WO2014163206A1 - Use of functional melanocytes readily differentiated from multilineage-differentiating stress-enduring (Muse) cells, distinct stem cells in human fibroblasts - Google Patents

Abstract

The induction of melanocytes from easy accessible stem cells has attracted attention for the treatment of pigment disorder or melanocyte dysfunctions such as vitiligo. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily differentiated into functional melanocytes. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by utilizing Muse cells, thereby contributing to transplantation for pigment disorder.

Description

Description

Title of the Invention:

Use of functional melanocytes readily differentiated from multilineage-differentiating stress-enduring (Muse) cells, distinct stem cells in human fibroblasts

Field of the Invention

[0001]

This application is a new U.S. patent application that claims benefit of US Provisional Patent Application

No. 61/807,465, filed on April 2, 2013, the entire content of which US Provisional Patent Application No. 61/807,465, is hereby incorporated by reference. [0002]

The present invention relates to use of Muse- melanocytes in regenerative medicine. Specifically, the present invention provides a pharmaceutical composition comprising the Muse-melanocytes and keratinocytes, and a method of treating pigment disorder. The present

invention is based on a finding of newly identified Muse cells in human dermal fibroblasts which are readily differentiated into functional melanocytes by using certain combinations of factors and cytokines.

Background Art

[0003]

Melanocytes produce melanin and deliver them to neighboring keratinocytes to protect the skin from ultraviolet (UV) rays (Kondo et al, 2011; Slominski et al, 2004). Melanocyte dysfunction results in a variety of pigment disorders, such as albinism and vitiligo, which cause not only cosmetic problems but also increase the risk of skin cancers due to incomplete protection from UV rays (Mabula et al, 2012) .

[0004]

Current treatments for vitiligo include topical treatment with corticosteroids or immunomodulators ; UV treatment, and autologous skin grafts (Alikhan et al, 2011; Felsten et al, 2011). The effectiveness of these treatments, however, is inadequate. Autologous cultured melanocyte transplantation is a potential cell therapy

(Fioramonti et al, 2012; van Geel et al, 2001), but is not widely used because human adult melanocytes are difficult to culture and amplify large scale in vitro. Several groups recently reported successful melanocyte induction from embryonic stem (ES) or induced pluripotent stem (iPS) cells (Nissan et al, 2011; Yang et al, 2011; Fang et al, 2006; Motohashi et al, 2006; Ohta et al, 2011; Yamane et al, 1999). Indeed, these are attractive cell sources for melanocyte induction, but the ethical problems of obtaining ES cells (Knoppers et al, 2009;

Manzar et al, 2011) and the risk of tumorigenesis for both ES and iPS cells are obstacles to clinical use (Fong et al, 2010; Goldring et al, 2011; Ben-David 2011; Okita et al, 2007) .

[0005]

Mesenchymal stem cells (MSCs) are adult stem cells with a lower risk of tumorigenesis that exist in

mesenchymal tissues such as the bone marrow, dermis, fat tissue, and dental pulp, and are used for the treatment of many kinds of diseases (Sng et al, 2012; Hare et al,

2009; Macchiarini et al, 2008; Jiang et al, 2011) . MSCs have attracted attention because of their ability to differentiate into a broad spectrum of cells. MSCs can differentiate not only into mesodermal lineage cells, but also into ectodermal or endodermal lineage cells (Phinney et al, 2007; Pittenger et al, 1999; Dezawa et al, 2004; Sakaida et al, 2004) . Melanocytes have also been induced from dental pulp stem cells (DPSCs) (Stevens et al, 2008, Paino et al, 2010) However, MSCs generally comprise crude cell populations and contain different cell types based on their cell surface antigens because they are usually harvested just as adherent cells from mesenchymal tissues. Therefore, the cells responsible for

differentiation across the oligolineage boundaries between mesodermal and ectodermal, namely mesenchymal to melanocytes, remain unknown.

[0006]

We recently reported a novel type of stem cell that exists among adult human MSCs that we named Multilineage- differentiating stress enduring cells (Muse cells)

(Kuroda et al, 2010; Wakao et al, 2011) . Muse cells normally exist in human mesenchymal cultured cells such as dermal fibroblasts and bone marrow stromal cells, and also in mesenchymal tissue, such as dermis and bone marrow. Muse cells have characteristics similar to both pluripotent stem cells and MSCs: they can be isolated by fluorescence-activated cell sorting (FACS) as cells double-positive for pluripotency [stage-specific

embryonic antigen-3 (SSEA-3), a marker for

undifferentiated human ES cells] and mesenchymal markers (CD105), and have the ability to self-renew and

differentiate into endodermal-, mesodermal-, and

ectodermal-lineage cells from a single cell. Unlike other pluripotent stem cells such as ES cells and iPS cells, however, Muse cells have low telomerase activity and do not form tumors in vivo. Thus, they have high potential for clinical application. Recently, skin-derived

precursor cells (SKPs) have been reported as multipotent mesenchymal stem cells in human foreskins (Toraa et al. 2005) . However, unlike SKPs, Muse cells are located sparsely in the connective tissue of dermis and adipose tissue and are not associated with any particular

structure such as dermal papilla, connective tissue sheath, or hair follicular epithelium. Additionally, Muse cells do not express the SKPs markers Snail and Slug (Wakao, et al. 2011). These data indicate that Muse cells are distinct from SKPs.

Summary of the Invention [0007]

We recently discovered that Muse cells in human dermal fibroblasts are readily differentiated into functional melanocytes by using certain combinations of factors and cytokines. Muse cell-derived melanocytes (Muse-melanocytes ) expressed melanocyte markers such as microphthalmia- associated transcription factor (MITF) , tyrosinase-related protein 1 (TRP-1), dopachrome

tautomerase (OCT) , KIT, gplOO, and tyrosinase, were positive for the 3, 4-dihydroxy-L- phenylalanine (L-DOPA) reaction assay, produced melanin, and integrated into the basal layer of epidermis. On the other hand, when the Muse cells were removed from human fibroblasts before melanocyte induction, the remaining cells (namely, "non- Muse cells") failed to become melanocytes and did not produce melanin. This finding indicates that Muse cells, the cells that already have triploblastic differentiation ability, can cross the oligolineage boundary between mesodermal and ectodermal lineages and become

differentiated into melanocytes, while the remaining fibroblasts do not participate in this event. Thus, Muse cells are an ideal cell source for generating functional melanocytes from adult human fibroblasts that can be applied to autologous or allogeneic transplantation for pigment disorders.

[0008]

In a first embodiment, the present invention

provides Muse-melanocytes which express tyrosinase and produce melanin. In one aspect, the Muse-melanocytes may further express at least one marker selected from the group consisting of DCT, Ki-67 and S100. According to the present invention, the Muse-melanocytes may be derived from Muse cells by use of two or more factors and/or cytokines selected from the group consisting of Wnt3a, stem cell factor (SCF) , endothelin-3 (ET-3), basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-0-tetradecanoyl-phorbol 13- acetate (TPA) , insulin-transferrin-selenium (ITS), and dexamethasone .

[0009]

In a second embodiment, the present invention provides a pharmaceutical composition comprising the Muse-melanocytes defined in the first embodiment. In one aspect, the present invention provides the pharmaceutical composition in which naive Muse cells remain.

[0010]

In a third embodiment, the present invention

provides a three dimensional skin comprising the above Muse-melanocytes. Optionally, keratinocytes may be comprised in the three dimensional skin. In one aspect, the present invention provides the three dimensional skin in which naive Muse cells remain.

[0011]

In a forth embodiment, the present invention

provides a method of producing a three dimensional skin, comprising the steps: (a) providing Muse cells from a mesenchymal tissue; (b) culturing the Muse cells in a differentiation medium; (c) culturing the Muse cell derived melanocytes with keratinocytes in a gel layer; and (d) obtaining a three dimensional skin. In one aspect, the mesenchymal tissue may be a dermal tissue. In another aspect, Muse cells may be separated from

fibroblasts in the mesenchymal tissue. Moreover, the differentiation medium used in the above method may contain two or more factors and/or cytokines selected from the group consisting of Wnt3a, stem cell factor

(SCF) , endothelin-3 (ET-3), basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-0-tetradecanoyl-phorbol 13-acetate (TPA) , insulin-transferrin-selenium (ITS), and dexamethasone.

[0012]

In a fifth embodiment, the present invention

provides a method of treating pigment disorder in

subjects in need of such a treatment, comprising applying the Muse-melanocytes according to the first embodiment, the pharmaceutical composition according to the second embodiment, or the three dimensional skin according to the third embodiment, to an affected area caused by the disorder.

[0013]

In a sixth embodiment, the present invention

provides a method of obtaining Muse-melanocytes by differentiating Muse cells with two or more factors and/or cytokines selected from the group consisting of

Wnt3a, stem cell factor (SCF) , endothelin-3 (ET-3) , basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-O-tetradecanoyl-phorbol 13- acetate (TPA) , insulin-transferrin-selenium (ITS), and dexamethasone .

