MD Yinjuan Wang

Ph.D Céline Viennet
email: celine.viennet@univ-fcomte.fr

Sophie Robin

Ph.D. c , Li He M.D Jean-Yves Berthon

M.D Philippe Humbert
email: philippe.humbert@univ-fcomte.fr

Li

PhD Adeline Jeudy

MD Ferial Fanian

MD Li He

Pr Li

Precise role of dermal fibroblasts on melanocyte pigmentation

Keywords: skin, clinical trial, sensitive, hydration, cream

Dermal fibroblasts are traditionally recognized as synthesizing, remodeling and depositing collagen and extracellular matrix, the structural framework for tissues, helping to bring thickness and firmness to the skin. However, the role of fibroblasts on skin pigmentation arouses concern recently. More is known about the interactions between epidermal melanocytes and keratinocytes. This review highlights the importance of fibroblast-derived melanogenic paracrine

For Prof. Philippe HUMBERT Thank you for believing me and giving me the possibility to do my PhD thesis in your lab.

Thank you for supporting me so much chances to learning and training in past 3-year. This 3-year is meaningful for career and make me have more confidence to choose the future life I love.

For Prof. Li HE

You are one of professors I admire most. You are my professor both in academic and in life.

Thank you for you always believing me and giving me freedom and space to finish my work individually. Your encouragement and trust support me stronger to face any difficult.

For Céline VIENNET

Thank you for staying with me in past 3-year, we experienced lots of difficult and challenge together. You spent too much spirit and time in conducting my thesis and helping me, always be patient and be polite. Thanks a lot for all your help and warm smile.

For Mr. Jean-Yves BERTHON,

Thank you for being my co-director and providing me the precious chance to wide my academic vision and present my thesis in SPIM. Thank you for the financial support from Greentech Biotechology Company.

For Prof. Li LI and Prof. Xiu-li WANG

Thank you for being the reviewers of my thesis. It is my great honor that you would like to be the juries in my defense, your coming will support me more confidence to defense and finish my thesis. To analyze the relative changes in gene expression from the real-time PCR experiments, the comparative 2 -△△ Ct method was used. The △Ct value for FL and FN was calculated using the equation: Ct (DKK1) -Ct (reference, Abl). The fold change in gene expression of DKK1 was finally obtained from the formula 2-△△ Ct , where the △△Ct value was the difference between △Ct (FL) and △Ct (FN) values. The relative gene expression is set to 1 for FN samples. DKK1 expression ↘ in FL compared to FN. The mean ± SD was determined from 9 independent experiments, each performed in duplicate. ***p<0,001. Table 1.

List of Figures

List of Tables

General Introduction

Our skin reflects our general health and well-being. It plays the role of interface between our body and the outside environment, and protective barrier against external aggressors. Therefore many people pay special attention to their skin. Among skin diseases, pigmentation disorders are now a very important area of research in cosmetology and dermopharmacy. Melanin plays key roles in determining human skin pigmentation.

Melanocytes are cells that possess the unique capacity to synthesize melanin within melanosomes. This thesis focuses on the study of skin pigmentation in different views: melanocyte, melanin synthesis, and related signaling pathway.

The first chapter introduces the process of melanogenesis and the factors that regulate melanin production in melanocytes. Transcription factors specifically expressed by melanocytes and keratinocytes-derived paracrine factors regulate the functions of melanocytes. Today, there is growing scientific evidence suggesting that fibroblasts secrete factors that are involved in the regulation of skin pigmentation. Therefore, a literature review was written to describe the recent findings on melanogenic factors secreted from fibroblasts.

The second chapter describes and validates an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. This method would be suitable for establishment of optimal primary melanocyte cultures for clinical application and research.

Lastly, the third chapter explores the role of fibroblast-derived paracrine factor DKK1 in melanogenesis with a focus on solar lentigo lesion. An hypothesis proposes that TGF-β1 mediates development of solar lentigo by reducing DKK1 expression in fibroblasts through the p38 MAP kinase pathway, which leads to an activation of the Wnt/b-catenin signaling cascade. The investigation is conducted with primary cultures of fibroblasts from solar lentigo, and dermal equivalent models exposed to UV radiation.

Abstract

This thesis focuses on the study of skin pigmentation in different views: melanocyte, melanin synthesis, and related signaling pathway.

The first part of the experimental work was to develop a new extraction method of melanocytes. Cell extraction is an inevitable and critical step in the development and production of Advanced Therapy Medicinal Products (ATMP) as for the establishment of primary cell bank. The techniques described in the literature are usually based on trypsin, alone or in combination with dispase. Such enzymes are used in order to separate dermis from epidermis and subsequently provide a suspension of epidermal cells. The implementation of these protocols is often operator-dependent, not suitable for small samples and requires animal derived products (not compatible with clinical approach). The objective of this work was to define and validate an epidermal cell extraction method being the simplest, the most effective and applicable to smaller samples and avoiding animal reagents. A new product (TrypLE, Lifescience), animal-free product, has been tested on skin biopsies. The extraction efficiency was judged on the following criteria: separation epidermis / dermis, cultured melanocytes, functionality of the extracted cells. Results showed the ease of separation between the dermis and epidermis on the one hand, and between the epidermal cells on the other hand. A minimum size of skin sample was defined to allow the extraction of functional melanocytes. In conclusion, this optimal method opens new perspectives for establishment optimal melanocyte cell lines suitable for cutaneous pathophysiology research and production of ATMP.

The second part of the experimental work was to investigate the mechanism underlying the lesion of solar lentigo by exploring the role of Dickkopf-related protein 1 (DKK1) in hyperpigmentation. Recent studies reported that high DKK1 expression by dermal fibroblasts is closely related to hypopigmented skin, as palmoplantar skin and vitiligo lesion. The skin is composed by three main layers: the epidermis, the dermis and the subcutaneous tissue (Figure 1).

I.I Epidermis

The epidermis is the outer layer of the skin which is consisted of 5 layers: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum basale. It contains 95% keratinocytes, melanocytes, Langerhans cells, Merkel cells. Keratinocytes originate and proliferates from deepest layer of the epidermis updated to the surface layer step by step. Melanocytes, which located in basal layer of epidermis, secrete melanin to protect the skin from UV radiation. Langerhans cell, are responsible to the skin immune system. Merkel cells display the feature of sensory receptor cells (Figure 2).

I.II Dermis

The dermis is connected to the epidermis through a basement membrane. It is divided in 2 layers: the superficial area known as papillary region, and deeper and thicker area known as reticular region. Its skeleton structure is mainly constituted by collagen and elastin fibers, extrafibrillar matrix. In addition, there are also hair follicles, sweat glands, sebaceous glands, lymph and blood vessels. Fibroblasts are the principal dermal cells.

I.III Subcutaneous tissue

Subcutaneous tissue also known as hypodermis, consists of loose connective tissue, fat, larger blood vessels and nerves.

II. Skin Pigmentation

II.I Melanocyte and its characteristics

Melanocytes derive from neural-crest cells located on the basal layer of the epidermis, the uvea of the eyes, inner ear, nervous system, heart, bones and so on. In epidermis, each melanocyte connects with 30-40 associated keratinocytes to form a unit as "Epidermal Melanin Unit". The proportion of melanocytes to keratinocytes in the epidermal basal layer is 1:10. 1200 melanocytes per mm 2 are approximately contained in human skin. However, the number of melanocytes in the skin of palms and soles are fivefold lower than melanocytes number at other part of the skin 2 . It also decreases by 10-20% per decades after 30 years old. With senescence, melanocytes trend to be larger and more dendritic, and to have less tyrosinase activity 3 . Melanocyte is not the only cell which could produce melanin in human body, other cells such as cells of pigmented epithelium of retina, epithelia of iris and ciliary body of the eye, some neurons, adipocytes could also synthesize melanin 4 .

II.II Melanogenesis and related enzymes and modulators

In melanocytes, melanin production takes place in cytoplasmic organelles named melanosomes. It experiences four stages to be mature: stage I, premelanosome, spherical form containing dense spot and few filaments; stage II, premelanosome, ellipsoidal form containing organized, structured fibrillar matrix; stage III, the beginning of the melanin production; stage IV, being full of melanin 5 (Figure 3). The process of melanin formation starts with the rate limiting enzyme, tyrosinase.

Tyrosine is hydroxylated to L-3,4-dihydroxyphenylalanine (DOPA) by tyrosinase (TYR), then rapidly oxidized to DOPAquinone. With cysteine and glutathione, cysteinylDOPA is formed then oxidized and polymerized to pheomelanin which presents yellow-red soluble melanin.

In the absence of cysteine and glutathione, DOPAquinone is cyclizated to DOPAchrome, then transfered to 5,6-dihydroxyindole (DHI) which is oxidized to the dark-brown-black named as DHI-melanin. On the other way, DOPAchrome transformes to DHI-2-carboxylic acid (DHICA) by DOPAchrome tautomerase (TYRP2/DCT), then TYRP1 catalyzes DHICA to form lighter brown color DHOCA-melanin. Both DHICA-melanin and DHI-melanin belonge to Eumelanin which type plays more important role in the skin color and diversity of ethnic groups. Comparing to pheomelanin, eumelanin is more effective in photoprotection with higher resistance to degradation, and ability to neutralize reactive oxygen species (ROS) 4,6 (Figure 4).

MITF (Microphthalmia-associated transcription factor) acts as a master regulator of melanin synthesis in melanocyte. It regulates melanocyte differentiation, proliferation and survival by adjusting various genes of differentiation and cell-cycle. MITF itself is also regulated by many other transcription factors, including PAX3, SOX9, SOX10, LEF-1/TCF, CREB (cAMP responsive element binding protein), and DICER. There are three factors affecting melanosome structure: Pmel17, MART-1 and GPNMB 7,8 . Enzymes and proteins modulate melanin synthesis in distinct way: TYR, TYRP-1, DCT, BLOC-1 (lysosome-related organelle complexe), OA1 (melanosomal G-protein-coupled receptor), P (membrane transporters), and SLC45A2 (implicated in the control of melanosome osmolarity) 1 . Some proteins involve melanosome trafficking or transport: microtubules, F-actin, kinesin, dynein, Rab27a, melanophilin, myosin Va, RILP 5,9 .

