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(12) Patent Application Publication (10) Pub. No.: US 2015/0086513 A1 Savkovic Et Al 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 Figs. A-D Patent Application Publication Mar. 26, 2015 Sheet 2 of 17 US 2015/0O86513 A1 Figs. 2A Patent Application Publication Mar. 26, 2015 Sheet 3 of 17 US 2015/0O86513 A1 Figs. 3A-F Patent Application Publication Mar. 26, 2015 Sheet 4 of 17 US 2015/0O86513 A1 Fig. 4 OO 90 : & 80 70 S SO s: ; 30 a 20 to ( ; Fig. 5 23 :::s Fibroblasts HW f -- 3. 3. D Patent Application Publication Mar. 26, 2015 Sheet 5 of 17 US 2015/0O86513 A1 Figs. 6A-B Patent Application Publication Mar. 26, 2015 Sheet 6 of 17 US 2015/0O86513 A1 Figs. IA: Patent Application Publication Mar. 26, 2015 Sheet 7 of 17 US 2015/0O86513 A1 E. S. Patent Application Publication Mar. 26, 2015 Sheet 8 of 17 US 2015/0O86513 A1 Figs. 9A-D Patent Application Publication Mar. 26, 2015 Sheet 9 of 17 US 2015/0O86513 A1 Figs. 10A-B ssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss PC Scafiki fire PC Safic thick PS age ent fire itre surface PC scaffic fire PC scaffod thic PS adhereft fibre fibre stiface Patent Application Publication Mar. 26, 2015 Sheet 10 of 17 US 2015/0086513 A1 Figs. 10C-D C 300 80 - 7 8 SO S. 3. 2) - O - PC scaffold fire C scaffod thick S seret ife fibre surface PC. Scaffe P. scaffic thick PS alert ife fire surface Patent Application Publication Mar. 26, 2015 Sheet 11 of 17 US 2015/0086513 A1 Patent Application Publication Mar. 26, 2015 Sheet 12 of 17 US 2015/0086513 A1 Figs. 12A-H sa g) +L-DOPA h) +IBMX +L-DOPA Patent Application Publication Mar. 26, 2015 Sheet 13 of 17 US 2015/0086513 A1 Fig. 13 Comparative changes in the Outer Root Sheath surface 180000 160000 140000 s E 120000 4. Clifret frethod St 100000 ieckman method 80000 60000 40000 i.o. day. day 7 day 4 Patent Application Publication Mar. 26, 2015 Sheet 14 of 17 US 2015/0086513 A1 Figs. 14A-F Graphic display of comparative ORS surface changes current rethod Dieckman method day 0 day 7 day 14 Patent Application Publication Mar. 26, 2015 Sheet 15 of 17 US 2015/0086513 A1 Fig. 15 Comparative growth in the adherent culture 100,000.000 go 10,000.000 op-1 or OOOOOO ...------------------------------------------/------------------------------------------ logarithmic linear 1.000.000 g A current method E Dieckmann method K. Dieckmann method + G418 . S Passage Patent Application Publication Mar. 26, 2015 Sheet 16 of 17 US 2015/0086513 A1 Fig. 16 Comparative growth in the adherent culture, cell number per hair foilicle log 10,000.000 gr. 1.000.000 ir:-- A current method 100.000 ----- 10,000 ru ( )ieckmain method 1.Q00 OO - a aaaaaaaaaaaaaaaaaaaaaaaaaaaaXaXa aaaaaaaaaaaaaaaaaaaaaaaaaa a a -a aaaaaaaaaaaaaaaaaaaaaaaaaa. 2%. Dieckmann method + G418 10 or 1 r. Passage Patent Application Publication Mar. 26, 2015 Sheet 17 of 17 US 2015/0086513 A1 Fig. 17 Different size of cell soma and dendrite length in NHEM and HM Oendrite length (b) - 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.
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