Brief Explanation of the Drawings

[0014]

FIG. 1 is data showing characterization of Muse cells .

(a) Fluorescence-activated cell sorting (FACS) analysis for stage-specific embryonic antigen-3 (SSEA-3)

expression in naive normal human dermal fibroblasts

(NHDF) . (b) M-cluster formed in single-cell suspension culture at Day 7. (c) ALP staining of an M-cluster. (d-f)

Immunocytochemistry for neurofilament (d) , alpha smooth muscle actin (a-SMA) (e) , and GATA4 (f ) in cells derived from a single M-cluster. (g) Schematic diagram of

melanocyte differentiation from Muse cells and non-Muse cells. (Scale bars: b-f, 50 μπι) . Abbreviations: b-FGF, basic fibroblast growth factor; ET-3, endothelin-3; SCF, stem cell factor; TPA, 12-O-tetradecanoyl-phorbol 13- acetate .

[0015]

FIG. 2 is data showing morphology of Muse cells and non-Muse cells after differentiation, (a) Microscopic images of Muse cells and non-Muse cells before, and 3, 5, 6 weeks after the differentiation. Microscopic images of naive normal human dermal fibroblasts (NHDF) and human melanocytes are also provided (Scale bars: 100 μτη) . (b) Enlarged images of Muse cells and non-Muse cells 6 weeks after the differentiation. Normal human melanocytes and NHDF are also shown.

[0016]

FIG. 3 is data showing characterization of Muse- melanocytes. (a) RT-PCR analysis of microphthalmia- associated transcription factor (MITF) , KIT, tyrosinase- related protein 1 (TRP-1), dopachrome tautomerase (DCT), gplOO, and tyrosinase at 3, 5, and 6 weeks of

differentiation both in Muse and non-Muse cells. The positive control was human melanocytes and the negative controls were naive normal human dermal fibroblasts

(NHDF) and no-template, (b) Immunocytochemical analysis of the melanocyte markers MITF, gplOO, and tyrosinase in Muse and non-Muse cells 6 weeks after differentiation. The positive control was human melanocytes and the negative control was naive Muse cells without primary antibody. (Scale bars: 50 μιπ) . (c) L-DOPA reaction assay of Muse-melanocytes (6 weeks), human melanocytes, and naive NHDF. The pigmented cells are L-DOPA positive cells. (Scale bars: 100 μτη) .

[0017]

FIG. is data showing effect of factors on

melanocyte differentiation, (a) Summary of the

proliferating capacity and melanocyte marker expression of Muse cells cultured in 7 different media, (b) RT-PCR for microphthalmia-associated transcription factor

(MITF), KIT, tyrosinase-related protein 1· (TRP-1), dopachrome tautomerase (DCT) , gplOO, and tyrosinase in Muse cells at 6 weeks of culture in 4 different media. The positive control was human melanocytes and the negative control was no-template, (c) Morphology of Muse cells in 4 different media at 6 weeks of culture. (Scale bars: 100 μπι) . Abbreviations: b-FGF, basic fibroblast growth factor; ET-3, endothelin 3; ITS, insulin- transferrin-selenium; RA, retinoic acid; SCF, stem cell factor

[0018]

FIG. 5 is data showing histologic analysis of 3D cultured skin, (a) Hematoxylin eosin (HE) staining of 3D cultured skin containing human melanocytes, Muse- melanocytes, non-Muse cells, and keratinocytes alone, (b) HE staining of the 3D cultured skin containing Muse- melanocytes. (Arrowheads indicate Muse-melanocytes). (c) Immunohistochemical analysis of microphthalmia-associated transcription factor (MITF) , tyrosinase, tyrosinase- related protein 1 (TRP-1) , gplOO, and S-100, and Fontana- Masson staining of the 3D cultured skin. (Scale bars: 50 μπι)

[0019]

FIG. 6 is data showing functional characterization of Muse-melanocytes after transplantation into SCID mouse back skin, (a) Macroscopic observation of skin grafts containing Muse-melanocytes, human melanocytes, and keratinocytes only 10 days after transplantation, (b) Hematoxylin eosin staining of each skin graft (Scale bars: upper panels, 100 μιη, lower panels, 50 μιη) . (c) Immunohistochemical analysis of microphthalmia- associated transcription factor (MITF) , tyrosinase, tyrosinase-related protein 1 (TRP-1), gplOO, S100, and Fontana-Masson staining in the skin grafts, (d)

Localization of Muse-melanocytes labeled with green fluorescent protein (GFP) detected by anti-GFP antibody and Alexa-568 (in red) was observed (arrow). (Scale bars:

50 μπι)

[0020]

FIG. 7 is data showing histologic analysis of three dimensional (3D) cultured human skin made by mixing naive

Muse cells with human keratinocytes for the epidermis. (a) Localization of naive Muse cells labeled with green fluorescent protein (GFP) was observed in the epidermis (arrow) . (b) Immunohistochemical analysis of tyrosinase (TYR) , SlOO, and tyrosinase related protein-1 (TRP-1) in naive Muse cells labeled with GFP. The positive control was human melanocytes. (Scale bars: 50 μπι) . Abbreviation:

DAPI, 4 ' , 6-diamidino-2-phenylindole

[0021]

FIG. 8 is data showing histologic analysis of three dimensional (3D) cultured skin made by including naive

Muse cells or Muse-melanocytes into the dermal

equivalent, (a) Localization of naive Muse cells labeled with (GFP) was observed both in the epidermis and the dermis of the 3D cultured human skin made by including naive Muse cells into the dermal equivalent (arrows) .

These naive Muse cells were negative for both SlOO and tyrosinase related protein-1 (TRP-1) (data not shown).

(b) Immunohistochemical analysis of SlOO and TRP-1 of 3D cultured human skin made by including Muse-melanocytes in the dermal equivalent. Muse-melanocytes positive for SlOO and TRP-1 were detected in the basal membrane of the epidermis. (Scale bars: 50 μπι) Abbreviation: DAPI, 4 ' , 6- diamidino-2-phenylindole

[0022]

FIG. 9 is data showing immunohistochemical analysis using anti Ki-67 antibody (Roche, Basel, Switzerland) and anti SlOO antibody in skin grafts containing Muse- melanocytes. Ki-67 expression was detected by an HPR-DAB system (brown color) (Wako, Osaka, Japan) . SlOO

expression was detected by an Alkaline Phosphatase-

Permanent Red kit (red color) (DAKO) . Cells double positive for Ki-67 and SlOO were detected in the basal layer of epidermis (inset) . (Scale bar: 50 μιη) .

Abbreviation: HRP, horseradish peroxidase; DAB,

Diaminobenzidine Mode for Carrying Out the Invention

[0023]

The present invention provides a pharmaceutical composition comprising melanocytes and keratinocytes, in which the melanocytes derived from multilineage- differentiating stress-enduring (Muse) cells by using certain combinations of factors and cytokines. The present invention also provides a method of producing a three dimensional skin with Muse cells and a method of treating pigment disorder.

[0024 ]

According to the present invention, a pharmaceutical composition comprising Muse cell-derived melanocytes (i.e., Muse-melanocytes ) and keratinocytes is provided in which the Muse-melanocytes are derived from Muse cells by use of two or more factors and/or cytokines, which include, but are not limited to, nt3a, stem cell factor (SCF), endothelin-3 (ET-3) , basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) , insulin-transferrin-selenium (ITS), and dexamethasone .

[0025]

According to the present invention, a three

dimensional skin comprising Muse-melanocytes and

optionally keratinocytes is provided. The three

dimensional skin may comprise any cells and/or proteins required for building the same.

[0026]

According to the present invention, a method of producing a three dimensional skin comprising the Muse- melanocytes and keratinocytes is provided. The method comprises the steps:

(a) providing Muse cells from normal dermal

fibroblast or other mesenchymal tissues;

(b) culturing the Muse cells in a differentiation medium containing two or more factors and/or cytokines, which include, but are not limited to, nt3a, stem cell factor (SCF), endothelin-3 (ET-3) , basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L- ascorbic acid, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) , insulin-transferrin-selenium (ITS), and

dexamethasone, to obtain Muse-melanocytes;

(c) culturing the Muse-melanocytes with

keratinocytes in a gel layer; and

(d) obtaining a three dimensional skin.

[0027]

According to the present invention, a method of treating with pigment disorder in subjects in need of such a treatment, comprising applying the above skin to an affected area caused by the disorder, is provided.

[0028]

According to the present invention, a method of obtaining Muse-melanocytes from Muse cells is provided, the method comprising the step of differentiating Muse cells with two or more factors and/or cytokines, which include, but are not limited to, Wnt3a, stem cell factor (SCF), endothelin-3 (ET-3), basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-0-tetradecanoyl-phorbol 13-acetate (TPA) , insulin-transferrin-selenium (ITS), and dexamethasone.

