WO2017064672A1 - A method and composition to differentiate mesenchymal stem cells into melanocytes - Google Patents

Abstract

The present invention provides an effective solution to the lack of an optimal method to obtain a sufficient amount of melanocytes in a short term that could be effective to treat pigmentation disorders. As shown herein, culture of MSCs in a differentiation medium followed by a boost of UV light, induces differentiation into melanocytes: cells acquire a dendritic-like shape, adipose derived MSCs (AD-MSCs) show dark granules similar to the active ones present in melanocytes, and show increased levels of the melanocyte marker HMB45 and the expression of genes associated with melanocyte differentiation also increased in AD-MSCs. Treatment with an improved differentiation medium in addition to an exposure to a mild UV light pulse, or to a change of culture medium to a melanocyte culture had a similar effect in AD- MSCs differentiation into melanocytes. In all cases, the protocol was shorter than 49 days. The high number of MSCs obtainable from the adipose tissue, allows to reduce the expansion process to 30 days as well. Overall, the present invention provides a novel method to obtain melanocytes in a shorter period of time with respect to previously described methods.

Description

A method and composition to differentiate mesenchymal stem cells into melanocytes

Filed of the invention

The present invention relates to the field of methods and compositions for directing mesenchymal stem cells cultivated in vitro to differentiate into melanocytes, to a substantially pure population of differentiated, or partially differentiated, melanocytes obtained or obtainable by a method of the invention and to medical uses thereof, in particular for their use to treat pigmentation disorders.

Background of the invention Melanocytes are pigment-producing cells that can be found in various organs throughout the body, especially in skin and hair follicles, but also in the inner ear, iris and choroid of the eye. Skin melanocytes a re located in the stratum basale of the epidermis and are responsible of the biosynthesis of melanin, which has a photoprotective role against UV irradiation. Melanin production occurs within specialized lysosome-related structures known as melanosomes which are subsequently transferred to neighboring keratinocytes, giving the skin its characteristic pigmentation.

Melanin is an end-product of L-tyrosine after complex multistep transformations. It is composed of eumelanin, pheomelanin, neuromelanin and mixed melanin pigment. Eumelanin has a dark brown color, and is very effective at absorbing the energy of UV radiation. Pheomelanin exhibits a yellow to reddish-brown color, and is less effective at preventing penetration of UV photons into the skin. Neuromelanin is produced in dopaminergic neurons in the substantia nigra and can also chelate redox active and toxic metals, protecting these cells against neurodegeneration.

Melanocytes are derived from the neural crest. During development, melanocyte precursors migrate from the neural crest to different target sites, like the dermis, hair follicles, the uveal tract of the eye, the stria vasculare, the vestibular organ, the endolymphatic sac of the ea r, and the leptomeninges of the brain. Once they reach these tissues, the fina l differentiation process and melanosome production begins. Melanosome development involves four stages (I -IV) determined by the structure and composition of the melanin produced. In stage I melanosomes are spherical vacuoles without internal structural components, and no tyrosinase (TYR) activity, the most important enzyme involved in melanin biosynthesis. However, at this stage, TYR can be detected in the Golgi vesicles. Pmel, an important melanosomal structural protein, determines the transformation of stage I to stage II of melanosomes, turning them into elongated and fibrillary organelles, with TYR activity and minimal deposition of melanin. At stage III, melanin synthesis starts and the pigment is uniformly deposited on the internal fibrils. Finally, at stage IV, melanocytes show an either ellipsoidal or elliptical shape and highly pigmented melanosomes. They are electron-opaque due to the complete melanization, and exhibit minimal TYR activity.

An abnormality in the differentiation and proliferation of melanocytes and on melanin synthesis may cause various diseases, like vitiligo which is characterized by the depigmentation of some areas of the skin. A mutation in MITF-M may cause the abnormal differentiation and proliferation of neural crest cells, resulting in Waardenburg' s syndrome type 2, which is characterized by white spot disease, iris heterochromia and deafness. Moreover, if an abnormality occurs in tyrosinase, which is an important enzyme for melanin synthesis, and its activity is lost, melanin is not generated at all, resulting in oculo-cutaneous albinism type I. At least due to the high visibility of the skin disorders, these diseases have a negative impact in the patients' quality of life.

Treatments for pigmentation disorders, especially for vitiligo, often involve the administration of steroids and calcinum inhibitors (i.e. tacrolimus), but long treatments are associated with undesirable secondary effects and they do not always achieve complete or partial pigmentation recovery. Another alternative is the exposure to ultravioltet (UV) light, however, it carries an elevated risk of DNA damage that increases with the duration of the treatment. Additionally, there is no consensus about the optimal duration of this therapy. For difficult cases of pigmentation disorders such as vitiligo, surgery is currently the most successful approach. The success of dermabrasion, which consists on epidermal surface removal, is near 50%. Another approach is to make skin grafts, whose success rate is near 70% (Quezada N. et al., Melanocytes and Keratinocytes Transfer Using Sandpaper Technique Combined with Dermabrasion for Stable Vitiligo. Dermatologic Surgery, 2011). Another alternative is the autologous transplant of melanocytes, however it shows highly variable success rates and depends on the number and quality of cells obtained from a minimal skin sample. Mesenchymal stem cells (MSCs) are a promising source of undifferentiated cells to obtain a large number of cell-types. In this sense, it has been reported that dental pulp MSCs can differentiate to melanocytes, but It requires a long stimulation process (120 days) (Paino F. et al., Ecto-mesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes. European cells and materials, 2010). Melanocytes could be obtained similarly from bone marrow mesenchymal stem cells (BMSCs), however the process takes 120-180 days of stimulation. Another limitation of this protocol is the limited amount of MSCs that can be obtained from each extraction. Dermal MSc (DSCs) also differentiate into melanocytes (Li et al., Human dermal stem cells differentiate into functional epidermal melanocytes. J Cell Sci, 2010 ), but initial samples are limited and are isolated from newborn foreskin which hinders the autologous use. Similar situations occur with melanocytes obtained from umbilical cord blood. Therefore, there is not an optimal protocol to obtain a sufficient amount of melanocytes in a short term that could be effective to treat pigmentation disorders.

Brief description of the invention

The present invention provides an effective solution to the problem exposed above, of a lack of an optimal method to obtain a sufficient amount of melanocytes in a short term that could be effective to treat pigmentation disorders. As shown herein, culture of MSCs in a differentiation medium for 28 days followed by a boost of UV light, that requires about 6 more days, induces a dendritic-like shape soon after the differentiation process begins. In addition, adipose derived MSCs (AD-MSCs) show dark granules similar to the active ones present in melanocytes. The expression of genes associated with melanosome formation and enzymatic activity, linked to multiple stages of differentiation, such as MITF-M, MC1R, Pmel, and TYR, was also increased in AD-MSCs after treatment with the differentiation protocol described above. Expression and distribution in AD-MSCs treated cells of the melanocyte marker HMB-45, was also similar to that of melanocytes. Consistently, mesenchymal markers, especially Cdl05, were inversely altered.

Treatment with an improved differentiation medium in addition to an exposure to a mild UV light pulse increased the expression of MC1R and Pmel genes, which are involved in later stages of differentiation, but not of those genes involved in earlier stages of differentiation (PAX3, MITF-T and MITF-M). Similar results were obtained when, instead of exposing cells to the mild UV light pulse, at least after a weak of treatment in the improved differentiation medium, the medium was changed to a standard melanocyte culture medium. In all cases, the characteristic melanocyte parameters were observed earlier then 49 days after starting the treatment.

Overall, the present invention provides a novel method to obtain melanocytes in a shorter period of time with respect to previously described methods. Additionally, since a high number of MSCs can be obtained from the adipose tissue, the cell expansion process in the present invention can be shortened to 30 days, compared to the 60-90 days of cell expansion required for other sources of MSCs.

Brief description of the figures

Figure 1. Figure 1 shows the ddifferentiation of human mesenchymal stem cells to melanocytes.