II.III Main signaling pathways regulating melanogenesis in melanocyte

a) MAPK/ERK (mitogen-activated protein kinases/ extracellular signal-regulated kinases) signaling is crucial to the proliferation and differentiation of melanocytes. The kinases MEK and ERK in MAPK signaling pathway involve the activation of melanocyte receptors via ligand binding to their extracellular domain (eg, receptor tyrosine kinase c-Kit).

With binding to their receptors, ligand activates complex mechanisms (Ras-Raf-MEK-ERK) that lead to up-regulate MITF. The mutation of MAPKs' inhibitor such as BRAF affecting MAPKs transduction gives a potential for melanoma 10 . The MAPK signaling pathway by the synergistic action of factors derived from melanocytes and keratinocytes induces ERK activation, which triggers melanocyte proliferation 13,14 (Figure 5).

In melanocytes, Wnt/β-catenin signaling is an important pathway in pigmentation process and melanocytes differentiation. Activation of Wnt/β-catenin signaling occurs upon binding of Wnt to frizzled receptors and LRP5/6. Signals are transduced through the inhibition of glycogen synthase kinase-3β (GSK-3β) activity, leading to stabilization and transport of βcatenin into the nucleus that regulates transcription of MITF through interactions with lymphoid enhancer-binding factor (LEF). Wnt signaling is modulated by secreted and transmembrane Wnt inhibitors and activators 13,15 . 18-20 .

II.IV Role of epidermal keratinocytes in melanogenesis

Melanosome is the only organelle which could load and transmit melanin in the basal layer from melanocyte to keratinocyte. Apart from the type of melanin, the melanin release and transfer are another significant factors influencing skin color. In light skin, melanosomes are released from melanocyte to keratinocyte in cluster form while in dark skin, they are distributed individually 21 (Figure 6).

In keratinocyte membrane, a 7-transmembrane G-protein-coupled receptor known as the protease-activated receptor-2 (PAR-2) controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation 22,23

II.V Role of dermal fibroblasts in melanogenesis

Increasing evidence has underlined the contribution of dermal components in the regulation of pigmentation. More recently, dermal fibroblasts were demonstrated to exert a regulatory role on pigmentation through the secretion of soluble factors.

Factors secreted from fibroblasts bind to membrane receptors of melanocytes, active different signaling pathways and modulate melanocyte functions such as melanin synthesis.

Stem cell factor (SCF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), neuregulin-1 (NRG-1), neurotrophin-3 (NT-3) and semaphorin7a (Sema7a) target CREB promotor via MAPK signaling pathway and active expression of the MITF 24-30 . DKK1, an antagonist of Wnt/β-catenin signaling pathway by disrupting the Wnt-induced Frizzle-Lrp5/6 complex, prevents the Wnt-induced stabilization of β-catenin and the binding to the LEF-1 transcription factor 31 . Secreted frizzle-related protein (sFRP) actives Wnt/β-catenin signaling pathway by binding to Frizzled receptor whereas Wnt inhibitory factor-1 inhibits it 32,33 . With the adjusted promotor of LEF-1, CREB or PAX3, MITF controls the activity of Tyrosinase, Tyrosinase-related protein -1 (TRP-1), dopachrome tautomerase (DCT) and thereby modulates melanin synthesis. NRG-1, Nerve growth factor (NGF) and NT-3 regulate PI3K/Akt signaling pathway, and Sema7a actives FAK and LIMKII, FAP-α and CCN which are associated with melanocyte proliferation, adhesion and migration 5 (Figure 7).

The role of dermal fibroblasts in solar lentigo (SL) lesion has been recently demonstrated. SL is a common pigmentation disorder presented as aging spot on the exposure skin. It is characterized by hyperpigmented macules, and the number of the spots is related to the age and skin phototype. The histological characterization of SL is defined by a higher melanin deposition in the basal layer, elongated epidermal ridges and large melanosomal complexes.

Differential gene-profiling analyses between SL and normal skin biopsies revealed that SL tissues are mainly composed of activated melanocytes 34 . There are some fibroblast-derived paracrine factors such as HGF (Hepatocyte growth Factor), KGF (Keratinocyte Growth Factor), SCF (Stem Cell factor) involved in SL lesion formation 69, 35 . Immuno-staining analyses of some growth factors and secreted proteins in the upper dermis of SL biopsies strongly suggest that dermal fibroblasts contribute to functionally dysregulating the epidermal cells 36 .

Moreover, the treatment with KGF and KGF associated with IL-1 induces hyperpigmented lesions in vivo 37 .

III.II Functions

In the function of effecting on Wnt signaling pathway, some differences exist. DKK1, 2 and 4 modulate Wnt signaling pathway as inhibitors while sometimes DKK2 acts as Wnt activator.

DKK3 and sgy did not show evidence that they have function in Wnt signaling pathway and they are known as divergent member of the DKKs family 39 . Related to Wnt trigger, there are several different pathways that could be effected such as Wnt/𝛽-catenin signaling pathway, Wnt/Jun kinase signaling pathway, Wnt/Ca 2+ Cascade. DKKs only show effect on Wnt/𝛽catenin signaling pathway 38,40 (Figure 9).

IV. DKK1

IV.I DKK1-LRP5/6 complex DKK1, as the founding member of the family, was identified as a secreted protein that is required for head formation during Xenopous embryogenesis. In Wnt signaling pathway, DKK1 is the only antagonist that interacts with co-receptors known as LRP5 and LRP6, contrary to Wnt inhibitory factor-1 and secreted frizzled-related protein which directly bind with Wnt 43 . LRP5 and LRP6 are specific coreceptors for DKK1. DKK1 binding to LRP6 interrupts the complex with Wnt-Fz and causes the accumulation of 𝛽 -catenin, thereby blocks the Wnt/ 𝛽-catenin signaling pathway.

It exists another type of DKK1 coreceptors known as Kremen1 and Kremen2, a single-pass transmembrane proteins.

IV.II DKK1 skin expression

It has recently be demonstrated that DKK1 secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, MITF and β-catenin. 2 The levels of DKK1 in palmoplantar dermal fibroblasts (at mRNA and protein levels) are physiologically higher than those observed in non-palmoplantar dermal fibroblasts. Thus DKK1 has a role on skin pigmentation and thickness by inducing the palmoplantar phenotype.

IV.III DKK1 effects on keratinocytes

In previous study, transfected-DKK1 keratinocytes showed a stimulation of growth and density and an inhibition of melanin uptaken (low expression of PAR-2) 44 . In addition, the thickness of a reconstructed skin model with keratinocytes treated by rhDKK1 (recombinant human DKK1) was significantly thicker 31,44 . Therefore, DKK1 regulates pigmentation not only on melanin synthesis and melanocyte activity but also on melanin uptaken by keratinocytes (Figure 10). enhances the thickness of the skin by upregulating keratin 9 in keratinocytes 31 In rhDKK1 treated-keratinocytes, upregulation of a-KLEIP and keratin 9 and downregulation of Tulp3 were observed. Keratin 9 is specifically expressed in suprabasal palmoplantar epidermis and is only observed in acrosyringia in nonpalmoplantar epidermis.

High expression of keratin 9 in rhDKK1 treated-keratinocyte confirms that DKK1 undirectly regulates the thickness of palmoplantar skin 45 . aKLEIP is a protein that is related to cell adhesion and cytokinesis. High expression of aKLEIP shows that DKK1 regulates proliferation and cell density of keratinocytes by increasing cell adhesion and cytokinesis 31 .

Tulp3 is a protein belong to Tubby-like protein family which involves cell apoptosis.

Downregulation of Tulp3 suggests that DKK1 regulates keratinocyte number by decreasing cell apoptosis 31 .

IV.IV DKK1 effects on melanocytes

Reconstructed skin model treated by rhDKK1 showed significant less pigmentation than control model 44 (Figure 11).

The decreased expression of DKK1 has an effect on melanocyte proliferation and function in the regulation of the related protein of melanosome and melanin (TYR, DCT, and MART1).

In melanocyte, some Wnt-related genes such as PKCb1, Krn1, and LRP6 have high response to DKK1 by upregulation. Some other genes encoding receptors such as LDLR, GPR51, and TNFRSF10A were responsive to DKK1 treatment 39,46 . LDLR has relationship with LRP6 known as the co-receptor of DKK1 47 . GPR51 is related to MC1R known as a specifically receptor in melanocyte 48 . TNFRSF10A actives p53-independent apoptosis, which indicates that DKK1 has closed relationship with melanocyte apoptosis. Besides, a research has also found that DKK1 upregulates Gadd45b and induces melanocyte apoptosis by p38 mitogenactivated protein kinase pathway 49 . These studies suggest that DKK1 is involved in the inhibition of melanocyte growth.

In terms of melanosomal proteins, DKK1 plays a significant role directly or indirectly in upregulation of some expression proteins such as SVF3B, caveolin, syntaxin 5A, and melanophilin. DKK1 also could directly influences tyrosinase by upregulating the critical protein myoacitve teradeca peptide (MATP) in melanocyte. Thereby, we could also suggest that DKK1 has effect on melanocyte differentiation 39 .

DKK1 also affects the HOX related genes that play a role in the growth and differentiation of melanocytes, by upregulating acid fibroblast growth factor-like protein 50 .

IV.V Role of DKK1 in vitiligo

Vitiligo is a hypopigmentation disorder induced by dysfunction or deficiency of melanocytes.

The etiology of vitiligo is still unclear and it could co-exist with other autoimmune diseases.

Most researches have concentrated on the abnormality of melanocytes and keratinocytes rather than the abnormality of fibroblasts. An imbalance of keratinocyte-derived cytokines and a dysregulation of interactions with their respective receptor have been demonstrated.