Muse cells used in the present inventions may be derived from dermal fibroblast or other mesenchymal tissues .

[0029]

1. Indication for treatment

The present invention is_. directed to use of Muse- melanocytes in regenerative medicine. Specifically, the present invention provides a pharmaceutical composition and a three dimensional skin for treating and/or

preventing skin pigment disorders using Muse-melanocytes. As used herein, the term "pigment disorder" refers to conditions that cause the skin to appear lighter or darker than normal, or blotchy and discolored. The pigment disorder includes albinism and vitiligo, which cause not only cosmetic problems but also increase the risk of skin cancers due to incomplete protection from UV rays. In addition, the pigment disorder includes a skin burn, a scald, a scar of a burn, a wound and a keloid scar.

[0030]

2. Muse-melanocytes

In accordance with the present invention, Muse- melanocytes can be obtained by differentiating Muse cells with specific, factors and/or cytokines.

[0031]

2-1. Muse cells (multilineage-differentiating stress- enduring cells)

According to the present invention, multilineage- differentiating stress-enduring (Muse) cells which are used for inducing melanocytes can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem

Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014), or skin tissue such as dermis connective tissue or other mesenchymal tissues. Muse cells are scattered around connective tissues in organs. Further, Muse cells have both characteristics of pluripotent stem cells and mesenchymal cells, and, for example, they can be

identified as "SSEA-3 (Stage-specific embryonic antigen- 3)" and "CD105" double positive cells. SSEA-3 and CD105 are cell surface markers of pluripotent stem cells and mesenchymal cells, respectively. Therefore, Muse cells or a cell population containing Muse cells can be separated from living tissues, for example, as a measure of these antigen markers. More detailed descriptions of a

separation method, an identification method and

characteristics of Muse cells, are disclosed in

WO2011/007900. Moreover, as reported by Wakao, et al. (2011., set forth above) , in the case where mesenchymal cells from bone marrow or skins are cultured and used as a parent population, it has been found that all of SSEA-3 positive cells are CD105 positive cell. Therefore, in the pharmaceutical composition according to the present invention, if Muse cells are separated from living mesenchymal tissues or cultured mesenchymal cells, Muse cells can be purified merely by use of SSEA-3 as an antigen marker. In this specification, pluripotent stem cells separated from the living mesenchymal tissues or cultured mesenchymal cells (Muse cells) or the cell population containing Muse cells are also referred to as "SSEA-3 positive cells". In addition, as used herein, the term "non-Muse cells" means cells contained in the living mesenchymal, tissues or cultured mesenchymal cells (other than "SSEA-3 positive cells").

[0032]

Briefly, Muse cells or a cell population containing Muse cells can be separated from a living tissue (for example, a mesenchymal tissue) using an antigen against SSEA-3 or both antibodies against SSEA-3 and CD105. The term "living body" refers to a living mammalian body. The living body does not include fertilized eggs or embryos at development stages before the blastula stage, but include embryos at development stages on and after the blastula stage, such as fetuses and blastulae. Examples of mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cattle, horses, donkeys, goats, and ferrets. Muse cells used in the pharmaceutical composition according to the present invention are clearly distinguished from

embryonic stem cells (ES cells) or iPS cells (EG cells) in that they are from living body tissue using a

marker (s). In addition, the term "mesenchymal tissue" refers to tissue such as bone, cartilage, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, and cord blood, and tissue reside in each of organs. For example, Muse cells can be obtained from bone marrow, skin or adipose tissue. It is preferred to obtain a mesenchymal tissue from a living body and separate and utilize Muse cells from this tissue. Moreover, Muse cells may be separated from cultured mesenchymal cells such as fibroblasts or bone marrow stromal cells by use of the above separating method. In the pharmaceutical composition according to the present invention, Muse cells used may be autologous or xenologous against recipients.

[0033]

As described above, Muse cells or the cell

population containing Muse cells can be separated, for example, using SSEA-3 positive marker or both SSEA-3 and CD105 double positive markers as an index. In addition, it is known that human adult dermis contains several types of stem cells and progenitor cells. However, Muse cells are not identical to these cells. Such stem cells and progenitor cells include skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs) ,

melanoblasts (MBs), perivascular cells (PCs), endothelial progenitors (EPs), and adipose-derived stem cells

(ADSCs) . Muse cells can be separated using "non- expression" of markers specific to these cells as an index. Specifically, Muse cells can be isolated using non-expression of at least 1, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, markers selected from the group

consisting of CD34 (a marker of EP and ADSC) , CD271

(NGFR) (a marker of NSCS)^', NG2 (a marker of PC), vWF factor (von Willebrand factor) (a marker of EP) , SoxlO (a marker of NCSC) , Snail (a marker of SKP) , Slug (a marker of SKP), Tyrpl (a marker of MB), and Dct (a marker of MB), as an index. For example, the separation is possible using non-expression of CD117 and CD146 as an index.

Furthermore, the separation can be performed using non- expression of CD117, CD146, NG2, CD34, vWF, and CD271 as an index. Moreover, the separation can be performed using non-expression of the above 11 markers as an index.

[0034]

Muse cells having the above characteristics, which are used in the pharmaceutical composition, may have at least one property selected from the group consisting of

(i) having low or no telomerase activity; (ii) having capability to differentiate into one of triploblastic cells; (iii) representing non-tumorigenic proliferation; and (iv) having self-renewal capability. In one aspect of the present invention, Muse cells used in the

pharmaceutical composition have all of the above

properties. With respect to (i) above, the expression "having no or low telomerase activity" refers to no or low telomerase activity being detected when such activity is detected using a TRAPEZE XL telomerase detection kit

(Millipore) , for example. The term "low telomerase activity" refers to a situation in which cells have telomerase activity to the same degree as that of human fibroblasts or have telomerase activity that is 1/5 or less and preferably 1/10 or less that of Hela cells. With respect to (ii) above, Muse cells have capability to differentiate into triploblastic cells (ectodermal, mesodermal, and endodermal cell lineages) in vitro and in vivo, and they can differentiate into hepatocytes, neuronal cells, skeletal muscle cells, smooth muscle cells, bone cells and adipose cells. In addition, even when transplanted in the testis, Muse cells are capable of differentiating into triploblastic cells. Further, Muse cells are capable of migrating, surviving and differentiating into organs (e.g., skin, spinal cord, liver, and muscle) when transplanted to the damaged organs via intravenous injection into a living body. With respect to (iii) above, Muse cells have a property that they grow at a growth rate of about 1.3 days/cell

division by suspension culture, they proliferate from one cell of thereof and form embryoid body-like cell

clusters, but stop the growth within about 14 days. However, when further culturing the embryoid body-like cell clusters by adhesion culture, the cell proliferation restarts and proliferated cells expand from the cell clusters. Moreover, when transplanted into the testis, Muse cells have a property that they do not become cancerous for at least a half year. In addition, with respect to (iv) above, Muse cells have self-renewal capability. The term "self-renewal" refers to a situation in which cells contained in embryoid body-like cell clusters obtained by suspension culture of one Muse cell, can differentiate into triploblastic cells, and

simultaneously the next-generation embryoid body-like cell clusters can form by suspension culture of one cell contained in the previous embryoid body-like cell

clusters, and then cells contained therein by suspension culture can differentiate into triploblastic cells and embryoid body-like cell clusters can form. Self-renewal may be performed by repeating a cycle once to several instances.

[0035]

2-2. Differentiation of Muse cells into melanocytes

According to the present invention, Muse cells can be differentiated into melanocytes (hereinafter referred to as "Muse-melanocytes" , if necessary), by use of a specific combination of factors and cytokines. These factors and cytokines include, but not limited to, Wnt3a, stem cell factor (SCF), endothelin-3 (ET-3) , basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-O-tetradecanoyl-phorbol 13- acetate (TPA) , insulin- transferrin-selenium (ITS), and dexamethasone . In order to differentiate Muse cells into melanocytes, it is preferred to use two or more factors and/or cytokines selected form the group consisting of the above ten factors. As used herein, the term "Wnt3a" refers to a member of WNT gene family consisting of structurally related genes that encode secreted signaling proteins. The term "stem cell factor (SCF)" refers to a cytokine that binds to c-Kit receptor (CD117). The term "endothelin-3 (ET-3)" refers to a potent vasoconstricting factor that is produced and released from vascular endothelial cell. The term "basic fibroblast growth factor (b-FGF) " refers to a growth factor associated with angiogenesis, wound healing and embryogenesis . The term "linoleic acid" is a naturally occurring unsaturated free acid. The term "cholera toxin" refers to a protein complex secreted by the bacterium Vibrio cholerae . The term "L-ascorbic acid" refers to a naturally occurring organic compound with antioxidant properties. The term "12-O-tetradecanoyl-phorbol 13-acetate (TPA) " refers to a tumor promoter. The term "insulin-transferrin-selenium (ITS)" refers to a basal medium supplement for cell culture. The term "dexamethasone" refers to a potent synthetic member of glucocorticoid class of steroid drugs that has anti-inflammatory and immunosuppressant effects. According to the present invention, the combination of the above factors and cytokines is not limited to

specific combination as long as Muse cells can be

differentiated into melanocytes using two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) factors and/or cytokines selected from the group consisting of nt3a, SCF, ET-3, b-FGF, linoleic acid, cholera toxin, L-ascorbic acid,

TPA, ITS, and dexamethasone.