The complete differentiation protocol drives cells from a fibroblast-like morphology, towards a more dendritic-like shape and the formation of dark granules similar to the ones seen on mature melanocytes. (A) Control and (B) differentiated human bone marrow MSCs, at the end of the differentiation period. (C) Control and (D) differentiated human adipose derived MSCs after the entire differentiation process. Differentiated AD-MSCs show dark granules after being differentiated (black arrow heads) similar to the ones observed in the actives melanocytes. (E) Phase contrast image of mature human melanocyte isolated from normal skin.

Figure 2. Figure 2 shows that differentiated AD-MSCs express melanocytes associated markers, which increase after UVB exposure.

After the complete differentiation process, AD-MSCs express diverse differentiation markers associated to the different stages of the melanocytes differentiation process. (A) Scheme of the different stages related to the natural development of melanocytes. As differentiation towards mature melanocytes proceeds, the gene expression changes going from stem cell related markers, like SOXIO, to mature melanocytes markers, like Tyrosinase (TYR). Relative expression of mRNA measured by quantitative PCR of SGX10 (B), MITF-M (C), MC1R (D), Pmel (E) and TYR (F). *p<0.05: **p<0.01, compared to control BM-MSCs. Figure 3. Figure 3 shows that UVB treatment increases the percentage of differentiated MSCs.

Detection of differentiated cells by immunofluorescent detection of HMB45 helps to identify the differentiated cells and the distribution of melanosomes in cells in culture. BM-MSCs without (A) or with (B); AD-MSCs without (C) or with (D) UV treatment. (E) Quantification cells showing expression of HMB45 represented in % of cells. Both BM-MScs and Ad-MSCs were treated or not (control) with differentiation medium and with or without UV light at 20 mJ/cm². (F) Mature human melanocytes unstained (G) Mature human melanocytes stained with L- DOPA. Scale bar; lOOum.

Figure 4. Figure 4 shows that UVB treatment increases the percentage of differentiated MSCs.

The number of HMB45 positive cells after culture in differentiation medium is increased upon UV treatment, which might be working as differentiation factor or a selection factor. (A) BM- MSCs culture in differentiation medium without UV treatment compared to (B) BM-MSCs cultured in differentiation medium treated with UV light do not show major differences in HMB45 expression, but show a reduction in cell survival. A similar situation was observed for the AD-MSCs cultured in differentiation medium without (C) and with UV (D) treatment. However, in this case, an increase of HMB45 positive cells was observed.

Figure 5. Figure 5 shows that differentiation of MSCs to melanocytes changes the expression profile of typical MSCs markers. The differentiation procedure not only seems to induce melanocyte markers, but it also seems to diminish the expression of some MSCs related markers, like CD105.

Figure 6. Figure 6 shows that the culture of AD-MSCs in the differentiation culture medium induces the expression of melanocyte-specific genes.

The levels of A) PAX3, B) MITF-T and C) MCIR where determined by q-PCR and normalized to GAPDH at 1,2,3, and 4 weeks after starting the differentiation process. As controls, Melanocytes and SKMEL cells (melanoma cell line) were used. Figure 7. Figure 7 shows that mild UVB radiation already stimulates the differentiation of AD- MSCs to melanocytes.

The expression levels of the following genes A) PAX3, B) MITF-T, C) MITF-M, D) MCIR and E) Pmel were analyzed by qRT-PCR and normalized by GAPDH in AD-MSCs, after 4 weeks of treatment with differentiation culture medium and with (+) or without (-) exposure to 3 mild boosts of UV light, one every week.

Figure 8. Figure 8 shows that a mild boost of UV light at the 2d and 3d week of culture in differentiation medium has a similar effect on the expression levels of PAX3, MITF-T and MCIR genes to 3 mild boosts of UV light.

AD-MSCS cultured in the differentiation culture medium were boosted (+) or not (-) with a mild UV light exposure at the end of the 1^st, 2d, 3d or 4^th week of culture. All samples were collected after 4 weeks of culture in the differentiation medium, except for the first column in each graph, which represents a sample collected after a week of culture in the differentiation medium without UV boost. The levels of A) PAX3, B) MITF-T, C) MCIR were determined by qRT- PCR and normalized by GAPDH.

Figure 9. Figure 9 shows that the culture of partially differentiated AD-MSCs in a melanocyte culture medium fosters the acquisition of a phenotype similar to that of melanocytes (based on qualitative observations). After four weeks in a differentiation culture medium, AD-MSCs were cultured for 10 days in a (A) common melanocyte culture medium, or (B) in a standard AD-MSC culture medium (DMEM 10% FBS).

Figure 10. Figure 10 shows that treatment with melanocyte culture medium has an effect on AD-MSCs differentiation into melanocytes similar to the UV boost. Cells were treated with the differentiation culture medium for 1,2,3,4 or 5 weeks, and then with the melanocyte culture medium for 2 additional weeks. Then, the gene expression levels of A) PAX3, B) MITF-T, C) MITF-M. D) MCIR and E) Pmel were quantified by qRT-PCR and normalized by GAPDH.

Description of the invention Definitions

- In the context of the present invention, the term "Mesenchymal Stem Cells (MSCs)" refers to multipotent mesoderm-derived progenitor cells. They have the capacity to differentiate into cells that compose adipose, bone, cartilage, and muscle tissue. The minimal criteria set by The International Society for Cellular Therapy in assuring MSC identity by using CD70, CD90, and CD105 as positive markers and CD34 as a negative marker. They can be found in nearly all tissues, preferably bone marrow (BM), peripheral blood (PB), adipose tissue (AT), neonatal birth-associated tissues including placenta (PL), umbilical cord (UC), and cord blood (CB). - In the context of the present invention, the term "melanocyte" refers to are pigment- producing cells that can be found in various organs throughout the body, especially in skin and hair follicles, but also in the inner ear, iris and choroid of the eye. Skin melanocytes are located in the stratum basale of the epidermis and are responsible of the biosynthesis of melanin, which has a photoprotective role against UV irradiation. Melanin production occurs within specialized lysosome-related structures known as melanosomes which are subsequently transferred to neighboring keratinocytes, giving the skin its characteristic pigmentation.

- In the context of the present invention, "complete differentiation protocol" refers to the protocol where MSCs cells are cultured in a differentiation culture medium for at least 1 week, and then exposed to UV light.

- In the context of the present invention, the term "keratinocyte" refers to the predominant cell type in the epidermis, the outermost layer of the skin, constituting 90% of the cells found there. The primary function of keratinocytes is the formation of a barrier against environmental damage by pathogenic bacteria, fungi, parasites, and viruses, heat, UV radiation and water loss.

- In the context of the present invention, the term "Melanin" refers to a group of natural pigments found in most organisms (arachnids are one of the few groups in which it has not been detected). Melanin production occurs within specialized lysosome-related structures known as melanosomes, present in the melanocytes, which are subsequently transferred to neighboring keratinocytes, giving the skin its characteristic pigmentation. Melanin is an end- product of L-tyrosine after complex multistep transformations. It is composed of eumelanin, pheomelanin, neuromelanin and mixed melanin pigment. Eumelanin has a dark brown color, and is very affective at a bsorbing the energy of the UV radiation. Pheomelanin exhibits a yellow to reddish-brown color, and is less effective at preventing penetration of UV photons into the skin. Neuromelanin is produced in dopaminergic neurons in the substantia nigra and can also chelate redox active and toxic metals, protecting these cells against neurodegeneration.

- In the context of the present invention, the term "cell culture" refers to the growth of cells in a medium in vitro. In such a culture, the cells proliferate, but they do not organize into tissue per se. - In the context of the present invention, the term "culture medium" is recognized in the art, and refers generally to any substance or preparation used for the cultivation of living cells.