There is only one study, based on fifteen skin biopsies, on the relationship between DKK1 and vitiligo. The immunohistochemistry staining revealed that the expression of DKK1 in dermis of vitiligo is significantly higher than in non-lesional dermis. The mRNA level of DKK1 in fibroblasts from vitiligo was also higher than fibroblast from non-lesion 51 . At the meanwhile, the expression of PAR-2 in dermis of vitiligo was significant lower than in nonlesion. These findings indicate that DKK1 in vitiligo not only decreases melanocyte activity but also melanin uptaken by keratinocyte. In addition, the lower expression of b-catenin in vitiligo epidermis compared to non-lesional part confirmed that DKK1 causes hypopigmentation by Wnt/b-catenin signaling pathway as previous studies 51-55 .

IV.VI Role of DKK1 in melanoma

Melanoma is the most serious type of human skin cancer that develops from melanocytes and occurs anywhere on the body exposed to direct sunlight. The main reason is caused by excessive UV exposure that damages DNA in melanocytes. Melanoma implicates not only malignant melanocytes, but also a heterogeneous mix of genetically stable non-cancer cells, including fibroblasts, endothelial and inflammatory cells. DKK1 was proved to be a diagnostic/prognostic serum biomarker in different human cancers 56 . Recently, DKK1 was found to be linked to melanoma development. The level of DKK1 in blood serum of melanoma patient is significantly higher than those in healthy control. In vitro, DKK1 inhibits the invasion of melanoma cells 40 . In B16F10 melanoma induced by viral-mediated tumor transfection, DKK1 was found to inhibit melanoma development by decreasing tumor angiogenesis and vascular perfusion 57 .

V. TGF-b family

V. I TGF-b and related signaling pathway

V.II TGF-bs and fibroblasts

TGF-b, on one hand, is a potent inducer of ECM accumulation; on the other hand, it reduces the turnover of ECM by inhibiting the expression of MMP-1 by dermal fibroblast 59 .

Accumulation of TGF-b is often found in cutaneous fibrosis diseases such as keloids, hypertrophic scars, and scleroderma 60-62 .

Autocrine TGF-b/Smad signaling regulates gene expression of contractility and matrix in dermal fibroblast. Smad7 plays inhibitor role in the formation of collagen I and a-smooth muscle actin 63 . TGF-b1 is highly expressed both in mRNA and protein levels in hypertrophic and scar tissue derived fibroblasts compared to normal fibroblasts, indicating the potential of TGF-b1 in the formation hypertrophic scar 64,65 . in gingival fibroblasts 66 .

V.III p38-MAPK

TGF-b is a key regulator of fibroblast activation that drives the synthesis of extracellular matrix in wound healing and fibrotic diseases 61,64,66,67 . The role of Wnt signaling pathway in fibrosis as well as scarring makes a considering about the crosstalk between two signaling pathways. In fibrotic fibroblast, TGF-b stimulates canonical Wnt signaling in a p38/MAPK manner by downregulating DKK1 68 .

mediators in the regulation of melanocyte activities. Fibroblasts act on melanocytes directly and indirectly through neighboring cells by secreting a large number of cytokines (SCF), proteins (DKK1, sFRP, Sema7a, CCN, FAP-α) and growth factors (KGF, HGF, bFGF, NT-3, NRG-1, TGF-β) which bind to receptors and modulate intracellular signaling cascades (MAPK/ERK, cAMP/PKA, Wnt/ -catenin, PI3K/Akt) related to melanocyte functions. These factors influence the growth, the pigmentation of melanocytes via the expression of melaninproducing enzymes and melanosome transfer, as well as their dendricity, mobility and adhesive properties. Thus, fibroblasts are implicated in both skin physiological and pathological pigmentation. In order to investigate their contribution, various in vitro models have been developed, based on cellular senescence. UV exposure, a major factor implicated in pigmentary disorders, may affect the secretory crosstalk between dermal and epithelial cells. Therefore, identification of the interactions between fibroblasts and melanocytes could provide novel insights not only for the development of melanogenic agents in the clinical and cosmetic fields, but also for a better understanding of the melanocyte biology and melanogenesis regulation.

Highlights

• Cutaneous pigmentation is regulated by a complex dermal-epidermal network.

• Fibroblasts interact with melanocytes via the synthesis of biochemical factors

• Fibroblasts-derived factors bind to melanocyte receptors and modulate intracellular melanogenic pathways.

•

The importance of fibroblasts-derived factors is demonstrated by skin pigmentary changes.

Introduction

Dermal fibroblasts are traditionally recognized as synthesizing, remodeling and depositing collagen and non-collagen extracellular matrix (ECM), the structural framework for tissues, This review outlines the role of dermal fibroblasts in constitutive pigmentation and in the development of pigmentary disorders.

Correlation between fibroblast and melanocyte pigmentation

Studies increasingly elucidated the significant role of fibroblast in pigmentation. Photoaged fibroblasts in reconstructed skin model stimulate pigmentation, including both melanin production and melanogenic gene expression, compared to unexposed fibroblasts [1]. In transcription factor 10 (SOX10) [5]. These induced-melanocytes produce melanosomes and melanin, deliver melanin to keratinocytes in 3D-skin model and, even generate pigmentation in vivo. They may provide a new efficient way to treat melanogenic dysfunctions. Therefore, all these findings confirm an important cross-talking between fibroblasts and melanocytes in pigmentation. Specifically, these are fibroblast-derived secreted factors which are involved in the fibroblast interactions with melanocytes.

Signaling pathways of fibroblast-derived factors in melanocytes

As known, the pigment melanin including different types, pheomelanin and eumelanin, is produced by melanocytes in a complex process called melanogenesis: melanin synthesis in melanocytes, melanin transport from melanocytes to keratinocytes by melanosome, and melanin distribution in epidermis. All factors related to this process can affect melanin synthesis, including structural proteins of melanosome (Pmel17, MART-1, GPNMB), enzymes required for melanin synthesis (tyrosinase (TYR), tyrosinase-related protein-1 (TYRP-1) and dopachrome tautomerase (DCT)), and proteins necessary for melanosome transport and distribution (Rab27A, myosin Va, Slac2-a/melanophilin). MITF plays a significant role in melanogenesis and melanocytes differentiation, dendricity, proliferation and apoptosis. Specifically, MITF regulates the expression of melanogenic enzymes (TYR, TYRP-1 and DCT), melanosomal matrix (Pmel17, Rab27) and anti-apoptotic proteins (bcl-2). Signaling pathways play a key role in relaying extracellular signals from factor binding to cell membrane receptor to cell nucleus via a cascade of phosphorylation events. Four crucial intracellular signaling pathways regulate melanocyte functions, and three of them are associated with the expression and function of MITF [6,7].

3.1MAPK/ERK

MAPK/ERK (mitogen-activated protein kinases/ extracellular signal-regulated kinases)

signaling is essential to the proliferation and differentiation of melanocytes. The kinases MEK and ERK in MAPK signal transduction pathway involve the activation of melanocyte receptors via ligand binding to their extracellular domain (eg, receptor tyrosine kinase c-Kit) [8]. With binding to their receptors, ligand activates complex mechanisms (Ras-Raf-MEK-ERK) that lead to up-regulate MITF [9,10]. A mutation in the gene that encodes the RAF kinase BRAF leads to constitutive activation of downstream signaling in the MAP kinase pathway [11,12].

Wnt/β-catenin

Wnt/β-catenin signaling is another important pathway in pigmentation process and melanocytes differentiation, also for melanocyte stem cell [13][14][15]. Activation of Wnt/βcatenin signaling occurs upon binding of Wnt to frizzled receptors and lipoprotein receptorrelated protein 5 and 6 (LRP5/6). Signals are transduced through the inhibition of glycogen synthase kinase-3β (GSK-3β) activity, leading to stabilization and transport of β-catenin into the nucleus, where it regulates transcription of MITF through interactions with lymphoid enhancer-binding factor (LEF). Wnt signaling is modulated by secreted and transmembrane Wnt inhibitors and activators [16].

cAMP/PKA

cAMP/PKA (cyclic adenosine monophosphate/protein kinase A) signaling can also contribute to MITF expression. Activation of some melanocyte receptors with their ligands (eg, melanocortin receptor MCR-1) results in increased levels of intracellular cAMP and activation of PKA [17]. PKA phosphorylates cAMP responsive element binding protein (CREB) which acts as a transcription factor of MITF. It has also been reported that activation of PKC can be associated with cAMP-dependent pathway [18]. Various intrinsic and extrinsic factors affect melanogenesis through this signal transduction pathway. They exert their actions directly on melanocytes or indirectly via mediators produced by surrounding skin cells [18,19].

PI3K/Akt

PI3K/Akt (phosphatidylinositol 3′-kinase/Akt) signaling pathway plays a critical role in melanocyte proliferation and apoptosis through the cell cycle regulation with GSK-3 and protein cyclin D1 [20,21], and the control of the proapoptotic protein BAD [22,23]. It could also cooperate with Ras-Raf-MEK-ERK signaling cascade on regulating melanocyte activity.

Fibroblast-derived factors involved in melanocyte activity

Fibroblasts release melanogenic factors which act both directly and indirectly on melanocytes. Numerous mediators secreted from fibroblasts play significant roles in the process of skin pigmentation through different signaling pathways. Some are involved in down-regulation (DKK1), modulation (sFRP) and induction (KGF, NRG-1) of pigmentation, some are involved in induction of proliferation and survival (SCF, bFGF, NT-3, Sema7a, TGFβ, CNN, FAP-α), and some are universal contributor (HGF) (Table . 1).

Inhibiting factor -DKK1

Dickkopf (DKK) family comprises 4 members (DKK1-4), and encodes secreted proteins that antagonize Wnt signaling by inhibiting Wnt coreceptors Lrp5 and 6. It takes part in numerous processes as bone formation, Alzheimer's disease, eye development and also skin pigmentation. DKK1 is produced by fibroblasts in skin and its regulatory role in melanogenesis was firstly described in 2004. Yamaguchi et al. reported that melanocyte density in palmoplantar human skin was five times lower than that in nonpalmoplantar part.

Fibroblasts express highly levels of DKK1 mRNA on the palms and soles, and highly levels mRNA of DKK-3 in nonpalmoplantar area [24]. In further study, Yamaguchi et al. proved that DKK1 has an inhibitory effect on MITF expression which results mainly from the decreased activity of GSK-3β and β-catenin [25]. In addition, DKK1 up-regulates the expression of myoactive tetradeca peptide (MATP) which reduce TYR activity [26]. Therefore, DKK1 acts on melanocytes by suppressing proliferation and melanin production. These combined effects explain the lower pigmentation observed on the palms and soles.