[0036]

In accordance with the present invention, Muse cells can be differentiated into melanocytes, for example, as follows: Muse cells are cultured in differentiation medium containing 1-lOOOng/ml Wnt3a, 0.1-500ng/ml SCF, 0.1-lOOnM ET-3, 0.1-lOOng/ml b-FGF, 0.01-lOOmg/ml

linoleic acid, 1-lOOOpM cholera toxin, 10^"6-10^"2M L- ascorbic acid, l-500nM TPA, 0.1-lOX ITS, and/or 0.01-lM dexamethasone. The basic medium that is essential for survival of cells, but is not limited to, a-Minimum

Essential Medium (cc-MEM) , Dulbecco ' s Modified Eagle Medium (D- E ) , RPMI 1640, Basal Medium Eagle (BME) , Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM/F-12) , Glasgow Minimum Essential Medium (Glasgow MEME) , Hank's balanced solution, and MCDB 153 medium. The above basic medium in which the above factors and/or cytokines are dissolved can be used as the

differentiation medium. In addition, Muse cells are maintained in the differentiation medium for 1-12 weeks, and further such a melanocyte induction from Muse cells may be repeated at least 2 times, preferably at least 3 times. Alternatively, the melanocyte induction may be carried out by adding the above factors and/or cytokines to the basic medium three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.

[0037]

According to the present invention, in order to confirm that Muse cells have been differentiated into melanocyte (i.e., Muse-melanocyte has been obtained), the expression of melanocyte-related markers can be examined by various kinds of detection methods such as reverse transcription-polymerase chain reaction (RT-PCR) and FACS. The melanocyte-related markers include, but are not limited to, MiTF, c-kit, TRP-1, gplOO, DCT, and

tyrosinase. If the induced Muse cells express at least one of MiTF, c-kit, TRP-1, gplOO, DCT, and tyrosinase, the cells may be considered as Muse-melanocytes.

[0038]

3. Preparing and use of the pharmaceutical composition according to the present invention

The pharmaceutical composition according to the present invention can be obtained, for example, by suspending the Muse-melanocytes and keratinocytes in a normal saline solution or an appropriate buffer such as a phosphate buffered saline (PBS) . In this connection, if the number of Muse-melanocytes is not sufficient to use for treatment or transplantation, the Muse-melanocytes or Muse cells (not induced) may be grown in a cell medium until the desired number of cells. As described in

WO2011/007900, since Muse cells do not undergo

tumorigenic transformation, a probability of canceration of the cells is low and can be safe, even when

undifferentiated cells obtained from a living tissue are contained in cell culture.

[0039]

When using Muse-melanocytes in a pharmaceutical composition, the pharmaceutical composition may contain, for example, DMSO and/or serum albumin for protection of the cells, and/or antibiotics for preventing the cells from contamination by bacteria. Further, various kinds of pharmaceutical acceptable components such as carriers, excipients, disintegrants , emulsifying agents, suspension agents, soothing agents, stabilizing agents, preserving agents, antiseptic agents and a normal saline solution, can be contained in the pharmaceutical composition.

[0040]

4. Three dimensional skin and production thereof

In one embodiment, the present invention provides a three dimensional skin comprising Muse-melanocytes. The three dimensional skin can further comprise keratinocytes and extracellular matrix such as collagen, fibronectin, laminin, heparan sulfate proteoglycan and Matrigel ®. According to the present invention, a method of producing such a three dimensional skin, comprising the steps:

(a) providing Muse cells from normal dermal

fibroblast or other mesenchymal tissues;

(b) culturing the Muse cells in a differentiation medium containing two or more factors and/or cytokines selected from the group consisting of Wnt3a, stem cell factor (SCF), endothelin-3 (ET-3), basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L- ascorbic acid, 13-0-tetradecanoyl-phorbol 13-acetate (TPA) , insulin-transferrin-selenium (ITS), and

dexamethasone, to obtain Muse-melanocytes;

(c) culturing the Muse-melanocytes with

keratinocytes in a gel layer; and

(d) obtaining a three dimensional skin.

[0041]

Specifically, a gel layer comprising extracellular matrix (e.g., collagen) and normal dermal fibroblast may be generated to mimic the dermis. Subsequently, Muse- melanocytes may be seeded onto the gel layer, optionally in combination with keratinocytes , and then they may be cultured until the desired tissue constructs can be obtained.

[0042]

To investigate whether Muse-melanocytes can survive and maintain their melanocyte functions for a certain period of time in vivo, the three dimensional skin according to the present invention can be transplanted on model animals, and evaluated. The evaluation of Muse cell pluripotency can be carried out, for example, by

immunocytochemistry, L-DOPA reaction assay,

immunohistochemistry, and Fontana-Masson stain, all of which are well known to a person skilled in the art.

[0043]

Unless defined otherwise, all technical and

scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein the following terms have the following meanings.

[0044]

As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" when used to define

compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic ( s ) of the claimed disclosure. "Consisting of" shall mean excluding more than trace elements of other ingredients and

substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thread" includes a plurality of threads.

EXAMPLES

[ 0045]

Hereinafter, the present invention is described in more detail by referenced to the Examples, but the present invention is not limited to the Examples.

[ 0046]

MATERIALS AND METHODS

All animal experiments were approved by the Animal Care and Experimentation Committee of Tohoku University Graduate School of Medicine.

[0047 ]

Cell culture

NHDF (Lonza, Walkersville, MD) were cultured in a - MEM (Invitrogen, Carlsbad, CA) containing 10% FBS

(Hyclone; Thermo Fisher Scientific, Logan, UT) . Human melanocytes (LifeLine Cell Technology, Frederick, MD) were maintained in DermaLife M LifeFactors medium

(LifeLine Cell Technology) . Human Neonatal Epidermal Keratinocytes (Gibco Invitrogen) were cultured in

Humedia-KG2 medium (Kurabo, Osaka, Japan) .

[ 0048]

Melanocyte induction

After sorting, Muse cells and non-Muse cells were seeded separately at a density of 10,000 cells per 6-well plate and cultured for 1 day in a-MEM. The cells were then cultured in differentiation medium containing 0.05 M dexamethasone (Sigma-Aldrich, St. Louis, MO), lx ITS (Invitrogen) , 1 mg/ml linoleic acid-bovine serum albumin (Sigma-Aldrich), 30% low-glucose DMEM, 20% MCDB-201 medium, 10^"4 M L-ascorbic acid (Sigma-Aldrich) , 50% DMEM conditioned by L-Wnt3a cells (American Type Culture

Collection, Manassas, VA) , 50 ng/ml SCF (R&D System Inc., Minneapolis, MN)_., 10 nM ET-3 (Sigma-Aldrich), 20 pM cholera toxin (Wako, Osaka, Japan), 50 nM TPA (Sigma-

Aldrich) , and 4 ng/ml b-FGF (Wako) . All chemical reagents were treated according to the product information sheets. Cells were maintained in this differentiation medium for 6 weeks. The medium was changed every 2 days. Cultures were passaged when cells reached 50% to 80% confluency.

Melanocyte induction from both Muse and non-Muse cells was repeated at least three times.

[0049]

The methods for cell culture, FACS, evaluation of Muse cell pluripotency, generating Wnt3a conditioned medium, RT-PCR, immunocytochemistry, L-DOPA reaction assay, immunohistochemistry, Fontana-Masson stain, generation of 3D cultured skin in vitro and skin

transplantation were described in supplementary materials online.

