features on the PCL scaffolds. media applied. The black bar represents epidermal melano (0271 FIG. 9: Both epidermal melanocytes (NHEM) and cyte control (differentiated NHEM), grown in a separate cul ORS melanocytes (HM) are forming melanosomes which are US 2015/0O86513 A1 Mar. 26, 2015 30 ready to be secreted from the end of the dendrites. The figure 0281. The hair follicles cultivated in the medium-air-inter shows NKI/beteb labelling of melanosomes in NHEM and face upon the current method (a,c,e) displayed higher HM. increase in ORS surface than those cultivated according to the 0272 A: NHEMs on 3 um PCL scaffold, B: NHEMs on 10 Dieckmann (loc. cit.) method (b.de). On day 0, the follicles um PCL scaffold C: HM on 3 um PCL scaffold D: HM on 10 are compact (a,b). On day 7, outgrowth and migration of the um PCL scaffold. The scale bars correspond to 50 lum, at cells was evident (c,d), as well as on day 14 (e.f). Stronger 40-fold magnification. outgrowth of the follicles cultivated according to the current 0273 FIG. 10: Melanin content and L-DOPA-tau method is visible. tomerase activity of NHEM and HM on PCL scaffolds and 0282 FIG. 15. Comparative cell growth in the adherent adherent Surfaces. All graphs are displaying clear trend of culture higher melanin production and L-DOPA-tautomerase activ 0283 Growth curves of ORS cells cultivated according to ity on PCL scaffolds than at PS-adherent surfaces. Black bars (A) the current method, (O) Dieckmann (loc. cit.) method, represent the NHEM and grey bars the HM. (A) Melanin (*) Dieckmann (loc. cit.) method with Geneticin supplement content in epidermal melanocytes on PCL Scaffolds com during the first 6 passages of adherent culture. The lower part pared to adherent Surface: Epidermal melanocytes produce of the graph is displayed linearity and the upper part logarith 4.59 more melanin on PCL scaffolds than on the adherent mically. The cells cultivated upon the method described polystyrene surfaces. (B) Melanin content in ORS melano herein reached the number of 1,000,000 cells by the second cytes on PCL scaffolds compared to adherent surface: HM passage of adherent culture. ORS melanocytes produce 2.58-fold more melanin on PCL 0284 FIG. 16. Comparative growth in the adherent cul scaffolds than on the adherent polystyrene surfaces. (C) ture, per hair follicle. L-DOPA activity in epidermal melanocytes on PCL scaffolds (0285 Growth curves of ORS cells persamled hair follicle compared to adherent Surface: Epidermal melanocytes con cultivated according to (A) the current method, (O) Dieck vert 6.65-fold more L-DOPA on the PCL Scaffolds than on the mann (loc. cit.) method, (*) Dieckmann (loc. cit.) method adherent polystyrene surfaces. (D) L-DOPA activity in ORS with Geneticin Supplement during the first 6 passages of melanocytes on PCL scaffolds compared to adherent surface: adherent culture. The graph is displayed logarithmically. HM ORS melanocytes convert 1.72-fold more L-DOPA on 0286 FIG. 17. PCL scaffolds than on the adherent polystyrene surfaces. All 0287. Different size of cell soma and dendrite length in values are shown relative to the corresponding values of a NHEM and HM particular cell type on adherent surface, which are taken as (a) Relative cell size of NHEM and HM measured by Forward 100%. All of the differences are highly statistically significant Scatter Plot (X-axis) (p<0.01 to very highly significant (p<0.005). (b) Cell size of NHEM and HM in um (0274 FIG. 11: Outer root sheath melanocytes (HMORS) (c) Dendrite length of NHEM and HM in um embedded in the keratinocyte epidermal equivalent remain in What is claimed is: the lower layer of the graft, equivalent to epidermal stratum 1. A method for generating melanocytes from stem cells basale, and express melanocyte markers. Tyrosinase and comprising the steps of nuclear DAPI signals are visible. A: Epidermal melanocytes, (i) removing a bulb of an epilated human hair; normal light; B: Epidermal melanocytes, fluorescence. (ii) incubating any remaining part of the epilated hair with (0275 Scale bar corresponds to 200 p.m. a collagen degrading agent to separate stem cells from its 0276 FIG. 12: Immunocytochemical characterization of outer root sheath; the controls: expression of NKI-beteb in normal human epi (iii) cultivating the separated Stem cells from step (ii) in a dermal melanocytes (NHEM) (a), Tyrosinase in epidermal medium that induces differentiation and stimulates melanocytes (b), CD-90 expression in pure dermal fibroblast growth of stem cells, melanocyte precursors and mel culture (c), lack of CD-90 expression in HM culture (d), HM anocytes, wherein the medium comprises one or more in culture without L-DOPA addition (e), IBMX-mediated growth factors for differentiation into melanotic mel changes in HM morphology and increased melanin content, anocytes. without L-DOPA (f), melanin content upon L-DOPA sub 2. The method according to claim 1, further comprising a strate addition in HM (g) and increase in melanin content step between step (ii) and (iii) of washing the remaining part upon L-DOPA addition in IBMX-pre-treated HM (h). Scale of the epilated hair with a washing solution after incubation bars (a-d) correspond to 50 uM, bars (e-f) to 100 p.m. with the collagen degrading agent. (0277 Figure FIG. 13. 