Modulating factor -sFRP

Secreted frizzle-related protein (sFRP) family consists of 5 secreted proteins in humans (sFRP1-5) that modulate Wnt signaling by binding Wnt proteins and Frizzled receptors.

Early studies found that sFRP binding to Wnt prevented the activation of Wnt receptors, leading to the initial classification of sFRPs as Wnt signaling inhibitors [27][28]. However, subsequent finding has suggested that sFRP2 functions as a melanogenic stimulator through Wnt/β-catenin signaling, but the precise mechanism needs to be clarified [29]. Kim et al.

highlighted a certain paracrine role of fibroblast-derived sFRP2 in pigmentation. A coculture experiment with sFRP2 over/downexpressed fibroblasts demonstrated that fibroblast-derived sFRP2 increased pigmentation in normal human melanocytes. Thereby, the term of Wnt-signaling modulator is preferentially attributed to sFRP. Wnt inhibitory factor-1 (WIF-1) belonging to sFRP family is also an agonist of Wnt signaling pathway. It was shown that WIF-1 increases pigmentation in melanocytes co-cultured with WIF-1 overexpressed fibroblasts [30].

Activating factors -KGF

The keratinocyte growth factor (KGF) derived from fibroblasts participates in melanogenesis process by inducing melanosome transfer. Interleukin-1 α (IL-1α), an inflammatory mediator produced by keratinocytes after UVB exposure, stimulates fibroblasts to generate KGF. In synergy with cAMP, transferrin, ET-1 and bFGF, KGF increases differentiation, cell body expansion, dendrites extension and melanosome transfer [31]. In addition, KGF alone or in synergy with IL-1α and bFGF, induces melanin deposition and elongated rete ridges [32].

-NGR-1

Neuregulin-1 (NRG-1), a nerve growth factor related to the differentiation and migration of neurons, is expressed differently among skin phototypes, and visibly increases skin pigmentation [33]. A higher level of NRG-1 is expressed in 3D-skin equivalents included fibroblasts from type VI (dark skin). In addition, the amount and size of melanocytes, as well as thickness of dendrites are increased [34]. NRG-1 binds specifically to ERBB3 and ERBB4 receptors and activates PI3K and MAPK signaling pathways in melanocytes [35].

-

SCF

The cytokine stem cell factor (SCF) is secreted constitutively by fibroblasts. The soluble form secreted from fibroblasts binds to the c-kit receptor of melanocytes and activates the MAPK/ERK signaling pathway. SCF increases proliferation and differentiation of melanocytes with or without factors produced by keratinocytes, as cAMP, ET-1 and bFGF [36]. However the absence of SCF is correlated to a dysfunction of melanocyte proliferation. The signaling SCF/c-kit is necessary to the viability of melanocytes. The use of an antibody neutralizing c-kit (ACK2) induces apoptosis of murine melanocytes [37].

-bFGF

The basic fibroblast growth factor (bFGF, FGF2), a member of the fibroblast growth factor family, is synthesized by fibroblasts and acts in a paracrine manner on melanocytes via its transmembrane receptor FGFR2 and the intracellular signaling MAPK pathway. bFGF is mitogenic and melanogenic for melanocytes [38].

-NT-3

Neurotrophin regulating melanocyte differentiation and survival respectively [39].

-Sema7a

Semaphorin 7a (Sema7a) from semaphorin family, a large class of secreted and membrane anchored proteins that is involved in numerous biological processes, stimulates dendrite outgrowth from melanocytes. Sema7a is a paracrine and UV irradiation-inducible ligand expressed by fibroblasts [40]. Plexin C1 and β1-integrins receptors are ligands for Sema7a, and signaling by these receptors has opposing effects on Sema7a-induced dendrite formation. Sema7a induces focal FAK and MAPK activation via β1-integrin, and stimulates melanocyte spreading and dendricity in human melanocytes. It regulates negatively melanocyte dendricity via the receptor Plexin C1.

-TGF-β

The Transforming growth factor-β (TGFβ) family regulates a multitude of cellular processes, including cell survival, proliferation and apoptosis [41]. It is secreted from various cells including fibroblasts. After binding to its type I and II cell surface receptors, TGF-β activates smads signaling cascades. TGFβ signaling has been shown to exhibit a repressive effect on both melanocyte differentiation and melanogenesis via downregulation of MITF and PAX3, and to influence quiescence of melanocyte stem cells [42][43][44]. It is interesting that both TGFβ1 and TGFβ2 are upregulated by PAX3, and PAX3 itself is repressed by TGFβ1, suggesting a negative feedback mechanism. On the other hand, TGFβ reduces CREBdependent transcription of MITF by repression of PKA [45]. In terms of paracrine action, TGF-β is a potent inhibitor of HGF secretion from fibroblasts.

-CCN

The CCN family is a group of multifunctional secreted proteins designated CCN1 to CCN6.

CCN proteins regulate crucial biological processes by connecting cell surface and ECM.

Although they appear not to have specific high-affinity receptors, they signal through integrins and proteoglycans. CCN2 and CCN5 are mostly expressed in the dermis [46,47].

CCN1 is increasingly associated with age growth [48]. A role in skin pigmentation has been recently discovered. UV radiation upregulates CCN1-2 whereas CCN3-6 are downregulated [49].

-FAP-α

Fibroblast activation protein-α (FAP-α), a member of serine protease family, is selectively expressed in fibroblasts. It plays an important role on tumor spreading and is highly expressed after UVR treatment [50]. This process could be upregulated by platelet derived growth factor-BB (PDGF-BB), TGF-β1, signaling protein Wnt5a released from melanocytes and plasminogen activator from melanoma cells [51,52].

-HGF

The hepatocyte growth factor (HGF, also known as scatter factor), highly expressed by fibroblasts, binds to melanocyte receptor c-MET and triggers the MAPK and the PI3K-Akt signaling pathways, modulating melanocyte proliferation, migration, and melanogenesis [53,54]. The MAPK activates the ribosomal S6 kinase (RSK) family and improves the phosphorylation of CREB protein. The secretion of HGF is stimulated by keratinocyte cytokines, IL-1α and tumor necrosis factor-α (TNF-α) [55].

Fibroblasts in the development of pigmentary disorders

Alteration in melanin production is the fundamental change in pigmentary disorders of the skin, which can be caused by defects of melanocytes, keratinocytes and fibroblasts.

Pigmentary anomalies can be classified according whether the pigment melanin is increased (lentigo, melasma, café-au-lait macules) or decreased (vitiligo), with localized or generalized distribution (Table .2).

The earlier scientists thought that keratinocytes play the leading roles in pigmentation, and fibroblasts have quite few influence on pigmentation. For example, membrane-bound SCF derived from keratinocytes is more likely to increase the melanin production and proliferation of melanocytes rather than soluble SCF derived from fibroblasts [56].

Fibroblasts are involved in pathological pigmentation, by an altered expression of various factors. Levels of HGF, SCF and KGF in dermis of café-au-lait macules and freckles are significantly higher than them in non-hyperpigmented dermis [57]. The tanning response to UV radiation exposure is mediated by a spectrum of locally produced cytokines and growth factors [58]. Mutations in genes encoding these regulators modify their expression and/or functionality, leading to altered signaling pathways, modified skin phenotypes, and development of benign lesions or tumors [59]. To better understand the dermal influence on skin pigmentation, this review focuses on some common pigmentary anomalies that involve fibroblasts-derived factors.

Melasma

Melasma is a common acquired hyperpigmentation disorder, occurred on forehead, cheeks and mandible. It affects appearance and has highly incidence in Latino, Asian and Dark-skin women. The number of melanocytes in melasma lesion is increasing however the activity of melanin synthesis enhanced. The pathogenesis and etiology of melasma have not been clearly identified, however, the previous researches identified the hormonal factors, family history, sun exposure and cosmetics as the four main triggering and aggravating factors for melasma development [60]. Some cytokines in epidermis such as SCF, PGE2, ET-1 are highly expressed in epidermis of melasma lesion [61]. The paracrine linkage between dermal fibroblasts and melanocytes also played an important role in the mechanism of hyperpigmentation in melasma. UV-repeated radiations stimulate directly or indirectly, through keratinocyte-derived cytokines, the secretion of soluble SCF by dermal fibroblasts.

UVA radiations induce weaker effect on SCF secretion than those of UVB. Kang et al. found that SCF expression in the dermis of melasma lesion is significantly increased, its receptor c-kit on melanocytes is upregulated in dermis of melasma as well by immunohistochemical staining and RT-PCR [61]. In melasma, CCN proteins could be regulated by some factors related to pigmentary diseases such as FGF2, ET, estrogen and its receptors, progesterone and its receptors. Fibroblasts extracted from melasma express more NGF-β compared to fibroblasts from perilesional skin. Recently, researchers showed that expression of WIF-1 is significantly reduced in melasma lesion. Downregulation of WIF-1 in fibroblasts was reported to stimulate tyrosinase expression and melanosome transfer [62].

Solar Lentigo

Solar lentigo (SL) is a common pigmentation disorder presented as aging spot on the exposure skin. It is characterized by hyperpigmented macules, and the number of the spots is related to the age and skin phototype. The histological characterization of SL is defined by a higher melanin deposition in the basal layer, elongated epidermal ridges and large melanosomal complexes. Differential gene-profiling analyses between SL and normal skin biopsies revealed that SL tissues are mainly composed of activated melanocytes [63].

Overexpression of HGF has been consistently proved in lentigo, and its receptor, c-MET, highly expressed in metastatic melanoma [64]. There are some fibroblast-derived paracrine factors such as HGF, KGF, SCF involved in SL lesion formation [65]. Immuno-staining analyses of some growth factors and secreted proteins in the upper dermis of SL biopsies strongly suggest that dermal fibroblasts contribute to functionally dysregulating the epidermal cells [66]. KGF synergy with IL-1 network described before effect was proved in solar lentigo as well [67]. KGF shows also efficiently ability to promote the production and secretion of SCF in keratinocytes [65].