- In the context of the present invention, the term "Basal mammalian cell culture medium" refers to an artificial or synthetic basal media prepared by adding nutrients (both organic and inorganic), vitamins, salts, 0₂ and C0₂ gas phases, serum proteins, carbohydrates, cofactors. Different basal media have been devised to serve one or more of the following purposes: 1) immediate survival (a balanced salt solution, with specific pH and osmotic pressure); 2) prolonged survival (a balanced salt solution supplemented with various formulation of organic compounds and/or serum); 3) indefinite growth; 4) specialized functions. Culture media contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and available either as a powder or as a liquid form from commercia l suppliers. The requirements for these components vary among cell lines and basal mammalian cell culture medium as mentioned in the present invention refers to a medium that fulfills the requirements of cells originally obtained from a mammalian organism. Here are some examples of common basal mammalian cell culture media:

Eagle's Minimum Essential Medium (EMEM)

EMEM was among the first widely used media and was formulated by Harry Eagle from a simpler basal medium (BME). EMEM contains balanced salt solution, nonessential amino acids, and sodium pyruvate. It is formulated with a reduced sodium bicarbonate concentration (1500 mg/l) for use with 5% C0₂. Since EMEM is a non-complex medium, it is generally fortified with additional supplements or higher levels of serum making it suitable for a wide range of mammalian cells.

Dulbecco's Modified Eagle's Medium (DMEM)

DMEM has almost twice the concentration of amino acids and four times the amount of vitamins as EMEM, as well as ferric nitrate, sodium pyruvate, and some supplementary amino acids. The original formulation contained 1,000 mg/L of glucose and was first reported for culturing embryonic mouse cells. A further variation with 4500 mg/L of glucose has been proved to be optimal for culture of various types of cells. DMEM is a basal medium and contains no proteins or growth promoting agents. Therefore, it requires supplementation to be a "complete" medium. It is most commonly supplemented with 5-10% Fetal Bovine Serum (FBS). DMEM utilizes a sodium bicarbonate buffer system (3.7 g/L) and therefore requires artificial levels of C0₂ to maintain the required pH. Powdered media is formulated without sodium bicarbonate because it tends to gas off in the powdered state. Powdered media requires the addition of 3.7 g/L of sodium bicarbonate upon dissolving it in water. DMEM was used initially for the culture of mouse embryonic stem cells. It has been found to be widely applicable in primary mouse and chicken cells, viral plaque formation and contact inhibition studies. RPMI-1640

RPMI-1640 is a general purpose media with a broad range of applications for mammalian cells, especially hematopoietic cells. RPMI-1640 was developed at Roswell Park Memorial Institute (RPMI) in Buffalo, New York. RPMI-1640 is a modification of McCoy's 5A and was developed for the long-term culture of peripheral blood lymphocytes. RPMI-1640 uses a bicarbonate buffering system and differs from the most ma mmalian cell culture media in its typical pH 8 formulation. RPMI-1640 supports the growth of a wide variety of cells in suspension and grown as monolayers. If properly supplemented with serum or an adequate serum replacement, RPMI- 1640 has a wide range of applications for mammalian cells, including the culture of fresh human lymphocytes, fusion protocols, and growth of hybrid cells.

Ham's Nutrient Mixtures

These were originally developed to support the clonal outgrowth of Chinese hamster ovary (CHO) cells. There have been numerous modifications to the original formulation including Harris's F-12 medium, a more complex formulation than the original F-10 suitable for serum- free propagation. Mixtures were formulated for use with or without serum supplementation, depending on the type of cells being cultured. Ham's F-10: It has been shown to support the growth of human diploid cells and white blood cells for chromosomal analysis.

Ham's F-12: It has been shown to support the growth of primary rat hepatocytes and rat prostate epithelial cells. Ham's F-12 supplemented with 25 mM HEPES provides more optimum buffering.

Coon's modification of Ham's F-12: It consists of almost two times the amount of amino acids and pyruvate as compared to F-12 and also includes ascorbic acid. It was developed for culturing hybrid cells produced by viral fusion. DMEM/F12: It is a mixture of DMEM and Ham's F-12 and is an extremely rich and complex medium. It supports the growth of a broad range of cell types in both serum and serum-free formulations. HEPES buffer is included in the formulation at a final concentration of 15 mM to compensate for the loss of buffering capacity incurred by eliminating serum. MCDB MCDB

media were designed for the low-protein or serum-free growth of specific cell types using hormones, growth factors, trace elements or low levels of dialyzed fetal bovine serum protein (FBSP). Each MCDB medium was formulated (qualitatively and quantitatively) to provide a defined and optimally balanced nutritional environment that selectively promoted growth of a specific cell type. MCDB 105 and 110 are modifications of MCDB 104 medium, optimized for long-term survival and rapid clonal growth of human diploid fibroblast-like cells (WI-38, MRC-5, I MR-90) and of low-passage human foreskin fibroblasts using FBSP or hormone and growth factor supplements. MCDB 151, 153, 201 and 302 are modifications of Ham's nutrient mixture F-12, designed for the growth of human keratinocytes, clonal growth of chicken embryo fibroblasts and Chinese hamster ovary (CHO) cells using low levels of FBSP, extensive trace elements or no serum protein Iscove's Modified Dulbecco's Medium (IMDM)

IMDM is a highly enriched synthetic media well suited for rapidly proliferating, high-density cell cultures. IMDM is a modification of DMEM containing selenium, and has additional amino acids, vitamins and inorganic salts as compared to DMEM. It has potassium nitrate instead of ferric nitrate and also contains HEPES and sodium pyruvate. It was formulated for the growth of lymphocytes and hybridomas. Studies have demonstrated that IMDM can support murine B lymphocytes, hemopoietic tissue from bone marrow, B cells stimulated with lipopolysaccharide, T lymphocytes, and a variety of hybrid cells. - In the context of the present invention, the term "hormone" refers any member of a class of signaling molecules produced by glands in multicellular organisms that are transported by the circulatory system to target distant organs to regulate physiology and behaviour. Hormones affect distant cells by binding to specific receptor proteins in the target cell resulting in a change in cell function. When a hormone binds to the receptor, it results in the activation of a signal transduction pathway. This may lead to cell type-specific responses that include rapid non-genomic effects or slower genomic responses where the hormones acting through their receptors activate gene transcription resulting in increased expression of target genes. Hormones are therefore required to regulate proper cell function and are usually required for the growth and proliferation of cells in culture (especially in the absence of serum). For instance, insulin can promote the use of glucose and amino acids in the cell. Some hormones are cell-type specific, as hydrocortisone that can promote the growth of epidermal cells and prolaction that induces the proliferation of mammary epithelial cell.

- In the context of the present invention, the term "growth factor" refers to a group of proteins that stimulate the growth of specific tissues. Growth factors play an important role in promoting cellular differentiation and cell division, and they occur in a wide range of organisms. Some growth factors are similar to hormones in that they can be secreted into the blood stream, which carries them to their target tissues. However, whereas the production of hormones is limited to glandular tissue, growth factors can be produced by many different types of tissue. Growth factors are usually required for the growth and proliferation of cells in culture

- In the context of the present invention, the term "cellular differentiation" refers to the normal process by which a cell becomes increasingly specialized in form and function. The classic example is the process by which a zygote develops from a single cell into a multicellular embryo that further develops into a more complex fetus. Differentiation is a common process in adults as well: adult stem cells divide and create fully-differentiated daughter cells during tissue repair and during normal cell turnover. Both cell differentiation and the development of body structures are regulated by intricate pathways of signaling transduction that coordinate the activities of individual cells and ultimately give rise to organisms as complex as human beings.

- In the context of the present invention, "signal transduction" refers to the transmission of a molecular signal in the form of a chemical modification by recruitment of protein complexes along a signaling pathway that ultimately triggers a biochemical event in the cell. Signal transduction occurs when an extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell (this extracellular molecule is an activator of the signal transduction pathway). In turn, this receptor triggers a biochemical chain of events inside the cell - known as a signaling cascade - that eventually elicits a response. Depending on the cell, the response may alter the cell's metabolism, shape, gene expression, or ability to divide. Therefore, a signal transduction pathway in the context of cell differentiation refers to a signal transduction pathway whose activation leads to the differentiation of the target cell.