[0050]

Fluorescence activated cell sorting (FACS)

To obtain Muse cells, normal dermal human

fibroblasts (NHDF, Lonza Walkersville, MD) were incubated with rat anti stage-specific embryonic antigen-3 (SSEA-3)

IgM antibody (1:50; Millipore, Billerica, MA; detected by fluorescein isothiocyanate-conjugated anti-rat IgM,

Jackson Immunoresearch, West Grove, PA) in the FACS antibody diluents and sorted by Special Order Research Products FACSAriall (Becton Dickinson, Franklin Lakes,

NJ) as described previously (Kuroda et al, 2010; Wakao et al, 2011) . [0051]

Evaluation of Muse cell pluripotency

According to a previous report (Kuroda et al, 2010), sorted Muse cells were individually plated in each well of 96-well ^' plates . by limiting dilution of the cells with a-MEM (Invitrogen, Carlsbad, CA) , and cultured in single- cell suspension culture for 7 days. The dishes were coated with poly-HEMA (Sigma Aldrich, St. Louis, MO) to prevent cell-attachment. M-cluster formation was observed on Day 7. Alkaline Phosphatase (ALP) staining was then performed using a Leukocyte Alkaline Phosphatase kit (Sigma Aldrich) . To analyze the differentiation ability of M-clusters in vitro, M-clusters were transferred individually onto gelatin-coated dishes. After 7 days of culture, immunocytochemistry was performed as described previously (Kuroda et al, 2010). Antibodies used in this study were a-SMA (Lab Vision, Fremont, CA) , neurofilament (Chemicon, Millipore) , and GATA-4 (Abeam, Cambridge, UK) .

[0052]

Conditioned media from L-Wnt3a cells (Wnt3a-CM)

The L-Wnt3a cell line was obtained from American Type Culture Collection (Manassas, VA) . First, L-Wnt3a cells were cultured in 10 cm dishes using high glucose DMEM (Invitrogen) containing 10% FBS (Hyclone; Thermo Fisher Scientific, Logan, UT) and 0.4mg/ml G418. After the cells grew to about 90% confluency, they were split 1:5 in 10 ml high glucose DMEM containing 10% FBS in 10 cm culture dishes and grown for 4 days. Then, the

conditioned medium in the dishes was collected and stored at 4° C after filtration with a 0.22μπι filter. This was the first batch of medium. Next, 10 ml high glucose DMEM + 10% FBS was added to the dishes and cultured for another 3 days. After 3 days, the conditioned medium was collected and filtered. This was the second batch of medium. The first and second batches were mixed. This was the Wnt3a-conditioned medium which can be stored at 4° C for up to 4 weeks. The presence of Wnt3a protein in the conditioned media was checked by detecting the signal in a Western-blot analysis.

[0053]

MCDB201 medium

CDB201 medium was obtained from Sigma-Aldrich.

MCDB201 medium is a modification of Ham's nutrient mixture F-12, designed for the clonal growth of chicken embryo fibroblasts using hormones, growth factors, trace elements, and low levels of fetal bovine serum protein.

MCDB201 medium was used to make differentiation medium with DMEM and 10 differentiation- inducing factors.

[0054]

Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was purified with^', an RNeasy Mini Kit

(QIAGEN, Venlo, The Netherlands) . cDNA synthesis was performed with Superscript II reverse transcriptase

(Invitrogen) primed with 01igo(dT)₁₅ primer (Promega, Madison, WI). PCR was performed as follows: one cycle at 94°C, 5 minutes, followed by 36 or 40 cycles at 94°C, 1 minute; gene-specific annealing temperature, 1 minute; 72°C, 1 minute; and then extension at 72°C, 7 minutes. The primers were designed as follows: KIT forward primer, 5 ' -GAAAGTGACGTCTGGTCCTATGG-3 ' ; reverse primer, 5'- GTGCTCTCTGAAATCTGCTTCTCA-3 ' ; dopachrome tautomerase (DCT) forward primer, 5¹ -TCCTTCCTGAACGGGACAAA-3 ' ; reverse primer, 5 ' -TGGCATAGCTGTAGCCAAGTTG-3 ' ; MITF forward primer, 5 ' -CGGGAACAGGACCATGGTTA-3¹ ; reverse primer, 5'- AGCTAGCCCCTGAAATGAATCC-3 ' ; gplOO forward primer, 5'- CCAGTGTATCCCCAGGAAACTG-3 ' ; reverse primer, 5'-

GAATGAGCAAGAGGCACATAGCT-3 ' ; TRP-1, forward primer, 5'- TGCACACCTTCACAGATGCA-3 ' ; reverse primer, 5'- AAGCGCCAACTACTGCTATGG-3¹ ; tyrosinase, forward primer, 5'- GAGGTCAGCACCCCACAAAT-3 ' ; reverse primer, 5'- GCAGCTTTATCCATGGAACCA-3 ' .

[0055]

Immunocytochemistry ^■ Cells were fixed with 4% paraformaldehyde in

phosphate buffered saline (PBS) and incubated with primary antibodies specific for MITF (Lab Vision), tyrosinase (Lab Vision) , and gplOO (Dako, Carpinteria, CA) . After washes, cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen) and counterstained with DAPI (Invitrogen) . Then they were examined using a clsi Nikon confocal microscope system (Nikon, Tokyo, Japan) .

[0056]

L-DOPA reaction assay

Cells were washed twice with 0.1 M sodium phosphate buffer pH 6.8 and fixed with 4% paraformaldehyde at room temperature for 20 minutes. Thereafter, the cells were washed three times with phosphate buffer and incubated with 5 mM L-DOPA (Sigma-Aldrich) in sodium phosphate buffer at 37°C for 18 hours in the dark. After incubation, the cells were washed with distilled water, fixed with 4% paraformaldehyde for 20 minutes, and photographed under light microscopy.

[0057]

3D cultured skin in vitro

Inserts of tissue culture trays (Corning Life

Sciences, Pittston, PA) were layered with a mixed

solution comprising collagen type 1 (Nitta Gelatin Inc,

Osaka, Japan), minimum essential medium with Hank's

Balanced Salts (Nitta Gelatin Inc.), and 3.5 x 10⁵ NHDF cells/ml. After incubation at 37°C for 3 days, Muse- melanocytes or non-Muse cell-derived cells (non-Muse cells treated for melanocyte induction) were seeded onto the collagen layer together with human epidermal

keratinocytes at a ratio of 1:2.5 Muse-melanocytes to keratinocytes. They were incubated in Humedia KG2 medium (Kurabo) for 5 days with a gradually increasing Ca⁺⁺ concentration. The Ca⁺⁺ concentration was 0.15 mM on Day

1, 1.0 mM on Day 2, and 1.5 mM for Days 3 through 5. The 3D cultured skins were cultivated for another 7 days at the air-liquid interface and then fixed with 10%

formaldehyde for evaluation or grafted onto SCID mice. The 3D cultured skin with human melanocytes was used as the positive control and without human melanocytes as the negative control.

[0058]

Immunohistochemistry of 3D cultured skin and skin grafts

3D cultured skin and skin grafts were fixed with 10% formaldehyde, embedded in paraffin, and cut into 3-μπι thick sections. The sections were treated with 0.3% hydrogen peroxide in methanol for 12 minutes to

inactivate the intrinsic peroxidase activity, and then stained with S100 (polyclonal, DAKO) , TRP-1 (polyclonal, Sigma) , MITF, tyrosinase, and gplOO antibodies using standard immunoperoxidase techniques.

[0059]

Skin grafts containing Muse-melanocytes labeled with GFP were fixed with freshly prepared periodate-lysine- paraformaldehyde for 6 hours at 4^°C, embedded in OCT compound (Sakura Finetechnical , Tokyo, Japan) , and cut into 8-μπι thick cryosections . The samples were washed with PBS and incubated with blocking solution at room temperature for 30 minutes. The slides were then stained with anti-GFP antibody (Abeam) at 4°C overnight. The slides were washed 3 times with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 568

(Invitrogen) at room temperature for 2 hours. They were then washed 3 times with PBS, counterstained with DAPI

(Invitrogen) for 30 minutes, and examined using a Nikon fluorescence microscope.

[0060]

Fontana-Masson staining

3D cultured skin and skin grafts were examined with Fontana-Masson staining for histologic detection of melanin. Paraffin-embedded sections were deparaffinized and rinsed in distilled water. Slides were treated for 24 hours at room temperature in the dark with Fontana silver nitrate solution (Muto Pure Chemicals Co., Ltd, Tokyo, Japan) . After washing with distilled water, the slides were treated with gold chloride solution (Muto Pure

Chemicals Co., Ltd) for 5 seconds and placed in acid hardening fixer (Muto Pure Chemicals Co., Ltd) for 1 minute. The slides were washed and counterstained with neutral red (Muto Pure Chemicals Co., Ltd) for 1 minute.

[0061]

Skin transplantation

3D cultured skins were grafted onto the back skin of 8 to 14-week-old SCID mice. The mice were anesthetized with an intraperitoneal injection of tribromoethanol (Nacalai Tesque, Kyoto, Japan) and graft beds (8 mm x 8 mm) were prepared on the mouse back skin. The 3D cultured skin was applied onto the graft beds and covered with Vaseline gauze, Steri-Strip ( 3M Company, St. Paul, MN) and a bandage. The bandage was removed on Day 3. Graft survival was observed for another 7 days. To trace Muse- melanocytes in vivo, Muse cells were infected with lentivirus containing green fluorescent protein (GFP) according to a previous report (Kuroda et al, 2010) , and then the cells were differentiated into Muse-melanocytes, subjected to 3D culture, and transplanted to the back skin of SCID mice. We used four SCID mice for the

transplantation of skin grafts with Muse-melanocytes, two SCID mice for the skin grafts with human melanocytes, and two with only keratinocytes . The skin graft samples were fixed with 10% formaldehyde on Day 10.