Vitiligo

Vitiligo is a hypopigmentation disorder induced by dysfunction or deficiency of melanocytes.

The etiology of vitiligo is still unclear. Most researches have concentrated on the abnormality of melanocytes and keratinocytes rather than the abnormality of fibroblasts. Low expression of SCF receptor c-kit in melanocyte at the edge of the lesion may indicate that downregulation of SCF/c-kit/MAPK signaling pathway contributes to dysfunction and loss of melanocytes [56]. It was reported that DKK1 secreted from fibroblast is highly expressed in vitiligo lesion and, β-catenin and PAR-2 expression in melanocyte is significantly lower in vitiligo lesion compared to non-lesion skin [68][69]. The dysregulation of Wnt signaling pathway in vitiligo lesion could prevent melanocyte differentiation. The occurrence of vitiligo could also be associated with some immune system disorders [70] such as thyroid disease, it has effectively been proved that DKK1 could increase the expression level of thyrotrophic embryonic factor and mitochondrial ribosomal proteins [71]. Furthermore, attachment of melanocytes to collagen IV is mediated through collagen-receptor DDR1

(Discoidin domain receptor 1) which is under the control of CCN3. The dysfunction of CCN3 and DDR1 interaction in vitiligo lesion causes weakly adhesion of melanocytes, and this process interacts with TGF-β and CCN2 [72].

Melanoma

Melanoma is the most serious type of human skin cancer that develops from melanocytes and occurs anywhere on the body especially in areas of the body that are exposed to direct sunlight. The main reason is caused by excessive UV exposure that damages DNA in melanocytes. Melanoma implicates not only malignant melanocytes, but also a heterogeneous mix of genetically stable non-cancer cells, including fibroblasts, endothelial and inflammatory cells. Infiltrated and surrounding stromal fibroblasts are recruited, perpetually activated through paracrine factors released from melanoma cells and transdifferentiated into cancer-associated fibroblasts (CAFs). One major source of CAFs in melanoma is resident normal skin fibroblasts. CAFs exhibit both morphological and functional differences compared to normal fibroblasts. They acquire properties of myofibroblasts, produce various growth factors, cytokines, and ECM proteins. Therefore

CAFs participate in the growth and invasion of the tumor cells by promoting angiogenesis, inflammation and metastasis [73]. However, the exact mechanisms of how normal skin fibroblasts interact with malignant melanoma cells and subsequently transform to CAFs, are poorly understood [74]. Melanoma cells and dermal fibroblasts communicate throughout the tumorigenic process, including expression of numerous chemokines (IL-6, IL-8, CXCL1-3, CXCL8, CCL5, CCL2...) and chemokine-receptors (CXCR4, CXCR2, CXCR3, CCR7 and CCR10 and CXCL1...), and deregulation of signaling pathways (MAPK -PI3K/AKT, Wnt/βcatenin) [75][76][77]. CAFs secretion of HGF results in activation of the c-MET receptor and signaling pathways [78]. Expression of SCF, bFGF and KGF is involved in melanoma genesis [79,80]. UV exposure induces upregulation of FAP-α in fibroblasts, and contributes to migration and invasion of melanoma cells [52]. HGF receptor, c-MET, highly expressed in metastatic melanoma pathway maintains the melanocyte survival by suppressing the proapoptotic Bad protein and increasing the anti-apoptotic Bcl-xl [38]. Nuclear Factor-kappa (NF-kB), a transcription factor involved in the immune response, has been shown to be upregulated in melanoma through deregulations in upstream signaling pathways such as Ras/Raf, PI3K/Akt. Therefore, targeting stromal fibroblasts and inhibiting stromal NF-kB signaling could be a possible treatment for melanoma [35,50,81,82]. NT-3 mediates cell invasion and ECM degradation in metastatic melanoma [81]. Additionally, development and progression of melanoma are associated with deregulation of Wnt signaling and loss of DKK-1 secreted by fibroblasts [83][84][85]. DKK-1 is known to be regulated by the canonical Wnt pathway. In normal melanocytes, Wnt pathway is activated by secretion of Wnt3a. However, in malignant melanocytes, Wnt5a which is an inhibitor of DKK-1, is expressed. It is also suggested that phenotypic changes of melanocytes in melanoma tumor are associated with senescence, particularly in the dermal compartment. The secretion of factors from senescent fibroblasts, such as MMP-3, IL-6, TGF-, alters epithelial differentiation, promotes endothelial cell motility and stimulates cancer cell growth and tumorigenesis [86]. In addition, senescent fibroblasts accumulate during aging. Secreted sFRP of aged dermal fibroblasts drive melanoma promoting both angiogenesis and metastasis [87]. Besides, loss of Sema7a receptor Plexin C1 may induce melanoma invasion and metastasis, and therefore Plexin C1 is known as a potential tumor suppressor for melanoma progression [88]. Downregulation of CCN3 in melanoma cells contributes to their invasive phenotype [89].

Dermatofibroma

Dermatofibroma (DF), also known as fibrous histiocytoma, is a benign common cutaneous nodule of fibroblast-like cells with unknown etiology. Histologically, DF lesion is characterized by a hyperpigmentation in the overlying epidermis with acanthosis and with an increased number of melanocytes. Proliferating fibroblast-like cells and histiocytes accumulate in the dermis and are surrounded by mature collagen and by increased capillaries. They secrete elevated gene and protein levels of SCF and HGF cytokines and stimulate melanocytes located in the adjacent epidermis, which results in the hyperpigmentation of the overlying skin [50]. The altered expression of SCF and HGF in DF lesions can be associated with the tumor cell proliferation and induction of DF. SCF or HGF derived from the fibroblastic tumor may function as a mitogen for melanocytes [90]. SCF is also known as mast cell growth factor and this explains why accumulation and degranulation of mast cells are observed with melanocyte activation in DF lesions. However, further studies are required to identify the factors that stimulate exclusively in abundance the secretion of SCF and HGF.

Conclusion

Cutaneous pigmentation is regulated by melanogenic factors, locally synthesized in the skin by both keratinocytes and fibroblasts, or produced by distant tissues and transported to the skin by the circulation. Many of those factors regulate constitutive and induced pigmentation. In this review, the contribution of dermal fibroblasts to the regulation of melanocyte activities is demonstrated. Fibroblasts interact with melanocytes directly and indirectly through keratinocytes. They release numerous biochemical factors that modulate the pigmented status of melanocytes by activating signaling cascades, gene expression and enzyme activity. Levels of fibroblast-derived melanogenic paracrine mediators depend on intrinsic and extrinsic factors such as skin type, genetic, sun exposition. Increased researches on the importance role of fibroblasts in melanocyte function under physiological or pathological conditions provide theory evidences for treatment of pigmentary disorders.

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Table .1. Effects of fibroblast-derived factors on melanocyte functions.

Table.2. Some common pigmentation disorders involving fibroblast-derived factors.

The only cells that produce melanin are melanocytes which are useful for studying many pigmentary disorders as well as mechanisms regulating melanogenesis, and for cell transplantation in treatment of vitiligo.

There are various methods to isolate melanocytes from skin, which are generally based on two-steps enzyme digestion (by dispase and trypsin) process (Table 1) 76-78 [START_REF] Tang | Primary culture of human face skin melanocytes for the study of hyperpigmentation[END_REF]). Although trypsin is utilized for a variety of cell culture applications, it is derived from animals and may contain toxic substances. Therefore most of the techniques described in the literature are a limiting factor in using cells for clinic.

This work describes a new method for isolation of melanocytes, based on one-step enzyme incubation using the TrypLE TM , an animal origin-free and recombinant enzyme. We reported the number of cells isolated from different limited sizes of skin tissue and characterized the cell population. In addition, we compared our novel procedure with an established technique [START_REF] Godwin | Isolation, culture, and transfection of melanocytes[END_REF], studying the functionality of the extracted melanocytes.

Materials and Methods

Collection of skin biopsies

Skin samples were obtained from discarded skin following abdominoplasty with informed donor consent (5 patients, 43±17 years, skin types II-IV). The hypodermis layer was removed. Subsequently, skin biopsies of 3 mm in diameter were realized and put for 10 min in phosphate-buffered saline (PBS) containing 100 IU/ml penicillin and 100 μg/ml streptomycin (PS) (all from Pan-Biotech, Dutscher, France). In total, 45 biopsies were collected per skin and 6 distinct groups were realized according to the number of biopsies (5 to 10). tissue culture flasks at a cell density of 2 to 4 x10 4 cells/cm 2 , and kept at 37°C in a humidified atmosphere with 5% CO2.

Dissociation of epidermis from dermis and separation of epidermal cells

Method II: double-enzymatic digestion (dispase and trypsin)A control group was set up by incubating biopsies in 10 mg/ml dispase II (GIBCO Ò , ThermoFisher Scientific, France) overnight (17 h) at 4°C. The epidermis was separated from the dermis using fine forceps.

Isolated epidermal layers were put in 0.05% trypsin/0.02% EDTA solution (Pan-Biotech, Dutscher, France) at 37 C for 10 min.

After incubation, melanocyte culture medium was added and epidermal cells were disaggregated mechanically by repeated pipetting up and down. The following steps were similar as TrypLE TM method.

Primary melanocyte cultures

Culture medium was firstly changed after 48 h, then every 3 days. When melanocytes reached 70% confluency, they were harvested by 3 min treatment at 37°C with 0.05% trypsin/0.02% EDTA solution. The cell suspension was neutralized with fetal bovine serum (Pan-Biotech, Dutscher, France) and centrifuged for 5 min at 1,100 rpm. The cell pellet was resuspended in melanocyte medium. Viable cell counts were performed before seeding melanocytes at 16 x10 3 cells/cm 2 into new culture flask for another round of incubation.

Images of cell cultures were acquired using an Olympus IX50 inverted microscope.

Melanocyte cultures at passage 2 were used for the following experiments.

Melanocyte proliferation assay

Melanocytes were seeded at a density of 6 x10 4 cells/well in 12-well plates. After 1, 2, 3 and 4 days of culture, cells were harvested by trypsinization and counted using trypan blue exclusion.