- In the context of the present invention, the term "population of partially differentiated cells" refers to a heterogeneous population of cells, where all cells derive from the same original cell type, but where each cell is in different stage of differentiation. - In the context of the present invention, the term "substantially pure population" refers to a population of cells that contains at least about 55%, at least about 60%, at least about 70%, at least about 75%, in some embodiments at least about 85% in some embodiments at least about 90%, and in some embodiments at least 95% of differentiated or partially differentiated MSCs into melanocytes. In other words, the sample is substantially pure if it contains less than about 50%, less than 40%, less than 30%, preferably less than 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of cells other than the differentiated or partially differentiated MSCs into melanocytes. Such percentage values refer to percentage by weight or of a population of cells.

- In the context of the present invention, the term "pigmentation disorder" refers to a disturbance of the skin color, either loss or reduction, which may be related to loss of melanocytes or the inability of melanocytes to produce melanin or transport melanosomes correctly.

- In the context of the present invention, the term "a-Melanocyte-stimulating hormone (a- MSH)" refers to an endogenous peptide hormone and neuropeptide of the melanocortin family, with a tridecapeptide structure and the amino acid sequence Ac-Ser-Tyr-Ser-Met-Glu- His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. It is the most important of the melanocyte-stimulating hormones (MSHs) (also known as melanotropins) known to stimulate melanogenesis, a process that in mammals is responsible for pigmentation, primarily of the hair and skin. It also plays a role in feeding behavior, energy homeostasis, sexual activity, and protection against ischemia and reperfusion injury. ct-MSH has been shown to increase intracellular level of cAMP and to induce melanogenesis in both murine and human pigment cells. After ct-MSH binds to its cell surface, MCl-R (melanocortin-1 receptor), a seven-transmembrane G-protein-coupled receptor that activates adenylate cyclase, the intracellular level of cAMP is elevated. The activation of the cAMP/PKA signaling pathway is one of the mechanisms known to induce the differentiation of BM-MSCs into melanocytes (Mei X. et al., In vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015).

- In the context of the present invention, the term "N6,2'-0-Dibufyryiadenosine 3',5'-cyclic monophosphate sodium (DBcAMP)", or Bucladesine refers to a cyclic nucleotide derivative which mimics the action of endogenous cAMP. DBcAMP is a cell permeable cAMP analog. The compound is used in a wide variety of research applications because it mimics cAMP and can induce normal physiological responses when added to cells in experimental conditions. dbcAMP, similarly to a-MSH, has been shown to induce melanogenesis in both murine and human pigment cells. The activation of the cAMP/PKA signaling pathway is one of the mechanisms known to induce the differentiation of BM-MSCs into melanocytes (Mei X. et al., In vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015).

- In the context of the present invention, the term "FGF" refers to FGF2, also known as bFGF, or FGF-β, which is a member of the fibroblast growth factor family. It binds heparin and has a broad range of cellular activities, including cell survival, division, differentiation and migration. FGF2 plays a key role in various processes, including limb and nervous system development, wound healing and tumor growth, as well as angiogenesis. It is expressed in normal ovarian tissue and in hepatocellular carcinoma, but not in normal liver cells. FGF2 interacts with FGF receptors FGFR1, -2, -3 and -4. FGF is also known to activate MAPK signaling transduction pathway which is another mechanism known to regulate the differentiation and proliferation of melanocytes (Mei X. et al., I n vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015).

- In the context of the present invention, the term "hydrocortisone" or Cortisol refers to a steroid hormone, in the glucocorticoid class of hormones. It is produced in humans by the zona fasciculata of the adrenal cortex within the adrenal gland. It is released in response to stress and low blood-glucose concentration. It functions to increase blood sugar through gluconeogenesis, to suppress the immune system, and to aid in the metabolism of fat, protein, and carbohydrates. Hydrocortisone is also known to activate the cAMP/PKA signaling pathway, shown to be one of the mechanisms that regulate the proliferation and differentiation of melanocytes (Mei X. et al., I n vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology I nternational ISSN, 2015).

- I n the context of the present invention, the term "Insulin" refers to a peptide hormone produced by beta cells of the pancreatic islets. It has important effects on the metabolism of carbohydrates, fats and protein by promoting the absorption of, especially, glucose from the blood into fat, liver and skeletal muscle cells. In these tissues the absorbed glucose is converted into either glycogen or fats (triglycerides), or, in the case of the liver, into both. In cell culture, insulin supports cell growth and regulates the cellular uptake and utilization of glucose, amino acids, and lipids. Insulin also has anti-apoptotic properties. The combination of insulin and transferrin allows the concentration of serum to be reduced in many cell culture application. - Transferrin refers to a plasma protein that transports iron through the blood to the liver, spleen and bone marrow. Transferrin is the physiologically appropriate method for providing iron to cells in culture. Given the central role of iron uptake in cell health, the inclusion of transferrin is absolutely critical in serum free cell culture media to ensure adequate cell proliferation and function ex vivo for most cell types. Transferrin is also known to activate the cAMP/PKA signal transduction pathway, one of the known mechanisms that regulate melanocyte differentiation and proliferation (Mei X. et al., I n vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015).

- I n the context of the present invention, the term "selenium" refers to a mineral with antioxidant properties. It can be toxic in large amounts, but trace amounts are necessary for cellular function in many organisms. In cell culture, selenium is an essential trace element that is normally provided by serum. Selenium is present in selenoproteins such as glutathione peroxidase and thioredoxin reductase, which contain the selenium analog of cysteine, selenocysteine. In particular, glutathione peroxidase has a role in detoxification in vivo as a scavenger of peroxides. - In the context of the present invention, the term "fetal bovine serum (FBS)" or fetal calf serum refers to the blood fraction remaining after the natural coagulation of blood, followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum comes from the blood drawn from a bovine fetus via a closed system of collection at the slaughterhouse. Fetal bovine serum is the most widely used serum-supplement for the in vitro cell culture of eukaryotic cells.

- I n the context of the present invention, the term "L-glutamine" refers to an unstable essential amino acid required in cell culture media formulations. Most commercia lly available media are formulated with free L-glutamine which is either included in the basal formula or added to liquid formulations at time of use. - I n the context fo the present invention, the term "penicillin" refers to are a group of antibiotics used to treat a large range of bacterial infections. They are derived from Penicillium fungi and are commonly used in cell culture protocol to inhibit bacterial contamination.

- Streptomycin refers to an antibiotic of the aminoglycosides family that acts against gram- negative and gram-positive bacteria. It is commonly used in cell culture protocols to inhibit bacterial contamination.

Description

A first aspect of the present invention, refers to a method for the differentiation of mesenchymal stem cells (MSCs) into melanocytes which comprises the culture of MSCs in a differentiation culture medium comprising a basal mammalian cell culture medium, at least one hormone, at least one growth factor, and at least an activator of a signal transduction pathway leading to melanocytes differentiation.

Melanocyte proliferation and differentiation have been reported to be primarily regulated by the actions of four different signal transduction pathways: the diacylglycerol/protein kinase C (DAG/PKC) pathway, nitric oxide/cyclic GMP/protein kinase G (NO/cGMP/PKG) pathway, mitogen-activated protein kinase (MAPK) cascade pathway, and cyclic AMP/protein kinase A (cAMP/PKA) pathway (Mei X. et al., In vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015). On the other hand a-Melanocyte-stimulating hormone (a-MSH) has been shown to elevate intracellular level of cAMP: after a-MSH binds to its cell surface, melanocortin-1 receptor (MCl-R), a seven- transmembrane G-protein-coupled receptor that activates adenylate cyclase, the intracellular level of cAMP is elevated (Cone R. D. et al., The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation. Recent Prog. Horm. Res. 1996). N6,2'-0- Dibufyryiadenosine 3',5'-cyclic monophosphate sodium (DBcAMP) is also known to induce an increase on the intracellular level of cAMP.