[0062]

Method of preparing Muse cells labeled with GFP (Muse- GFP)

The plasmids pMD.2G, pCMV-dR8.74, and pWPXL-GFP were kindly provided by Dr. Trono (Geneva, CH, Switzerland) . High-titer GFP lentiviral supernatants were generated by transient co-transfection of the three plasmids in 293FT cells using Lipofectamine2000 (Invitrogen) and Opti-MEM I medium (GIBCO, Invitrogen) . 293FT cells (90% to 95% confluency in a 100 mm dish) were transfected with 3 μg of pWPXL-EGFP, 3 g of pCMV-dR8.74, and 3 μg of pMD.2G. Supernatants of transfected 293T cells were collected 2 and 3 days after transfection . The supernatants were filtered through 0.45 mm pore-size filters (Millipore) and were concentrated by using Amicon-Ultral5

(Millipore) . The viral solution was mixed with 10 ml of aMEM containing 10% FBS. To label NHDF with GFP, NHDF was cultured in the mixed medium for 24 to 48 hours. Muse-GFP was isolated from NHDF-GFP as SSES-3/GFP double-positive cells using FACS.

[0063]

Example 1

Isolation of Muse cells from human fibroblasts

Muse cells were collected from normal human dermal fibroblasts (NHDF, Lonza Walkersville, MD) . Because 100% of SSEA-3 positive cells from NHDF are positive for

CD105, as described previously (Kuroda et al, 2010) , we isolated Muse cells by FACS as SSEA-3 positive cells. The ratio of SSEA-3 positive cells in NHDF was in the range of 2% to 3%, consistent with previous reports (Fig. la).

[0064]

We evaluated the differentiation ability of the collected Muse cells. When each Muse cell was cultured in a single-cell suspension culture after limiting dilution, cell clusters very similar to ES cell-derived embryoid bodies, namely Muse cell-derived cell cluster (M- clusters), were generated by Day 7 (Fig. lb). These clusters were positive for alkaline phosphatase (ALP) staining (Fig. lc) ; expressed pluripotency markers such as Nanog, Oct 3/4, Sox2; and could self-renew, as

reported previously (data not shown) (Wakao et al, 2011) . To observe their differentiation ability, single M- clusters were individually transferred onto gelatin- coated dishes. After 7 days, spontaneous differentiation of the cells expanded from the M-cluster into cells positive for neurofilament (ectodermal marker) , oc-smooth muscle actin (oc-SMA, mesodermal marker) , and GATA4

(endodermal marker) were detected (Fig. ld-f) . On the other hand, non-Muse cells did not form clusters in a single cell-suspension culture, and therefore

differentiation of non-Muse cells into endodermal-, mesodermal-, and ectodermal-lineage cells could not be observed. These results are consistent with our previous report (Wakao et al, 2011) .

[0065]

Example 2

Differentiation of Muse cells into melanocytes

NHDF was separated into Muse and non-Muse cells by FACS and both were cultured separately in a specific differentiation medium containing 10 factors: Wnt3a, stem cell factor (SCF) , endothelin-3 (ET-3) , basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L- ascorbic acid, 12-0- tetradecanoyl-phorbol 13-acetate (TPA) , insulin-transferrin- selenium (ITS), and

dexamethasone (Fig. lg) . The morphology of the Muse cells began to change and cells with dendrites appeared within 3 weeks. Cell size was slightly reduced by 5 weeks, and by 6 weeks, the cells had morphology similar to that of human melanocytes (Fig. 2) . Such changes did not occur in non-Muse cells, however, and most of the_. non-Muse cell- derived cells remained fibroblast-like, even after 6 weeks of differentiation (Fig. 2).

[0066]

Example 3 ^" ~

Characterization of Muse-melanocytes

The expression of melanocyte-related markers was examined in Muse-melanocytes and non-Muse cell-derived cells after 6 weeks of differentiation. Human melanocytes were used as a positive control. In reverse

transcription-polymerase chain reaction (RT-PCR) , cells induced from Muse cells expressed MITF, KIT, TRP-1, and gplOO at 3 weeks (Fig. 3a) . In addition, Muse cells expressed DCT at 5 weeks and tyrosinase at 6 weeks. We previously reported that naive Muse cells do not express either DCT or TRP-1 (Wakao et al, 2011); thus, the expression of these melanocyte-related markers in Muse cells is thought to be induced by differentiation. While non-Muse cell-derived cells expressed MITF, KIT, and TRP- 1 after 3 weeks, DCT, gplOO, and tyrosinase were not expressed in these cells even after 6 weeks of

differentiation (Fig. 3a) .

[0067]

In immunocytochemistry, cells positive for

tyrosinase, gplOO, and MITF were detected in Muse- melanocytes (6 weeks), as in the case of human

melanocytes, while none of the cells positive for these melanocyte markers were observed in non-Muse cell-derived cells at the same time point (Fig. 3b) . Although a MITF signal was detected in non-Muse cell-derived cells in RT- PCR (Fig. 3a), the protein expression level was not high enough to be detected by immunocytochemistry.

[0068]

Muse-melanocytes were further evaluated by the L- DOPA reaction assay to examine melanin productivity. Many cells were positive for the L-DOPA reaction assay (Fig. 3c) . These results suggested that cells with

characteristics similar to those of human melanocytes were induced from Muse cells, but not from non-Muse cells.

[0069]

Example 4

Effect of factors on melanocyte differentiation

To investigate the factors essential for melanocyte induction and to estimate whether the number of factors could be reduced from 10, 7 combinations of factors were created (Fig. 4a) . Muse cells cultured in media 1, 2, 3,

4, and 7 grew well, but they did not become similar to authentic human melanocytes. Muse cells cultured in media 5 and 6 did not proliferate well and all of them died within 20 days (Fig. 4a) . RT-PCR analysis revealed no expression of tyrosinase in all 7 sets of medium at 6 weeks, and thus these media were not superior to the medium with 10 factors in melanocyte induction (Fig. 4a, b) . Muse cells cultured in media 1 and 2 expressed only MITF and/or KIT. In media 3, 4, and 7, Muse cells

expressed several melanocyte-related markers, but not tyrosinase (Fig. 4a, b) .

[0070]

Example 5

Generation of 3D cultured skin by using Muse-melanocytes

We tried to construct 3D cultured skin using Muse- melanocytes as well as other cell types. A gel layer was created comprising collagen type 1 and NHDF to mimic the dermis. For construction of the epidermis, either culture 1) keratinocytes + Muse-melanocytes, culture 2)

keratinocytes only, culture 3) keratinocytes + human melanocytes, or culture 4) keratinocytes ^"+ non-Muse cell- derived cells was seeded onto the gel layer. After 15 days, pigmented cells were observed in the basal layer of the epidermis of cultures 1) and 3) (Fig. 5a-b) . Because culture 1) comprised human keratinocytes and Muse- melanocytes, and keratinocytes do not normally produce melanin, the cells producing melanin in culture 1) were considered to be Muse-melanocytes (Fig. 5b). In addition, cells positive for MITF, tyrosinase, TRP-1, gplOO, and S100 were identified in both cultures 1) and 3), and Fontana-Masson staining revealed the presence of melanin in the epidermis of both cultures (Fig. 5c). In contrast, no pigmented cells or cells positive for melanocyte- related markers and Fontana-Masson staining were observed in the cultured skin from cultures 2) and 4) (Fig. 5a, c) .

[0071]

The spontaneous differentiation potential of Muse cells in the 3D cultured skin was also examined. We mixed green fluorescent protein ( GFP) -labeled undifferentiated Muse cells (naive Muse cells) with keratinocytes to construct the epidermis of the 3D cultured skin. After 15 days, GFP-positive naive Muse cells were identified in the epidermis but none of them expressed melanocyte markers S100, TRP-1, or tyrosinase (Fig. 7a-b) . This result indicated naive Muse cells do not spontaneously differentiate into melanocytes even if they are

integrated into the epidermal layer of 3D cultured skin.

[0072]

Since some groups reported that the 3D cultured human skin model reflects the physiological situation of human melanocytes in human skin more accurately than the experiment by using mouse skin (Haake and Scott, 1991; Meier et al, 2000), we used 3D cultured skin to evaluate the migration potential of naive Muse cells and Muse- melanocytes. GFP-positive naive Muse cells or Muse- melanocytes mixed with NHDF were embedded into the dermal equivalent of 3D cultured skins and then seeded human keratinocytes only on top of the dermal equivalent. After

15 days, GFP-labeled naive Muse cells were detected in the epidermis of 3D cultured skin (Fig. 8a), although they did not express S100, TRP-1, or tyrosinase, as stated above (Fig. 7) . Some of the Muse-melanocytes migrated from the dermal equivalent, integrated into the epidermal layer, and expressed S100 and TRP-1 (Fig. 8b), showing that Muse-melanocytes have the potential of migrating into the epidermis where human melanocytes normally reside.