Measurement of melanin content

Melanocytes were plated at 400000 cells on 25 cm 2 flask and grown for 5 days to 70% confluence. They were washed with PBS, collected by scraping and pelleted. Intracellular melanin was extracted by solubilizing frozen cell pellets in 1 N NaOH containing 10% DMSO for 2 h at 90 °C. The samples were then centrifuged at 1100 rpm for 10 min and the supernatants were transferred to fresh tubes. The melanin content in these supernatants

was measured spectrophotometrically at an absorbance of 420 nm against a standard curve of known concentrations of synthetic melanin (0-50 μg/mL, Sigma-Aldrich, France).

The protein level in cell pellet was determined using the Bradford assay. The intracellular melanin content was adjusted by the amount of protein in the same sample.

Immunocytochemistry

Melanocytes were grown in 8-well chamber slides (Nunc Lab-Tek Chamber Slides, Dutscher, France) at a density of 1×10 4 cells/well. After 24 h, cells were fixed in 3% paraformaldehyde in PBS, then rinsed in PBS and permeabilised for 15 min in 0.1% Triton X-100 in PBS.

Nonspecific binding sites were blocked at room temperature by a 10 min incubation in 1% glycine in PBS followed by a 1 h incubation in 3% bovine serum albumin (BSA) solution in PBS containing 10% normal goat serum and 0.1% Triton X100. Cells were incubated at 4°C overnight with polyclonal rabbit anti-human microphthalmia-associated transcription factor (MITF) at 1:1000 dilution, then 1 h at room temperature with goat anti-rabbit antibody conjugated with rhodamine diluted 1:2000. They were double-stained with FITCconjugated phalloidin for 15 min at room temperature (all antibodies from Sigma-Aldrich, France). PBS with 1% BSA and 0.1% Triton X-100 was used for washes between incubations and antibody dilutions. Samples were mounted on glass slides with Fluoromount TM medium and observed under a Zeiss Axioskop 40 fluorescence microscope.

Statistical Analysis

All values were expressed as mean ± SE. Statistical analyses between skin biopsy groups were performed using ANOVA with SigmaStat. Differences were considered significant if the P value was < 0.05.

Results and discussion

Primary cultures of human melanocytes are appropriate models to study the pathomechanisms of pigmentation, the genetic disorders of melanocyte development or the efficacy of melanogenic compounds. Unfortunately, the clinical size and the anatomical location of melanocytic lesions often prevent a wide excision of skin and, thus make difficult the establishment of melanocyte cultures. Many methods for extracting and growing melanocytes are described (Hsu et

Enzymatic dissociation of human skin tissue

Melanocytes were isolated from very limited size of human skin, equivalent of multiple 3mm punch biopsies (not exceeding 70 mm 2 in total surface area). We used one-step enzyme incubation with TrypLE TM , an animal origin free and recombinant enzyme, for isolating epidermis from dermis and release epidermal cells. We compared our method with a standard dissociation protocol which is performed in two-step enzyme incubation. As shown in Figure . 2, the incubation in TrypLE TM resulted in a total separation of the epidermis from the dermis. The incubation with dispase was less effective with a partial separation of the 2 skin layers on the outside edge of biopsies. Epidermal sheets were peeled from the dermis with forceps. Following TrypLE TM treatment, the intact epidermal layer was easily dissociated mechanically into a cell suspension. With the standard protocol, the digestion of epidermis required a second enzyme incubation using trypsin, supported by a mechanical action.

In addition, contrary to the traditional method, the advantage of our method is the absence of animal derived enzyme, a major condition for the production of advanced therapy medicinal products. Kormos et al. ( 2016) described the use of the Mel-mix medium supplemented with human serum for obtaining pure melanocyte cultures. Therefore, the combination of different techniques offers valuable opportunity in cultured melanocytesbased transplantation therapies.

Growth pattern of extracted cells

As Normand and Karasek (1995) who focused on keratinocytes, endothelial cells and fibroblasts, our method established melanocyte cultures from small amount of tissue. There was no significant difference in number of cell extracted among groups of 6 to 10 biopsies, and whatever the method used (Figure . 3). Surface of skin tissue less than or equal to 5

biopsies (e.g., 35 mm 2 ) didn't allow survival and proliferation of extracted cells (data not shown). Extracted cells attached to the culture flask within 48 h and melanocytes initiated a dendritic appearance (Figure . 4). After 10 days of incubation, it was observed that melanocytes were 40-50% confluent and displayed typical dendritic morphology. Cells became highly proliferative. In case of TrypLE TM method, we obtained a relatively pure primary melanocyte culture. In contrast, primary cultures obtained from dispase and trypsin incubations were contaminated with keratinocytes, suggesting a significant increase of adhesion and growth of extracted keratinocytes compared to TrypLE TM . Incubation for 17 h at 4°C to separate epidermis from dermis with TrypLE TM method gave better results with less keratinocytes. However in subsequent passages these clusters of keratinocytes disappeared, inducing the decrease of cell number between extraction day and end of passage 1. Extracted cells reached 70% confluency around 14 days and they were subcultured at a 1:2 split ratio approximately every 10 to 14 days. There is no difference in the cell yield of early passage melanocytes using both methods and from skin samples with more than 42 and less than 70 mm 2 of tissue.

Functionality of melanocytesPrimary melanocytes in culture might to be optimal to examine in vivo pathomechanisms, to assess biological effect of compounds or to treat pigmentary lesions. Previous work showed that non-enzymatic tissue incubation method for cell

Conclusion

We report in this study a novel and simple extraction method of human skin melanocytes based on the use of TrypLE TM . This protocol provided pure populations of melanocyte after the extraction process and preserved cell morphology and functional characteristics. It maximized the number of melanocytes obtained from small skin surface (greater than 42 mm 2 or 6 x 3-mm punch biopsies), and therefore generated sufficient cells to start a primary culture. In addition, our procedure had the significant advantage to use an animal-free cell culture reagent and could be applicable to the development of advanced cell therapy product After TrypLE TM incubation, epidermis (arrow) was completely separated from dermis, whereas after dispase treatment, it was not perfectly detached.

I. Aims of the Study

Recent studies reported that high DKK1 expression by dermal fibroblasts is closely related to hypopigmented skin, as palmoplantar skin and vitiligo lesion. DKK1 encoded by DKK1 gene is an antagonistic inhibitor of the Wnt signaling pathway, by isolating the LRP5/6 co-receptor and preventing the Frizzeled-Wnt-LRP5/6 complex formation. Wnt/b catenin signaling regulates the transcription of melanocyte-specific genes like MITF, a gene involved in melanin synthesis.

The aims of the study were firstly, to determine the cellular level of DKK1 on hyperpigmented skin including solar lentigo; secondly to conduct a comprehensive investigation of the mechanisms underlying this lesion 72 .

In vitro models that mimic the in vivo environment were used: fibroblasts isolated from solar lentigo and peri-lesional biopsies, and normal fibroblasts embedded in 3D collagen gel and exposed to repeated doses of UVA.

II. Hypothesis

TGF-β1 mediates development of solar lentigo by reducing DKK1 expression in fibroblasts through the p38-MAP kinase pathway, which leads to an activation of the Wnt/b-catenin signaling cascade. This process may result in an uneven distribution of active melanocytes, with areas of hyperpigmentation in the skin (Figure 13).

III. Methods and Materials

III.I. Part I -Solar lentigo biopsies

III.I.I. Collection of skin biopsies

Two biopsies of 3-mm punches (one from solar lentigo lesional skin (SL), one from adjacent normal skin (SN)) were obtained from nine female volunteers on the back of hands The PCR reaction mixture comprised 2 μL of cDNA sample, 10 μL of TaqMan universal PCR master mix, 1 μL of each of TaqMan gene expression assay and RNase-free water. Reactions were performed and monitored using the Biorad CFX96 Real-Time PCR System. Thermal cycling conditions were initiated with 2 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 95°C for 10 s (denaturation) and 60°C for 1 min (annealing/extension).

For quantification, gene expression levels were calculated by the comparative Ct method (also known as the 2 -DD Ct ). Duplicate reactions per sample were run. Primary melanocytes cultures were prepared from adult human abdominal skin.

Melanocytes (passage 2-3) were seeded in 24-well plates at a density of 40,000 cells/mL and cultured for 48 h in melanocyte medium which consists of M254 culture medium supplemented with human melanocytes growth supplement (HMGS, Life Technologies, ThermoFisher Scientific, France), 100 IU/mL penicillin and 100 µg/mL streptomycin. The fibroblast-conditioned medium was obtained by culturing FL and FS for 4 days at density 150,000 cells/well in full DMEM medium. It was then concentrated by lyophilization. Each lyophilisate was added to full medium M254 and the mixture was added on melanocyte cultures (Table 3). Triton X100, the secondary antibody (goat anti-rabbit IgG-rhodamine or TRITC, 18772, Sigma-Aldrich) diluted 1:200 in PBS containing 1% BSA and 0.1% Triton X100, was applied for 1 h. After washing in PBS, samples were incubated 15 min in rhodamine-conjugated FITC (Fluorescein isothiocyanate, P5282, Sigma-Aldrich) for F-actin staining (2.5 µg/mL).

Samples were then mounted in Dako fluorescent medium, observed under fluorescence microscopy (Axioskop 40, Zeiss).

III.I.X. Melanin assay

Melanocytes were cultured at 400,000 cells on 25 cm 2 flask for 24 h, and treated with fibroblast-conditioned medium (15% v/v). After 48 h, they were washed with PBS, collected by scraping and pelleted. Intracellular melanin was extracted by solubilizing frozen cell pellets in 1 N NaOH containing 10% DMSO for 2 h at 90 °C. The samples were then centrifuged at 1100 rpm for 10 min and the supernatants were transferred to fresh tubes.

The intracellular melanin content in supernatants was measured spectrophotometrically at an absorbance of 420 nm against a standard curve of known concentrations of synthetic melanin (0-50 µg/mL, Sigma-Aldrich, France). Experiments were performed in duplicate.

Results were expressed in µg melanin/mL.

III.II. 3D biological model

III.II.I. Fibroblasts-populated tense collagen lattice preparation (FPCL)

Primary fibroblast cultures were prepared from adult human facial skin (passage 4-6).