Therefore, in a particular embodiment of the first aspect of the present invention, said activator of the signal transduction pathway leading to melanocyte differentiation is a-melanocyte stimulating hormone (a-MSH), N6,2'-0-Dibufyryiadenosine 3',5'-cyclic monophosphate sodium (DBcAMP) and a combination thereof. a-MSH may be found at a concentration between 5 and 50 ng/ml, preferably between 10 and 40 nd/ml, more preferably between 15 and 30 ng/ml, and/or DBcAMP may be found at a concentration between 10 and 100 μΜ, preferably between 25 and 80 μΜ, more preferably between 40 and 60 μΜ.

Growth factors used in the cell culture may be fibroblast growth factor (FGF), epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). In a particular method, the growth factor comprised in the differentiation culture medium is fibroblast growth factor. FGF is also known to activate MAPK signaling pathway (which as mentioned above regulates the differentiation and proliferation of melanocytes) (Mei X. et al.. In vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015) The growth factor may be at a concentration between 1 and 25 ng/ml, preferably between 2 and 15 ng/ml, more preferably between 3 and 12 ng/ml.

Hydrocortisone has also been reported to activate the cAMP/PKA signaling pathway, which was also reported to regulate the proliferation and differentiation of melanocytes (Mei X. et al., In vitro-induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015). Preferably, the hormone comprised in the differentiation culture medium is hydrocortisone. Hydrocortisone may be found at a concentration between 0.1 and 5 μg/ml, preferably between 0.2 and 3 μg ml, more preferably between 0.3 and 2 Mg ml. The differentiation culture medium may further comprise insulin. Preferably insulin is at a concentration between 0.5 and 25 μg/ml, preferably between 1 and 15 μg/ml, more preferably between 3 and 12 μg/ml.

Transferrin was reported to activate the cAMP/PKA signal transduction pathway, which was also reported to regulate melanocyte differentiation and proliferation (Mei X. et al., In vitro- induced differentiation of bone marrow mesenchymal stem cells into melanocytes; Cell Biology International ISSN, 2015). In addition, the differentiation culture medium may further comprise transferrin, preferably at a concentration between 1 and 15 ug/ml, more preferably between 2 and 10 ug/ml, even more preferably between 4 and 8 ug/ml. Moreover, the differentiation culture medium may further comprise selenium, preferably at a concentration between 1 and 15 mg/ml, preferably between 2 and 10 ng/ml, more preferably between 4 and 8 ng/ml.

As shown in example 6, culture of AD-MSCs in a differentiation culture medium as used in the method according to the first aspect, notably comprising a basal mammalian cell culture medium, ct-MSH, DBcAMP, FGF, hydrocortisone, insulin, transferrin and selenium, induced an increase in the expression of several melanocyte-specific genes. For instance, the expression of PAX3 tends to increase with the number of weeks of culture in this medium. The expression of microphthalmia-associated transcription factor - T (MITF-T), a master regulator of melanocyte development, also tends to increase since the second week of culture in the improved differentiation medium, and after the third week of culture, the expression of MITF-T increased significantly. Similar results were obtained when measuring the expression levels of MC1R, the receptor of ct-MSHl (Figure 6). Thus, results indicate that there is compromise of AD-MSCs towards differentiation. Therefore, a preferred embodiment of the first aspect of the present invention refers to a differentiation culture medium which comprises a basal mammalian cell culture medium, a- MSH, DBcAMP, FGF, hydrocortisone, insulin, transferrin and selenium. Said culture medium is referred all along this document as the "improved differentiation culture medium". In a more preferred embodiment, the differentiation culture medium comprises a basal mammalian cell culture medium, preferably MCDB 153, α-MSH at a concentration between 5 and 50 ng/ml, DBcAMP at a concentration between 10 and 100 μΜ, FGF at a concentration between 1 and 25 ng/ml, hydrocortisone at a concentration between 0.1 and 5 μg ml, insulin at a concentration between 0.5 and 25 μg/ml, transferrin at a concentration between 1 and 15 μg/ml, and selenium at a concentration between 1 and 15 ng/ml.

On the other hand, as shown in example 3, the culture of AD-MSCs in the absence of transferrin and/or selenium, also induced a significant increased expression of several melanocyte-specific genes, notably MC1R and Tyr (Figure 2). However, even if not in a statistically significant level, the expression of additional melanocyte-specific genes tended to increase (SRY-related HMG- box 10 (SOX10), MITF, and premelanosome protein (Pmel)). Therefore, a differentiation culture medium that does not comprise transferrin and/or selenium may also induce a differentiation of AD-MSCs into melanocytes. Therefore, in another preferred embodiment of the first aspect of the present invention, the differentiation culture medium does not contain transferrin and/or selenium. An ordinary skilled person in the art will know that cell culture medium may require additional reagents to allow healthy proliferation of the cells in culture. Therefore, in an additional preferred embodiment of the first aspect of the present invention the differentiation culture medium described in any of the previous embodiments, further comprises fetal bovine serum (FBS) at a volume percentage between 0,1 and 50 %, and/or L-glutamine at a concentration between 0,1 and 5 mM, and/or penicillin at a concentration between 20 and 200 units/ml, and/or streptomycin at a concentration between 20 and 200 μg/ml.

As described in example 2, the culture of AD-MSCs and BM-MSCs with differentiation culture medium and the exposure to a UV light pulse three times with a radiation energy of 20mJ/cm² for 45 seconds once every 48h induced a morphological change from a typical fibroblast-like shape of MSCs to a more dendritic-like shape, typical of a melanocyte. Additionally, treated AD- MSCs showed dark granules, similar to the ones observed in active melanocytes (Figure 1). Therefore, the culture of MSCs in a differentiation culture medium followed by an exposure to UV light pulses seems to induce the differentiation of MSCs, especially AD-MSCs, into melanocytes.

As described in example 3, when comparing samples exposed or not to the UV pulse, it is clear that the addition of the UV pulse to the differentiation protocol increased the expression levels of the melanocyte-specific genes analyzed (SOX10, MITF, MC1R, Pmel and Tyr) (Figure 2).

As described in example 4, Figure 3E also shows that culture of the AD-MSCs in the differentiation medium increases the expression of HMB45. However when including the exposure to the UV pulse, the expression of HMB45 increases considerably compared with the sample that was not exposed to the UV light.

As described in example 7, AD-MSCs cultured for 4 weeks in improved differentiation culture medium and exposed to a mild pulse of UV light (once per week) also showed a higher compromise towards differentiation into melanocytes than AD-MSCs not exposed to this UV light pulse. Indeed, the expression levels of genes encoding transcription factors involved in the first stages of differentiation (PAX3, MITF-T and MITF-M) (figure 7 A-C) were reduced, whereas those of the target genes of these transcription factors, were increased (MC1R and Pmel) (Figure D-E).

As described in example 8, a similar protocol to the one mentioned in example 7 but exposing cells to only one UV light pulse (at the end of the first, second, third, or fourth week) showed similar results to the ones mentioned above (Figure 8).

Overall, results indicate that the exposure of cells to a UV light pulse, in combination with the culture in any of the differentiation medium described in the previous embodiments, fosters the differentiation of MSCs, especially AD-MScs, into melanocytes.

Therefore, in a particular embodiment the present invention is that MSCs treated with the method/s described in any of the previous embodiments are exposed to a UV light pulse. According to examples 2 to 4, in a preferred embodiment the exposure to a UV light pulse has a radiant energy of about 20 mJ/cm² and said exposure is preferably carried no more than 3 times, preferably for no more than 45 seconds with a frequency preferably of no more than once every 48h. According to examples 7 and 8, in further preferred embodiment the exposure to a UV light pulse has a radiant energy of about 20 mJ/cm² and said exposure is carried preferably no more than 3 times, for preferably no more than 15 seconds with a frequency preferably of no more than once a week.