[0073]

Example 6

Functional evaluation of Muse-melanocytes in vivo

To investigate whether Muse-melanocytes can survive and maintain their melanocyte functions for a certain period of time in vivo, we transplanted 3D cultured skins containing Muse-melanocytes onto the back skin of severe combined immune deficiency (SCID) mice. 3D cultured skin containing human melanocytes and 3D cultured skin

containing^■ only keratinocytes were transplanted as positive and negative controls, respectively. Ten days after transplantation of the skin containing human melanocytes, some melanocytes were histologically

detected in the basal layer of the graft (Fig. 6a, b) . In the negative control, the skin graft did not appear pigmented and no pigmented cells were observed

histologically (Fig. 6a, b) . Histologic evaluation revealed that skin grafts with Muse-melanocytes contained

Muse-melanocytes located in the basal layer that were brown in color (Fig. 6a, b) , and immunohistochemical analysis revealed that they were positive for MITF, tyrosinase, TRP-1, gplOO, and S100, the same as grafted human melanocytes (Fig. 6c) . Furthermore, Muse- melanocytes and the neighboring keratinocytes were positive for Fontana-Masson staining, as seen in human melanocytes (Fig. 6c). Following transplantation of 3D cultured skin containing GFP-labeled Muse-melanocytes, GFP-positive Muse-melanocytes were confirmed to be located within the grafted skin, demonstrating that these GFP-positive cells were transplanted cells and not derived from the host (Fig. 6d) . These findings indicated that Muse-melanocytes homed to the basal layer of the epidermis, produced melanin, and delivered it to the neighboring keratinocytes in vivo. The negative control, skin graft with keratinocytes only, was negative for all melanocyte-related markers and Fontana-Masson staining (Fig. 6c) .

[0074]

We performed double staining for the melanocyte marker S100 and the proliferative marker Ki-67 to

investigate the proliferation capacity of Muse- melanocytes; 9.5% of Muse-melanocytes expressed both Ki- 67 and S100 (Fig. 9) .

[0075]

DISCUSSION The findings of the present study revealed that a specific type of stem cell, Muse cells, among NHDF can be readily differentiated into functional melanocytes by using a specific combination of factors and cytokines, while other cells among NHDF, non-Muse cells, cannot.

Muse-melanocytes expressed melanocyte markers in

immunocytochemistry and RT-PCR showed a positive reaction to L-DOPA in vitro, could grow in 3D cultured skin, survived in vivo, expressed melanocyte markers, and produced melanin after transplantation to SCID mice back skin. Thus, Muse-melanocytes are considered to be

equivalent to melanocytes.

[0076]

DPSCs, a mesenchymal stem cell type, are reported to differentiate into melanocytes (Stevens et al, 2008;

Paino et al, 2010) . Stevens et al successfully induced cells that expressed the melanocyte marker, melanoma antigen, recognized by T-cells 1 (Mart-1) from CD34 (- )/CD271(+) DPSCs. Although Muse cells in NHDF are also CD3 (-) , they might be distinct from dental pulp cells because NHDF-derived Muse cells are negative for CD271, as reported previously. Paino et al reported that DPSCs spontaneously differentiate into melanocytes without any stimulation. The spontaneously differentiated cells express DCT, TRP-1, and Mart-1, and are positive for the

L-DOPA reaction assay. While spontaneous differentiation is indeed attractive, more than 150 days were required. Compared with melanocytes derived from DPSCs, Muse cells could differentiate into melanocytes within 6 weeks and expressed tyrosinase, the essential enzyme for producing melanin. For these reasons, Muse-melanocytes are expected to be practical for clinical use.

[0077]

Several groups have already reported that

melanocytes can be induced from ES cells and iPS cells

(Nissan et al, 2011; Yang et al, 2011; Fang et al, 2006; Motohashi et al, 2006; Ohta et al, 2011; Yamane et al, 1999) . These melanocytes, like Muse-melanocytes , express several melanocyte-related markers containing tyrosinase and produce melanin in 3D cultured skin. The techniques for inducing melanocytes from Muse cells are important in relation to the clinical application. ES cells and iPS cells increase the risk for tumorigenesis ( Fong et al, 2010; Goldring et al, 2011; Ben-David et al, 2011; Okita et al, 2007), while MSCs, which contain Muse cells, have a low risk of tumorigenesis and have already been applied to patients in many clinical trials (Kuroda et al, 2011).

Therefore, since Muse cells have a property that they do not become cancerous, both differentiated Muse- melanocytes and Muse cells that have not been

differentiated into melanocytes can be used for

constructing a three dimensional skin, and they can be administered to a living body, without removing the Muse cells that have not been differentiated thereinto.

Because ES cells are obtained from fertilized eggs, treating ES cells requires much more effort and poses more ethical problems (Knoppers et al, 2009; Manzar et al, 2011). iPS cells are obtained from somatic cells such as fibroblasts, so the ethical problems are avoided, but they require artificial gene transduction to generate pluripotent stem cells (Takahashi et al, 2006; Takahashi et al, 2007). Muse cells on the other hand, normally reside in accessible mesenchymal tissue such as the dermis and in commercially available fibroblasts, so that they are an attractive cell source for clinical and industrial uses. In addition, Muse cells are easily isolated from mesenchymal cells by simple labeling with

SSEA-3 in a cell-sorting system, and this is beneficial, particularly for industrial use. As Muse cells can be obtained from accessible mesenchymal tissues, autologous or allogeneic transplantation of Muse-melanocytes can also be expected.

[0078]

Human melanocyte stem cells are known to reside in hair follicles, have self-renewal capacity, and play a role for maintaining the number of melanocytes in the epidermis. Both DCT and PAX3 were known as markers of melanocyte stem cells. We have demonstrated DCT

expression of Muse-melanocytes at the mRNA level. In order to investigate the proliferative capacity of Muse- melanocytes, we examined the Ki-67 expression of Muse- melanocytes (Fig. 9). The result showed that 9.5% of Muse-melanocytes expressed Ki-67. In addition, these Ki- 67-positive cells were also positive for S-100, a marker for melanocytes. These results indirectly suggested the self-renewal capacity of Muse-melanocytes after

transplantation .

[0079]

. The Wnt3a, ET-3, SCF, b-FGF, and cAMP inducers

(cholera toxin and TPA) used in the differentiation medium were known to promote the expression of

transcription factors PAX3, SOX10, CREB, LEF1 through intracellular signaling ( Steingrimsson et al, 2004, Dong et al, 2012, Kondo et al, 2011) . These four transcription factors regulate the promoter of MITF-M, which is one of the MITF variants specific for melanocytes and has a crucial role in melanocyte differentiation,

proliferation, survival, and melanogenesis . In view of this, Wnt3a, ET-3, SCF, b-FGF and cAMP inducers in the. differentiation medium are considered to induce

melanocyte-related factors in Muse cells through MITF-M activation. Ascorbic acid is known to stimulate the activity and synthesis of tyrosinase (Lee SA et al, 2011) . Dexamethasone was recently reported to promote the generation of melanocytic cells from mouse ES cells

(Yamane et al, 1999) . Both linoleic acid and ITS

supplement were used as supplemental factors in low serum medium. Collectively, it. is supposed that these factors cooperatively worked on the efficient induction of Muse cells into melanocytes. To investigate the essential factors for melanocyte differentiation, we created 7 combinations of factors based on articles reporting melanocyte induction from various stem cells (Yamane et al, 1999; Paino et al, 2010; Motohashi et al, 2007) . Fang et al described a combination of three factors (Wnt3a, ET-3, and SCF) is sufficient to induce melanocytes from pluripotent stem cells. In our experiment, when Muse cells were cultured in medium lacking any of those three factors, they expressed very few melanocyte-related markers and their morphology was different^■ from that of authentic melanocytes. In addition, Muse cells cultured in medium containing only those three factors expressed MITF, KIT, TRP-1, DCT, and gplOO, but not tyrosinase, suggesting that while, these three factors are preferable for differentiation into melanocytes, additional

differentiation- enhancing factors such as cholera toxin, TPA, and linoleic acid may be used in order to

differentiate Muse cells into mature melanocytes.

[0080]

The entire disclosures of all references and of the corresponding application are hereby incorporated by reference in their entirety.

[0081]

REFERENCES

1. Alikhan A, Felsten LM, Daly M, et al. (2011)

Vitiligo: a comprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, histopathology, etiology, and work-up. J Am Acad Dermatol 65:473-91.

2. Ben-David U, Benvenisty N (2011) The tumorigenicity of human embryonic and induced pluripotent stem cells.

Nat Rev Cancer 11:268-77.