Fibroblasts were brought to a concentration of 8 x 10 5 cells/mL in full DMEM medium. They were embedded in three-dimensional hydrated collagen gels 74,75 . Each collagen lattice was prepared by mixing, in the following order: 3 mL of 1.76X concentrated DMEM (containing 0.5% NaHCO3, 0.1N NaOH, 15% FBS), 1.5 mL of rat tail type I collagen solution (2 mg/mL, Institut de Biotechnologies Jacques Boy, France) and 0.5 mL of fibroblast suspension. The mixture was quickly poured into a 16 X 60-mm petri-dish containing a sterilized nylon mesh ring (Sefar Nitex 03-150/50, Heiden Switzerland) (Figure 14). After polymerization at 37°C for 1 h, 3 mL of full DMEM medium were added. After 5 days of culture, collagen gels were washed with PBS (until no phenol-red color) and covered with thin layer of PBS prior to UVA irradiation (6 J/cm 2 ). The UVA source was a bank of 8 fluorescent black light lamps (F15T8, Sylvania, Danvers, MA, USA). The peak emission was at 370 nm, and was checked using a UV radiometer IL-1700 with UVA filter (Dexter Industrial Green, Newburyport, MA, USA). UVA radiation process was performed three times at an interval of 48 h (Figures 15 and16). After the last irradiation, collagen gels were cultivated for 24 h in full DMEM medium. Control FPCL were kept in the same culture conditions without UVA exposure. All experimental conditions were made in triplicate.

III.III. ELISA

ELISA analyzes for Dkk1 and TGF-b1 in the supernatants of tense FPCL were performed following the same protocols as described before.

III.IV. Statistical analysis

All quantitative data are expressed as means ± SD. Paired Student's test (SPSS 24.0) was used to compare groups. p < 0.05 was considered significant. (* p<0.05, ** p<0.01, *** p<0.001) independent experiments, each performed in duplicate. DKK1 protein levels ↘ in the medium of FL compared to FN but no significant difference was detected between both groups.

IV. Results

IV.I. Comparative analyses of staining in SL versus

IV.III. Secretion of TGF-β1 from FL and FL

ELISA was used to measure secretion of TGF-β1 into the medium of fibroblast cultures. As shown in Figure 23, secretion of TGF-β1 in FL cultures was significantly higher than FN cultures (Figure 20).

IV.IV. Effects of conditioned medium from FL and FN on normal melanocyte cultures

IV.IV.I. Melanocyte viability

To determine the cytotoxic effect of fibroblasts-conditioned medium, normal melanocytes were incubated with various concentrations of FL/FN-conditioned medium (v/v).

Cytotoxicity was rated in accordance with ISO-standard 10993-5 as non-cytotoxic (cell viability higher than 75%), slightly cytotoxic (cell viability ranging from 50% to 75%), moderately cytotoxic (cell viability ranging from 25% to 50%), and severely cytotoxic (cell viability lower than 25%). As shown in Table 4, fibroblasts-conditioned medium from both FL and FN, at concentration less than or equal to 15% v/v, showed relatively no cytotoxic effect on melanocytes. showed no cytotoxic effect on melanocytes.

IV.IV.II. Melanocyte characteristics

In accordance with MTT results, the concentration of 15% v/v was chosen to evaluate melanocyte morphology and MITF expression.

Following FL-conditioned medium treatment, melanocytes showed increased dendritic morphology and intracellular melanin (Figure 21). These findings were supported by staining of MITF and F-actin, used respectively as a characteristic melanocyte marker and a cell shape marker, revealing a positive MITF expression and a classical dendritic morphology (Figure 22).

V. Discussion-Perspectives

The aging process in human skin results not only in elasticity loss, dry and thin skin but keratinocytes and, therefore may contribute to the hyperpigmentation of SL. The previous research in our lab has identified cellular senescence-like features of fibroblasts derived from SL 76 . Other factors are known to be involved in skin pigmentation, such as DKK1 which regulates hypopigmentation in vitiligo and palmoplantar skin, and inhibits cell invasion and angiogenesis in melanoma development 31,40,54,77,78 .

This study investigates a new possible role of DKK1 in hyperpigmentation, by using solar lentigo (SL) and normal adjacent (SN) skin biopsies from the same patient, which provides a more precision evidence for the research. Our experimental data indicate a reduced expression and secretion of DKK1 in fibroblasts from solar lentigo (FL) compared to fibroblasts from normal adjacent skin (FN). As previous studies described, DKK1 is an antagonistic inhibitor of the Wnt signaling pathway, by isolating the LRP5/6 co-receptor and preventing the Frizzeled-Wnt-LRP5/6 complex formation both in melanocyte and keratinocyte 31,77 . The down-expression of DKK1 in FL compared to FN highlights the potential role of DKK1 in the hyperpigmentation process. This decrease of DKK1 from FL may be caused by different endogenous and exogenous factors, such as pro-inflammatory cytokines.

It was found that TGF-b1 stimulates canonical Wnt signaling in p38-MAPK by downregulating DKK1 in fibrotic fibroblast. We aimed to explore the hypothesis that high levels of TGF-b1 may downregulate DKK1 by p38-MAPK signaling pathway in SL. We reported that the secretion of TGF-b1 from FL was higher than from FN.

To further verify the hypothesis, we tried to mimic in vitro the environment of solar lentigo and reproduce the skin photoaging process under pro-inflammatory conditions via TGF-b1.

We have chosen the model of UVA-irradiated 3D tense collagen gel. In 3D tense collagen lattices, fibroblasts undergo mechanical stress, differentiate in myofibroblasts and secrete TGF-b1. Besides, UVA-irradiation was set up to reproduce UV exposure which is the most exogenous factor inducing solar lentigo 36 . Experimental data indicated that UVA treatment could stimulate TGF-b1 secretion and inhibit DKK1 expression by fibroblasts embedded in collagen matrix. To verify more precisely our hypothesis, future experiments will focus on the inhibition of the p38 MAP kinase pathway.

DKK1 regulates melanocyte function via the regulation of two important signaling factors, MITF and β-catenin. A decrease of DKK1 expression induces an activation of Wnt/β-catenin signaling associated with an increase of melanin and β-catenin, which was confirmed by our histological observations.

In addition, undefined conditioned medium of fibroblast derived from SL can stimulate melanin synthesis in melanocytes. These results confirmed that melanogenic factors are secreted by fibroblasts.

Therefore all these data reveal a link between TGF-β1, DKK1 and the Wnt/β catenin pathway:

UV irradiation Þ ↗ TGF-β1 Þ ↘ DKK1 Þ upregulation of Wnt/β catenin cascade Þ ↗ MITF Þ ↗ melanin Þ SL lesion.
This process may result in an uneven distribution of active melanocytes, with areas of hyperpigmentation in the skin.

In the future, it would be a good practice to do co-cultures of melanocyte and keratinocyte in fibroblast-conditioned medium to further explore the influence of DKK1 in hyperpigmentation both in melanin synthesis in melanocyte and melanin taken by keratinocyte.

Introduction

Sensitive skin is a frequent complaint in dermatology consultation. It is defined by some clinical manifestations including stinging, redness, burning, pruritus, tingling that happen mainly on the face but also on other areas such as scalp and hands. 1 The potential mechanisms of sensitive skin involve skin neurosensory dysfunction, 2,3 neurogenic inflammation, 4 epidermal barrier disruption (increasing transepidermal water loss) 5-7 and immune cells activity (transient receptor potential channels). Various conditions induce the symptoms of sensitive skin, such as environmental factors (e.g. UV radiation, heat, cold, hash winds, pollution), lifestyle factors (e.g. cosmetic usage, diet and alcohol), and even psychological factors (e.g. stress) underlying dermatological diseases as atopic dermatitis. 3,8,9 The diagnosis of sensitive skin could be assisted by stinging tests, occlusion tests, behind-the-knee tests, washing and exaggerated immersion tests, to the evaluation of itching and quantitative sensory testing (QST). 10 In Europe, approximately 39% of the population are bothered by sensitive skin, and female skin is declared more sensitive than male skin. Apparently sensitive and very sensitive skin showed high incidence in France and Italy. 11 In response to this prevalence, the effective treatment of sensitive skin becomes challenging. Continuous and tropic care plays a significant role in treatment of sensitive skin whether in daily care or in the combination of drug treatment. Mildness, UV protection, hydration are the fundamental demanding for tropic daily care of sensitive skin, and there are several cosmetics and cosmeceuticals for sensitive skin according to this principle.

In this article, we evaluated an anti-sensitive moisturizing tolerance-extreme cream that is an innovative formulation mainly composed by China Yunnan Portulaca olreacea extract, Prinsepia utilis oil, and beta-glucan, sodium hyaluronate extracted from mushroom.

Portulaca oleracea has anti-inflammatory and anti-allergy effects, it can relieve skin irritation and enhance skin tolerance. 12 Prinsepia utilis promotes the synthesis of ceramide by stratum corneum cells, and therefore can induce repair of the skin barrier. 13 Mushroom beta-glucan stimulates proliferation of keratinocytes and, thus promotes healing of damaged skin. 14 We performed a monocentric, double-blinded, controlled and randomized study in a panel of 20 volunteers using clinical and instrumental evaluations.

Material and Methods

Study design

This baseline (T0) and 28 days (T1) after twice daily application. Volunteers stayed in a controlled room (temperature 22±2°C, humidity 50±10%) for at least 20 minutes before each evaluation. They were evaluated in the standard skin situation (last face washing the night before visit, without any cosmetic, water and makeup application until the measures).

Measurements were recorded using a flexible transparent plastic mask. This allowed measurements to be done in exactly the same place at each visit (Thermocell-Test® professional kit, Monaderm).

Evaluation

Clinical scores

Visual clinical scores were realized including dryness, roughness, desquamation, erythema with a 5-point scale (0=none, 1=slight, 2=mild, 3=moderate, 4=severe).

Self-assessment

Subjects completed a self-assessment questionnaire at T0 and T1 (Table 2).

Photographies

At T0 and T1, digital images were taken using a standardized photography system (SSA, Courage-Khazaka Electronic) connected to a Canon PowerShot A620 camera. All frontal and profile photographs were standardized for light and position.