According to examples 1 to 5, a particular embodiment of the present invention refers to a method for the differentiation of mesenchymal stem cells (MSCs) into melanocytes which comprises the culture of MSCs in a differentiation culture medium comprising a basal mammalian cell culture medium, preferably DMEM, α-MSH at a concentration between 5 and 50 ng/ml, DBcAMP at a concentration between 10 and 100 μΜ, FGF at a concentration between 1 and 25 ng/ml, hydrocortisone at a concentration between 0.1 and 5 μg/ml, insulin at a concentration between 0.5 and 25 μ /ιηΙ, FBS at a volume percentage of between 0.1 and 50%, L-glutamine at a concentration between 0.1 and 5 mM, penicillin at a concentration between 20 and 200 unit/ml, streptomycin at a concentration between 20 and 200 μg/ml for at least 1 week, followed by an exposure to a UV pulse with a radiant energy of about 20 mJ/cm² and said exposure is preferably carried no more than 3 times, preferably for no more than 45 seconds with a frequency preferably of no more than once every 48h. According to examples 6 to 9, another particular embodiment refers to a method for the differentiation of mesenchymal stem cells (MSCs) into melanocytes which comprises the culture of MSCs in a differentiation culture medium comprising a basal mammalian cell culture medium, preferably MCDB 153, a-MSH at a concentration between 5 and 50 ng/ml, DBcAMP at a concentration between 10 and 100 μΜ, FGF at a concentration between 1 and 25 ng/ml, hydrocortisone at a concentration between 0.1 and 5 g/ml, insulin at a concentration between 0.5 and 25 μg/ml, transferrin at a concentration between 1 and 15 μg/ml, selenium at a concentration between 1 and 15 ng/ml, FBS at a volume percentage of between 0.1 and 50%, L- glutamine at a concentration between 0.1 and 5 mM, penicillin at a concentration between 20 and 200 unit/ml, streptomycin at a concentration between 20 and 200 μg/ml for at least 1 week, followed by an exposure to a UV pulse with a radiant energy of about 20 mJ/cm² and said exposure is carried no more than 3 times, for no more than 15 seconds with a frequency preferably lower than once a week.

According to examples 3, 4 and 6 (figures 2, 3 and 6), treatment of AD-MSCs in a differentiation culture medium described in any of the previous embodiments without exposure to a UV light pulse, also shows a tendency to induce differentiation of AD-MScs into melanocytes. In particular embodiment, cells cultured in a differentiation medium described in any of the embodiments above, are not exposed to a UV light pulse.

As described in example 9, cells cultured in a melanocyte culture medium (MCM) for 10 days after culture in the improved differentiation medium for 4 weeks induced a morpohological change of AD-MSCs resulting in cells with long cellular extensions, similar to completely differentiated melanocytes (fig 9). The gene expression profile of melanocyte-specific genes upon culture of AD-MSCs for more than a week in the improved differentiation medium followed by 2 weeks of culture in the MCM resembled that of cells treated with the mild UV light pulse (initially increased expression of PAX3, MITF-T and MITF-M, followed by a decrease in their expression level, and increased expression of MC1 and Pmel) (Figure 10). These results indicate that the culture in MCM fosters the maturation of AD-MSCs into melanocytes, similarly to the UV light pulses.

Therefore, in another particular embodiment, after culture of cells following any of the methods described in the previous embodiments, cells are further cultured in a melanocyte culture medium, preferably for at least one week.

In another particular embodiment, the method of differentiation described so far uses MSCs, preferably MScs derived from adipose tissue (AD-MSCs).

A second aspect of the present invention refers to a substantially pure population of differentiated, or partially differentiated, melanocytes obtained or obtainable by a method according to any of the preceding aspects.

In a third aspect of the present invention, relates to the substantially pure population of differentiated, or partially differentiated, melanocytes for use as medicament. In a particular embodiment, the present invention refers to a pharmaceutical composition comprising a substantially pure population of differentiated, or partially differentiated, melanocytes, as just described.

An abnormality in the differentiation and proliferation of melanocytes and on melanin synthesis may cause various diseases, especially pigmentation disorders.

Therefore, in a fourth aspect, the present invention relates to a substantially pure population of differentiated, or partially differentiated, melanocytes, for use in the treatment of pigmentation disorders. Some common pigmentation disorders a re vitiligo which is characterized by the depigmentation of some areas of the skin. A mutation in MITF-M may cause the abnormal differentiation and proliferation of neural crest cells, resulting in Waardenburg' s syndrome type 2, which is characterized by white spot disease, iris heterochromia and deafness. Moreover, if an abnormality occurs in tyrosinase, which is an important enzyme for melanin synthesis, and its activity is lost, melanin is not generated at all, resulting in oculo-cutaneous albinism type I. At least due to the high visibility of the skin disorders, these diseases have a negative impact in the patients' quality of life.

Therefore, in a preferred embodiment, the substantially pure population of differentiated, or partially differentiated, melanocytes, for use in the treatment of pigmentation disorders is selected from the list consisting of vitiligo, melasma, lentigines, albinism, white spot disease and progressive pigmentary purpura.

Examples Materials and methods Cell Culture

Mesenchymal stem cells were isolated from human subcutaneous adipose tissue, um bilical cord, and dental pulp, and maintained in DMEM (Gibco^®), supplemented with 10% of FBS (Gibco^®), 2mM of L-glutamine (Gibco©), a nd 100 units/mL of Penicillin and 100 ug/mL of Streptomycin (Gibco^®).

Once isolated and characterized, MSCs from the different sources were seeded at 3*10³ cells/cm², and differentiation was induced with DMEM (Gibco^®), supplemented with 2% of FBS (Gibco^®), 2 mM of L-glutamine (Gibco^®), a nd 100 units/mL of Penicillin and 100 pg/mL of Streptomycin (Gibco^®), 0,5ug/ml of Hydrocortisone (Sigma), 50μΜ DBcAMP (Sigma), 4μg/ml of insulin (Gibco^®), 10 ng/ml of bFGF (Gibco^®), and 20ng/ml of aMSH (Sigma).

I n examples 6 to 10, differentiation was induced with an "improved" differentiation medium containing additional factors directed to further support the differentiation of cells into melanocytes. It comprises MCDB153, supplemented with 2%FBS (Gibco^®), 2mM of L-glutamine (Gibco^®), and 100 units/mL of Penicillin and 100 pg/mL of Streptomycin (Gibco^®), 0,5ug/ml of Hydrocortisone (Sigma), 50uM DBcAMP (Sigma), 10 ug/ml of insulin (Gibco^®), 5.5 ug/ml of transferrin, 6.7 ng/ml of selenium, 4 ng/ml of bFGF (Gibco^®) and 20ng/ml of aMSH (Sigma). Media was changed once every 3 days, and cells were maintained for 28 days. After the 28 days differentiation, differentiation was boosted by irradiating with 20mJ/cm² of UVB light.

Immunocytocheroical detection of HMB45 After 28 days of differentiation, MSCs were washed with phosphate-buffered saline (PBS), fixed in Methanol for 20 min at 4°C, permeabilized in PBS containing 0.25% Triton X-100 for 10 min, and then blocked with PBS containing 0.25% Triton X-100 and 1% bovine serum albumin for 1 h. After that, cells were incubated with an antibody anti-human HMB45 from mouse (Abeam) at a dilution of 1:100 at 4°C over night. After the incubation the cells were washed in PBS, and then stained with Alexa Fluor^® 488 goat anti-mouse IgG antibody (life Technologies).

Quantitative real-time PCR analysis

Total mRNA was extracted from control and differentiated cells at different time points using Trizol Reagent (Invitrogen) and cDNA was synthesized by reverse transcription. The cDNA samples were analyzed to determined expression levels of different melanocyte markers by qRT-PCR.

Results

Example 1. Adipose Derived MSCs are more prone to differentiate towards Melanocytes.