3. Dezawa M, Kanno H, Hoshino M, et al. (2004) Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest 113:1701-10.

4. Dong L, Li Y, Cao J, et al . (2012) FGF2 regulates melanocytes viability through the STAT3-transactivated PAX3 transcription. Cell Death Differ. 19:616-22

5. Fang D, Leishear K, Nguyen TK, et al. (2006)

Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells 24:1668-77. 6. Felsten LM, Alikhan A, Petronic-Rosic V (2011)

Vitiligo: a comprehensive overview Part II: treatment options and approach to treatment. J Am Acad Dermatol 65: 493-51 .

7. Fioramonti P, Onesti MG, Marchese C, et al. (2012) Autologous cultured melanocytes in vitiligo treatment comparison of two techniques to prepare the recipient site: erbium-doped yttrium aluminum garnet laser versus dermabrasion. Dermatol Surg 38:809-12.

8. Fong CY, Gauthaman K, Bongso A (2010) Teratomas from pluripotent stem cells: A clinical hurdle. J Cell Biochem

111:769-81.

9. Goldring CE, Duffy PA, Benvenisty N, et al. (2011) Assessing the safety of stem cell therapeutics. Cell stem cell 8:618-28.

10. Haake AR, Scott GA (1991) Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents. J Invest Dermatol 96:71-7.

11. Hare JM, Traverse JH, Henry TD, et al. (2009) A randomized, double-blind, placebo-controlled, dose- escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J Am Coll Cardiol 54:2277-86.

12. Jeon S, Kim NH, Koo BS, et al. (2009) Lotus (Nelumbo nuficera) flower essential oil increased melanogenesis in normal human melanocytes. Exp Mol Med 41:517-25.

13. Jiang R, Han Z, Zhuo G, et al. (2011)

Transplantation of placenta-derived mesenchymal stem cells in type 2 diabetes: a pilot study. Front Med 5:94- 100.

14. Knoppers BM, Bordet S, Is^'asi R (2009) The human embryo: ethical and legal aspects. Methods Mol Biol

550:281-305. 15. Kondo T, Hearing VJ (2011) Update on the regulation of mammalian melanocyte function and skin pigmentation. Expert rev Dermatol 6:97-108.

16. Kuroda Y, Kitada , akao S, et al. (2011) Bone marrow mesenchymal cells: how do they contribute to tissue repair and are they really stem cells? Arch

Immunol Ther Exp 59:369-78.

17. Kuroda Y, Kitada M, Wakao S, et al. (2010) Unique multipotent cells in adult human mesenchymal cell

populations. Proc Natl Acad Sci U S A 107:8639-43.

18. Lee SA, Son YO, Kook SH, et al. (2011) Ascorbic acid increases the activity and synthesis of tyrosinase in B16F10 cells through activation of p38 mitogen-activated protein kinase. Arch Dermatol Res 303:669-78.

19. Mabula JB, Chalya PL, McHembe MD, et al. (2012) Skin cancers among Albinos at a University teaching hospital in Northwestern Tanzania: a retrospective review of 64 cases. BMC Dermatol, 8 Jun 2012; 10.1186/1471-5945-12-5

20. Macchiarini P, Jungebluth P, Go T, et al. (2008) Clinical transplantation of a tissue-engineered airway.

Lancet 372:2023-30.

21. Manzar N, Manzar B, Hussain N, et al. (2011) The Ethical Dilemma of Embryonic Stem Cell Research. Sci Eng Ethics. 29 Oct 2011; 10.1007/sll948-011-9326-7

22. Motohashi T, Aoki H, Chiba K, et al. (2007)

Multipotent cell fate of neural crest-like cells derived from embryonic stem cells. Stem Cells 25:402-10.

23. Motohashi T, Aoki H, Yoshimura N, et al. (2006) Induction of melanocytes from embryonic stem cells and their therapeutic potential. Pigment Cell Res 19:284-9.

24. Meier F, Nesbit M, Hsu MY, et al. (2000) Human melanoma progression in skin reconstructs : biological significance of bFGF. Am J Pathol 156:193-200.

25. Nissan X, Larribere L, Saidani M, et al. (2011) Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis. Proc Natl Acad Sci U S A 108:14861-6. 26. Ohta S, Imaizumi Y, Okada Y, et al. (2011)

Generation of human melanocytes from induced pluripotent stem cells. PloS one 6:el6182.

27. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature

448:313-7.

28. Paino F, Ricci G, De Rosa A, et al. (2010) Ecto- mesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes. Eur Cell Mater 20:295-305.

29. Phinney DG, Prockop DJ (2007) Concise review:

mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stein Cells 25:2896-902.

30. Pittenger MF, Mackay AM, Beck SC, et al. (1999)

Multilineage potential of adult human mesenchymal stem cells. Science 284:143-7.

31. Sakaida I, Terai S, Yamamoto N, et al. (2004)

Transplantation of bone marrow cells reduces CC14-induced liver fibrosis in mice. Hepatology 40:1304-11.

32. Slominski A, Tobin DJ, Shibahara S,- et al. (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155-228.

33. Sng J, Lufkin T (2012) Emerging stem cell therapies: treatment, safety, and biology. Stem Cells Int , 2 Aug

2012; 10.1155/2012/521343.

34. Steingrimsson E, Copeland NG, Jenkins NA. (2004) Melanocytes and the microphthalmia transcription factor network. Annu Rev Genet 38:365-411.

35. Stevens A, Zuliani T, Olejnik C, et al. (2008) Human dental pulp stem cells differentiate into neural crest- derived melanocytes and have label-retaining and sphere- forming abilities. Stem Cells Dev 17:1175-84.

36. Takahashi K, Tanabe K, Ohnuki M, et al. (2007)

Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861-72.

37. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-76.

38. Toma JG, cKenzie IA, Bagli D, et al (2005)

Isolation and characterization of multipotent skin- derived precursors from human skin. Stem Cells. 23:727- 37.

39. van Geel N, Ongenae K, Naeyaert JM (2001) Surgical techniques for vitiligo: a review. Dermatology 202:162-6.

40. Wakao S, Kitada M, Kuroda Y, et al. (2011)

Multilineage-differentiating str.ess-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc Natl Acad Sci U S A 108:9875-80.

41. Yamane T, Hayashi S, Mizoguchi M, et al. (1999) Derivation of melanocytes from embryonic stem cells in culture. Dev Dyn 216:450-8.

42. Yang R, Jiang M, Kumar SM, et al. (2011) Generation of melanocytes from induced pluripotent stem cells. J Invest Dermatol 131:2458-66.

Claims

Claims

1. Muse-melanocytes which express tyrosinase and produce melanin.

2. The Muse-melanocytes according to claim 1, wherein said Muse-melanocytes further express at least one marker selected from the group consisting of DCT, Ki- 67 and S100.

3. A pharmaceutical composition comprising Muse- melanocytes which express tyrosinase and produce melanin.

4. The pharmaceutical composition according to claim 3, wherein said Muse-melanocytes further express at least one marker selected from the group consisting of DCT, Ki-67 and S100.

5. The pharmaceutical composition according to claim 3 or.4, wherein naive Muse cells remain in said composition.

6. A three dimensional skin comprising Muse- melanocytes, and optionally keratinocytes .

7. The three dimensional skin according to claim 6, wherein naive Muse cells remain in said skin.

8. A method of producing a three dimensional skin, comprising the steps:

(a) providing Muse cells from a mesenchymal tissue;

(b) culturing the Muse cells in a differentiation medium;

(c) culturing the Muse cell derived melanocytes with keratinocytes in a gel layer; and

(d) obtaining a three dimensional skin.

9. The method according to claim 8, wherein said mesenchymal tissue is a dermal tissue.

10. The method according to claim 8, wherein said Muse cells are separated from fibroblasts in said

mesenchymal tissue.

11. The method according to any one of claims 8-10, wherein said differentiation medium contains two or more factors and/or cytokines selected from the group

consisting of Wnt3a, stem cell factor (SCF) , endothelin-3

(ET-3), basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-O-tetradecanoyl- phorbol 13-acetate (TPA) , insulin-transferrin-selenium

(ITS), and dexamethasone .

12. A method of treating pigment disorder in subjects in need of such a treatment, comprising applying said Muse-melanocytes according to claim 1, said

pharmaceutical composition according to claim 2, or said three dimensional skin according to claim 3, to an affected area caused by the disorder.

13. A method of obtaining Muse-melanocytes by differentiating Muse cells with two or more factors and/or cytokines selected from the group consisting of Wnt3a, stem cell factor (SCF) , endothelin-3 (ET-3) , basic fibroblast growth factor (b-FGF) , linoleic acid, cholera toxin, L-ascorbic acid, 12-0-tetradecanoyl-phorbol 13- acetate (TPA), insulin-transferrin-selenium (ITS), and dexamethasone.