3. The method according to claim 1, further comprising a 0278 Comparative changes of the Outer Root Sheath sur step of: (iv) at least one of selecting or isolating the differen face tiated melanotic melanocytes in the culture. (0279 Both the current method (0) and the method of 4. The method according to claim 3, wherein the melano Dieckmann et al. (loc. cit.) () comprised an increase of the cytes are isolated in step (iv) using at least one of anatomic ORS surface. The surface increase is significantly higher in selection of hair root subsections or differential trypsiniza ORS of the follicles cultivated according to the current pro tion. tocol in comparison with that of the Dieckmann method (loc. 5. The method according to claim 3, wherein melanocytes cit.), as measured at day 7 and day 14. are isolated using a Geneticin treatment. 0280 FIG. 14. Graphic display of comparative ORS sur 6. The method according to claim 3, wherein the isolation face changes. comprises isolating the melanocytes using differential (a) current method, day 0 (b) Dieckmann method (loc. cit.), trypsinization and Subsequently Geneticin treatment. day 0 (c) current method, day 7 (d) Dieckmann method (loc. 7. The method according to claim 1, wherein the collagen cit.), day 7 (e) current method, day 14(f) Dieckmann method degrading agent is selected from the group consisting of (loc. cit.), day 14. collagenase I, collagenase IV and collagenase V. US 2015/0O86513 A1 Mar. 26, 2015

8. The method according to claim 1, wherein the differen 16. The method according to claim 12, wherein the culti tiation medium further comprises one or more compounds Vation of keratinocytes over the melanocytes is performed in selected from the group consisting of B-adrenergic receptor a medium-air interface until formation of a stratum corneum ligands, epinephrine and derivatives thereof, Ca", in step (iv). L-glutamine, insulin, fetal calf serum, human serum, and 17. The method according claim 11, wherein the kerati bovine pituitary gland extract. nocytes to melanocytes are provided in a ratio of 10:1. 9. The method according to claim 1, wherein the growth 18. A graft of melanocytes generated by the method of factors are selected from the group consisting of ethanola claim 10. mine, phosphoethanolamine, hydrocortisone, basic fibroblast 19. Melanocytes generated by the method of claim 1. growth factor (bFGF), dibutyryl cyclic adenosine monophos 20. A graft according to claim 18 for use in treatment of a phate (dbcAMP), melanocyte growth factor (MeCF), Kapo disease selected from the group consisting of leukoderma, sis sarcoma derived FGF-like factor (hst/K-FGF), hepato vitiligo, quadrichrome vitiligo, vitiligo ponctué, syndromic cyte growth factor (HGF), stem cell growth factor (SCF). Albinism, Such as AleZZandrini syndrome, Hermansky-Pud endothelin 1, alpha-melanocyte stimulating hormone lak syndrome, Chédiak-Higashi syndrome, Griscelli syn (C-MSH), and mixtures of native factors from the medium drome (Elejalde syndrome, Griscelli syndrome type 2 and conditioned by cultured human keratinocytes, from bovine Griscelli syndrome type 3), Waardenburg syndrome, Tietz pituitary extract (BPE), bovine brain extract, or human syndrome, Cross-McKusick-Breen syndrome, ABCD syn S. drome, Albinism-deafness syndrome and Vogt-Koyanagi 10. A method for producing a graft comprising melano Harada syndrome, oculocutaneous albinism, hypomelanosis cytes, said method comprising the steps of: (idiopathic guttate hypomelanosis, phylloid hypomelanosis, (i) providing a Suspension comprising melanocytes obtain progressive macular hypomelanosis), piebaldism, nevus able by a method for generating melanocytes from stem depigmentosus, postinflammatory hypopigmentation, pityri cells according to claim 1: asis alba, Vagabonds leukomelanoderma, Yemenite deaf (ii) providing a biocompatible scaffold; and blind hypopigmentation syndrome, Wende-Bauckus Syn (iii) cultivating the melanocytes on said biocompatible drome, Woronoffs ring, amelanism, Leucism, and diseases scaffold. associated with depigmentation of the skin. 11. The method according to claim 10, wherein the sus 21. A method for treating a disease using the melanocytes pension of step (i) further comprises introducing kerati of claim 19 comprising treating a disease selected from the nocytes. group consisting of leukoderma, vitiligo, quadrichrome viti 12. The method according to claim 10, wherein the method ligo, vitiligo ponctué, syndromic Albinism, such as Alezzan further comprises the following steps: drini syndrome, Hermansky-Pudlak syndrome, Chédiak-Hi (iv) stratifying keratinocytes on the melanocytes cultivated gashi syndrome, Griscelli syndrome (Elejalde syndrome, on the biocompatible scaffold; and Griscelli syndrome type 2 and Griscelli syndrome type 3), (V) cultivating the keratinocytes and the melanocytes cul Waardenburg syndrome, Tietz syndrome, Cross-McKusick tivated on the biocompatible scaffold. Breen syndrome, ABCD syndrome, Albinism-deafness syn 13. The method according to claim 10 to 12, wherein the drome and Vogt-Koyanagi-Harada syndrome, oculocutane biocompatible scaffold consists of a three-dimensional fiber ous albinism, hypomelanosis (idiopathic guttate network. hypomelanosis, phylloid hypomelanosis, progressive macu 14. The method according to claim 10, wherein the bio lar hypomelanosis), piebaldism, nevus depigmentosus, compatible scaffold consists of at least one of the materials postinflammatory hypopigmentation, pityriasis alba, Vaga selected from the group consisting of polycaprolacton (PCL), bonds leukomelanoderma, Yemenite deaf-blind hypopig collagen, human collagen I, human collagen IV, human col mentation syndrome, Wende-Bauckus syndrome, Woronoffs lagen V, fibrin and gelatine. ring, amelanism, Leucism, and diseases associated with 15. The method according to claim 11, wherein the mel depigmentation of the skin. anocytes are cultivated until the formation of a stratumbasale. k k k k k