Diverse sources of MSCs have been reported as able to be differentiated towards melanocytes. I n order to determine the proper protocol of differentiation into melanocytes, we first wondered which source of MSCs could be a better candidate to obtain melanocytes in vitro. We induced differentiation of MSCs obtained from different sources: bone marrow, adipose tissue, dental pulp, umbilical cord, chorion and menstrual fluid (data not shown). After 28 days under differentiation medium, plus three UVB doses of 20mJ/cm² for 45 seconds each, one every 48 hours, we observed morphological changes and positive staining for the melanocyte marker HMB45, an antibody that recognizes the melanosomal glycoprotein gplOO (Pmel) (Fig 4). We concluded that AD-MSCs were a promising source to induce differentiation of melanocytes under our proposed settings (Figures 1 and 2).

Example 2. Morphological changes after in vitro differentiation.

Melanocytes have been described to have 4 different stages of differentiation, related principally to the formation of the organelles called melanosomes. A mature melanocyte typically shows a dendritic-like shape with dark granules corresponding to melanosomes producing melanin. Therefore, we first assessed our differentiation protocol looking to these morphological changes. The complete protocol consists of 28 days under differentiation media, plus three UVB doses of 20mJ/cm² for 45 seconds each, one every 48 hours. Mature human epidermal melanocytes were used as positive control for all the experiments, which showed the characteristics previously mentioned (Figure IE). Both control bone marrow (Figure 1A) and control adipose derived (Figure 1C) mesenchymal cells presented the typical fibroblast-like shape of MSCs in culture. Both BM-MSCs (Figure IB) and AD-MSCs (Fig ID) acquired a dendritic- like shape soon after the differentiation process begins. However, dark granules (black arrow heads) were mostly observed in differentiated AD-MSCs (Figure ID), similar to the ones observed in the actives melanocytes. Thus, the differentiation protocol described above seems to be efficient to induce differentiation of MSCs cells, and especially to differentiate AD-MSCs into melanocytes.

Example 3. Differentiated AD-MSCs express Melanocytes associated markers, which increase after UVB exposure.

Throughout the development of melanocytes, different genes are expressed that are associated to

melanosome formation and enzymatic activity, each one linked to the multiple stages of differentiation (Figure 2A). We quantified the expression of genes associated to melanocytes differentiation by qRT-PCR. After the complete differentiation process, AD-MSCs showed a tendency to increase diverse differentiation markers, however, that increase was significant only after the UV-B pulses in all cases: Sox-10 (Figure 2B), MITF-M (Figure 2C), MC1R (Figure 2D), Pmel (Figure 2E) and TYR (Figure 2F). This significant increase was not observed in BM- MSCs cultured in the differentiation medium, supporting the hypothesis that AD-MSCs are more sensitive to this differentiation protocol. The expression of the genes analyzed seems to be indifferently high for the treated AD-MSCs, which may suggest that the population of differentiated cells might be going through different stages of differentiation. The formation of multiple differentiated subpopulations might be assessed by imaging of protein expression. Overall, gene expression analysis supports previous results suggesting that the differentiation protocol described above induces MSCs, especially AD-MSCs, to differentiate into melanocytes.

Example 4. UVB treatment increases differentiated MSCs percentage by inducing differentiation and selecting differentiated cells

Human melanoma black 45 (HMB-45) is a marker for melanocyte tumors such as melanomas. However, in a non-pathological context, it can be used to recognize a melanosome structural protein called Pmel 17. The detection of that protein in differentiated cells by immunofluorescence helps to identify the differentiated cells and the distribution of melanosomes within the cultured cells. In mature normal epidermal melanocytes, HMB45 distributes through the whole cell, including the dendritic extensions. Quantification of AD- MSCs treated with the differentiation protocol (which consists on the culture of cells in differentiation medium followed by UV light boosts) indicated that the expression of HMB45 in AD-MSCs is increased compared to non-treated AD-MSCs (fig 3 C-D), but not in BM-MSCs (fig 3 A-B). These results indicate that AD-MSCs are more prone to differentiate than BM-MSCs, which is significantly increased with the UV-B treatment (Figure 3E). Importantly, besides observing an increased percentage of positive cells, the UV treatment seems to induce a major distribution of the HMB45 positive melanosomes through the whole cell, especially when comparing differentiated AD-MSCs without UV (Figure 3C) and with UV treatment (Figure 3D), where we can see how the melanosomes distribution changed from a more nuclear subcellular localization, to be present in the dendritic extensions too. No significant changes were observed between BM-MSCs treated with UV light (Figure 3B) or without UV light (3A).

Overall, results indicate that the differentiation protocol has the potential to induce the differentiation of AD-MSCs into melanocytes.

When we compared BM-MSCs cultured in the differentiation medium but not treated with UV light pulse (Figure 4 A) and BM-MSCs cultured in differentiation medium treated with UV-B light (Figure 4B), we observed a reduction in the amount of cells in culture and no increase in the population of HMB-45 positive cells. When comparing AD-MSCs cultured in differentiation medium not treated (Figure 4C) and treated (figure 4D) with UV light, a reduction in the amount of cells was observed as well, however, the number of HMB-45 positive cells increased. This suggests that the UV-B treatment might work as a differentiation factor, but that in addition, it could be used as a selection method for the differentiated cells.

Staining with L-3,4-dihydroxyphenvla !anirie (L-DOPA) has been shown to be effective to recognize

active melanocytes. This could be used to identify differentiated MSCs with tyrosinase positive activity. Mature human melanocytes unstained (Figure 3.F) and stained with L-DOPA (Figure 3G)

are shown as example of this characterization.

Example 5. Differentiation of MSCs to melanocytes changes the expression of typical MSCs markers.

As previously mentioned, the differentiation treatment induces the expression of several melanocyte related markers, specifically in AD-MSCs, but it also seems to change the expression of some MSCs related markers, such as CD105 (Figure 5). This further supports the observation that the cell population has differentiated.

Example 6. Treatment of MSCs with an improved differentiation medium already induces the expression of some melanocyte-specific genes

AD-MScs were cultured in the improved differentiation medium containing additional reagents directed to further support the differentiation of cells into melanocytes. Cells were cultured for 1, 2, 3, or 4 weeks in this medium and the mRNA expression levels of several genes related with the differentiation of cells into melanocytes were measured. Results showed that the expression of PAX3 (a gene involved in the early stages of differentiation) tends to increase (Figure 6A). The expression of MITF-T, a master regulator of melanocyte development, is significantly increased after 3 and 4 weeks of treatment in the improved differentiation medium (Figure 6B). Similarly, the expression of MC1R, the receptor of aMSH, significantly increased at the third week of treatment (Fig 6C). Thus, results indicate that there is commitment of AD-MSCs to differentiate into melanocytes. Overall results suggest that AD-MSCs are committed towards differentiation after treatment with the improved differentiation medium.

Example 7. Treatment of SCs with the improved differentiation medium and with a mild UV light pulse supports the commitment of cells to differentiate

Cells were cultured for 4 weeks in the improved differentiation medium. The energy applied to cells with UV light was also improved compared to previous examples reducing the frequency from once every 48h to once per week. The time of the UV pulse was also reduced from 45 seconds to 15 seconds. Therefore, a mild UV light pulse was applied compared to previous examples. The gene expression of several genes was then measured (figure 7).

Results show that the expression of PAX3, MITF-T and MITF- genes was reduced upon stimulation with the UV pulse (Figure 7 A-C). However, the expression of MC1R and Pmel increased (Fig 7 D-E). Taken together, results show that genes encoding transcription factors that regulate the differentiation process and that are melanoblast-specific (involved in early stages of differentiation) are less expressed, whereas the expression of the target genes of these transcription factors is increased. Thus, results suggest that the mild UV pulse favors the commitment of cells towards differentiation into melanocytes.

Example 8. A lower frequency of the mild UV light pulse has a similar effect on differentiation.

Cells were treated with the improved differentiation medium for 4 weeks and treated with the mild UV light pulse but only once: only at the end of the first week, or only at the end of the second week, or only at the end of the third week or only at the end of the fourth week. The gene expression of PAX3 and MITF-T tend to decrease, but the expression of MCR1 increased, similarly to what was observed in example 7. These results were observed when cells were treated with the UV lights pulse at the end of the third and fourth week (Figure 8). Results suggest that only one mild UV pulse would be sufficient to promote the differentiation of AD- MSCs into melanocytes. Example 9. Maintaining cells in a common commercial melanocyte culture medium (MCM) after treatment in the improved differentiation medium has an effect on differentiation into melanocytes similar to that of a mild UV light pulse.

Ad-MSCs were cultured in the melanocyte culture medium (MCM), or in a standard medium to culture AD-MSCs (DMEM + 10% FBS). The morphological changes of the cells were then evaluated. Results showed that, compared to the standard medium to culture AD-MSCs, the MCM fostered the phenotypic changes resulting in cells with long cellular extensions, similar to completely differentiated melanocytes (Figure 9).

We then evaluated how the MCM affects differentiation. In this sense, AD-MSCs were treated with the improved differentiation medium for 1, 2, 3, 4 or 5 weeks and then in the MCM for 2 weeks.

After 7 weeks , the gene expression levels were analyzed. The expression levels of PAX3, MITF-T and MITF-M increased after treating the cells for 2 weeks in differentiation medium followed by the culture in MCM, and those of MITF-T and MITF-M also after 3 weeks of treatment. Treating cells with the improved differentiation medium for more than 3 weeks promoted a gene expression decrease of these genes (Figure 10 A-C). The levels of MC1R and Pmel increased in all cases after 2 weeks of treatment with the differentiation medium followed by the 2 weeks of culture in MCM (Figure 10 D-E). Overall we can conclude that the culture in MCM fosters the maturation of AD-MSCs into melanocytes, similarly to the UV light pulses.

Claims

Claims

A method for the differentiation of mesenchymal stem cells (MSCs) into melanocytes which comprises the culture of MSCs in a differentiation culture medium comprising a basal mammalian cell culture medium, at least one hormone, at least one growth factor, and at least an activator of a signal transduction pathway leading to melanocytes differentiation.

The method according to claim 1, wherein an activator of the signal transduction pathway leading to melanocyte differentiation is selected from the list consisting of a- melanocyte stimulating hormone (a-MSH), N6,2'-0-Dibufyryiadenosine 3',5'-cyclic monophosphate sodium (DBcAMP) and a combination thereof.

The method according to claim 2, wherein a-MSH is at a concentration between 5 and 50 ng/ml and/or DBcAMP is at a concentration between 10 and 100 μΜ.

The method according to claims 2 to 3, wherein the growth factor is fibroblast growth factor (FGF).

The method according to claim 4 wherein FGF is at a concentration between 1 and 25 ng/ml.

6. The method according to claims 2 to 5, wherein the hormone is hydrocortisone.

The method according to claim 6, wherein the concentration of hydrocortisone between 0.1 and 5 μg/ml.

8. The method according to claims 1 to 7, wherein the differentiation culture medium further comprises insulin. 9. The method according to claim 8, wherein the concentration of insulin is between 0.5 and 25 μg/ml.

10. The method according to any of claims 1 to 9, wherein the differentiation culture medium comprises a basal mammalian cell culture medium, a-MSH, DBcAMP, FGF, hydrocortisone, and insulin. 11. The method according to claims 1 to 10, wherein the differentiation culture medium further comprises transferrin and/or selenium.

12. The method according to claim 10 wherein transferrin is at a concentration between 1 and 15 μg/ml, and/or selenium is at a concentration between 1 and 15 ng/ml.

13. The method according to claims 1 to 10, wherein the differentiation culture medium does not comprise transferrin and/or selenium.

14. The method according to any of claims 1 to 13, wherein the differentiation culture medium further comprises fetal bovine serum (FBS) at a volume percentage between

0.1 and 50 %, and/or L-glutamine at a concentration between 0.1 and 5 mM, and/or penicillin at a concentration between 20 and 200 units/ml, and/or streptomycin at a concentration between 20 and 200 μg/ml. 15. The method according to any of claims 1 to 14 wherein cells are exposed to a UV light pulse.

16. The method according to claim 15 wherein said exposure to a UV light pulse has a radiant energy of about 20 mJ/cm² and said exposure is preferably carried no more than 3 times, preferably for no more than 45 seconds with a frequency preferably of no more than once every 48h.

17. A method for the differentiation of mesenchymal stem cells (MSCs) into melanocytes which comprises the culture of MSCs in a differentiation culture medium comprising a basal mammalian cell culture medium, a-MSH at a concentration between 5 and 50 ng/ml, DBcAMP at a concentration between 10 and 100 μΜ, FGF at a concentration between 1 and 25 ng/ml, hydrocortisone at a concentration between 0.1 and 5 μg/ml, insulin at a concentration between 0.5 and 25 μg/ml, FBS at a volume percentage of between 0,1 and 50%, L-glutamine at a concentration between 0.1 and 5 mM, penicillin at a concentration between 20 and 200 unit/ml, streptomycin at a concentration between 20 and 200 μg/ml for at least 1 week, followed by an exposure to a UV pulse with a radiant energy of about 20 mJ/cm² and said exposure is preferably carried no more than 3 times, preferably for no more than 45 seconds with a frequency preferably of no more than once every 48h.

18. The method according to claim 15 wherein said exposure to a UV light pulse has a radiant energy of about 20 mJ/cm² and said exposure is carried preferably no more than 3 times, for preferably no more than 15 seconds with a frequency preferably of no more than once a week.

19. A method for the differentiation of mesenchymal stem cells (MSCs) into melanocytes which comprises the culture of MSCs in a differentiation culture medium comprising a basal mammalian cell culture medium, a-MSH at a concentration between 5 and 50 ng/ml, DBcAMP at a concentration between 10 and 100 μΜ, FGF at a concentration between 1 and 25 ng/ml, hydrocortisone at a concentration between 0.1 and 5 μg/ml, insulin at a concentration between 0.5 and 25 μg/ml, transferrin at a concentration between 1 and 15 μg/ml, selenium at a concentration between 1 and 15 ng/ml, FBS at a volume percentage of between 0,1 and 50%, L-glutamine at a concentration between

0,1 and 5 mM, penicillin at a concentration between 20 and 200 unit/ml, streptomycin at a concentration between 20 and 200 μg/ml for at least 1 week, followed by an exposure to a UV pulse with a radiant energy of about 20 mJ/cm² and said exposure is carried preferably no more than 3 times, for preferably no more than 15 seconds with a frequency preferably of no more than once a week.

20. The method according to any of claims 1 to 14 wherein cells are not exposed to a UV light pulse. 21. The method according to claims 1 to 20, wherein cells are further cultured in a melanocyte culture medium.

22. The method according to claim 21, wherein cells are cultured in a melanocyte culture medium after being cultured in the differentiation culture medium according to any of claims 1 to 14 for at least one week. 23. The method according to any of claims 1 to 22, wherein the MSCs are derived from adipose tissue (AD-MSCs).

24. A substantially pure population of differentiated, or partially differentiated, melanocytes obtained or obtainable by a method according to any of the preceding claims.

25. A substantially pure population of differentiated, or partially differentiated, melanocytes according to claim 24 for use as medicament. 26. A pharmaceutical composition comprising a substantially pure population of differentiated, or partially differentiated, melanocytes, according to claim 24.

27. A substantially pure population of differentiated, or partially differentiated, melanocytes, according to claim 24, or a pharmaceutical composition according to claim 26, for use in the treatment of pigmentation disorders.

28. The substantially pure population of differentiated, or partially differentiated, melanocytes, for use or the pharmaceutical composition according to claim 27, wherein said pigmentation disorders are selected from the list consisting of vitiligo, melasma, lentigines, albinism, white spot disease and progressive pigmentary purpura.