Sensitivity

Stinging test is now accepted as a routinely test and is used for the recruitment of subjects with sensitive skin. It measures the skin reactivity particularly in the face. It consists of applying a 10% lactic acid at the nasolabial fold compared with physiological serum applied simultaneously to the other side. Subjects assessed stinging based to a 4-point scale (0=nothing, 1=slighting tingling, 2=moderate tingling, 3= intense tingling) at 10s, 2.5 min and 5 min.

Statistical analysis

The statistical analysis was conducted using Statistica Version 7. Differences with p < 0.05 were considered to be statistically significant.

Results

Test and control creams were well tolerated. For all parameters studied, no significant difference was observed between test and control creams. Only data for test cream are shown.

High resolution digital images were captured for all subjects at baseline and after 28 days of treatment. Representative example of one subject is shown in Figure 1. Visual grading analysis demonstrated that after test cream application, a significant improvement of dryness, erythema, and roughness was observed (Figure 2). At T0, 85% of volunteers exhibited slight and mild skin dryness whereas at T1 after test cream treatment, 90% of volunteers had none and slight skin dryness (P<0.0001). 25% of volunteers showed moderate skin roughness before treatment, which population disappeared after treatment.

At T1, roughness scores decreased with only none to mild levels (P=0.0107). At T0, nearly 50% of volunteers exhibited mild erythema whereas at T1, this percentage decreased to 14%. In addition after 28 days of treatment, 10% and 60% of volunteers had respectively no and slight erythema (P=0.0210). However, for skin desquamation, no significant difference was noted between T0 and T1.

The hydration index of skin was scoring from 48.7±9.5 at baseline T0 to 57.2±10.6 at T1 after test cream application, which reflected a significant increase of the skin surface hydration (P=0.0017) (Figure 3).

Skin texture parameters including roughness (Ser) and smoothness (Sesm) showed significant decline (respectively P=0.0333 and P=0.0111) after test cream treatment, and the texture parameter of energy showed significant increase (P=0.0045) (Figure 4).

However, no significant effect was observed for skin barrier function (TEWL), sebum recovery (lipid index), wettability, color and stinging test (data not shown).

Results of the self-assessment questionnaire showed that test cream was significantly favourable for 3 questions (Figure 5). Subjects considered their facial skin less tightness (p=0.0169), more hydrated (p=0.0004) and comfortable (p=0.0127) after test cream application.

Questionnaire revealed self-perceived benefits consistent with expert visual grading.

Discussion

The treatment of sensitive skin is challenging and generally based on continuous and topical application of anti-sensitive moisturizing tolerance extreme product that improves skin features associated with itching, stinging, dryness, tightness, burning or pain. 10,17-19 To maintain a healthy skin, it is recommended to hydrate and protect it. In this study, an antisensitive moisturizing tolerance-extreme cream from Beitaini Bio-technological Co.,Ltd. in China, was found to be an effective cosmetic cream to improve skin hydration and texture.

Its effect was similar as the control cream. The clinical assessment of quality of facial skin using visual scoring system, the instrumental assessment and the self-assessment by subject using questionnaire reported that after 28 days of twice daily application of test cream, dryness, smoothness, roughness and erythema of skin were significantly improved. These interesting skin benefits are due to the components of the innovative formulation. The improvement of skin texture is likely contributed by Prinsepia utilis oil, beta-glucan and sodium hyaluronate.

Prinsepia utilis is a genus of trees in the Rosaceae and mainly distributed in China, India, Bangladesh and Farmosa. In China, Prinsepia utilis is rich in the area above 2000 meters altitude of Yunnan province with pollution-free. Its kernel is composed of around 40% oil which contains a majority of unsaturated fatty acid and vitamin. In previous study, Prinsepia utilis oil enhanced the synthesis of ceramide in culture of keratinocytes. In nude mice model, its topical application during 7 days significantly decreased TEWL, increased epidermal hydration and skin surface lipids. 20,21 Therefore, Prinsepia utilis oil seems to be an ideal active ingredient for recovery the skin barrier in anti-sensitive formulation.

Beta-glucans are glucose polymers, extracted from cell walls of numerous fungal species.

They are found to contribute to wound healing, depressing immune system, and are even recommended for treatment of atopic dermatitis. 22-25 Sodium hyaluronate is the sodium salt of hyaluronic acid which is a component of the dermal extracellular matrix. Its hydration property makes it an excellent moisturizing ingredient. Concerning Portulaca oleracea, it belongs to Portulacaceae family and is distributed in mainly warm-climate area. There are many studies on its functions (anti-inflammatory, 26,27 anti-oxydant, 27 anti-ulcerogenic, 28 anti-bacterial, 29 anti-cancer, 30 and in wound healing 31,32 ). In addition, Portulaca oleracea extracts could prevent the apoptosis of keratinocyte and fibroblast cultures and has an assistant function in reversing the keratinocyte differentiation and skin barrier dysfunction. 33,34 In this study, the clinical scoring of erythema after test cream treatment firmly proved the anti-sensitive function of Portulaca oleracea in the innovative formulation.

Conclusion

The combination of different actives in this new anti-sensitive moisturizing toleranceextreme cream improved skin hydration and texture after 28 days of treatment. This

Figure 1 . 23 Figure 2 . 24 Figure 3 .

 1232243 Figure 1. Structure of the skin (Source from Pearson Education) ............................................... 23

Figure 4 . 29 Figure 5 . 32 Figure 6 .

 4295326 Figure 4. Melanin synthesis in melanocytes 100 ................................................................................. 29

Figure 7 . 37 Figure 8 . 38 Figure 9 . 39 Figure 10 . 42 Figure 11 . 44 Figure 12 . 48 Figure 13 .Figure 14 .Figure 15 .Figure 16 .Figure 17 .Figure 18 .

 737838939104211441248131415161718 Figure 7. Schematic representation of relationships between fibroblasts and

Figure 19 .Figure 20 .Figure 21 .Figure 22 .Figure 23 .

 1920212223 Figure 19. DKK1 secretion into the medium by ELISA. DKK1 secretion is normalized to

Table 2 .Table 3 .Table 4 . 3 NRG- 1 :- 1 PAR- 2 : 1 :

 23431121 Comparison of three classic melanocyte extraction methods ......................................... Primers and Taqman probes used for qRT-PCR ................................................................ Preparation of different concentrations of fibroblast-conditioned medium ......... Optical density measurements for MTT assay of viability of melanocytes cultured with different concentrations of fibroblast-conditioned medium. The mean ± SD was determined from 3 independent experiments, each performed 4 times. Fibroblastsconditioned medium from both FL and FN in concentration ranging from 0 to 15% v/v showed no cytotoxic effect on melanocytes. ................................................................................. List of Abbreviations AMH: Anti-Müllerian Hormone APC: Adenomatous Polyposis Coli bFGF: basic Fibroblast Growth Factor BMK1: Big MAP Kinase 1 BMPs: Bone Morphogenetic Proteins cAMP: cyclic Adenosine Monophosphate CREB: cAMP Responsive Element Binding protein DCT: DOPA Chrome Tautomerase DHI: 5,6-dihydroxyindole DHICA: DHI-2-carboxylic Acid DKK: Dickkopf DMEM: Dulbecco's Modified Eagle's Medium DMSO: Dimethyl Sulfoxide DOPA: L-3,4-dihydroxyphenylalanine Dsh: Dishevelled ERKs: Extracellular Signal-regulated Kinases FBS: Fetal Bovine Serum FPCL: Fibroblast-populated Collagen Lattice GDF: Growth and Differentiation Factors Gsk-3b: Glycogen Synthase Kinase-3𝛽 HGF: Hepatocyte Growth Factor JNK/SAPK: c-jun N-terminal kinases KGF: Keratinocyte Ggrowth Factor LDLR: Low-density Lipoprotein Receptor LEF: Lymphoid Enhancer-binding Factor MAPK: Mitogen-activated Protein Kinase MATP: Myoacitve Teradeca Peptide MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MITF: Microphthalmia-associated Transcription Factor NT-3: Neurotrophin-NeuregulinProtease-activated Receptor-2 PBS: Phosphate-buffered Saline PI3K/Akt: phosphatidylinositol 3′-kinase/Akt PKA: Protein Kinase A P/S: Penicillin/Streptomycin R-Smads: Receptor-activated Smad rhDKK1: recombinant Human DKK1 ROS: Reactive Oxygen Species Sema7a: Semaphorin7a SCF: Stem Cell Factor SFRP: Secreted Frizzled-related Proteins TGF-b:Transforming Growth Factor-beta TRP-1: Tyrosinase-related Protein -1 TYR: Tyrosinase WIf-Wnt Inhibitory Factor 1

  DKK1 encoded by DKK1 gene is an antagonistic inhibitor of the Wnt signaling pathway, by isolating the LRP5/6 co-receptor and preventing the Frizzeled-Wnt-LRP5/6 complex formation. Wnt/b catenin signaling regulates the transcription of melanocyte-specific genes like MITF, a gene involved in melanin synthesis. We hypothesized that TGF-β1 mediates development of solar lentigo by reducing DKK1 expression in fibroblasts through the p38 MAP kinase pathway, which leads to an activation of the Wnt/b-catenin signaling cascade. In vitro models that mimic the in vivo environment were used: fibroblasts isolated from solar lentigo and peri-lesional biopsies, and normal fibroblasts embedded in 3D collagen gel and exposed to repeated doses of UVA. Q-PCR and ELISA techniques showed that fibroblasts from solar lentigo and fibroblasts irradiated to UVA, express low level of DKK1.Immunohistochemical studies revealed a strong staining of b catenin and melanin in solar lentigo skin, which indicates an activation of Wnt/b catenin signaling and melanocyte function. Our previous data demonstrated the senescent-like phenotype of fibroblasts from solar lentigo with particularly a high secretion of TGF-β1, suggesting its role in the development of the lesion. Fibroblasts should respond to many inflammatory mediators released during hyperpigmentary disorder by increasing TGF-β1 secretion. TGF-β1 is known to suppress the expression of DKK1 in a p38-dependent manner. Therefore, our hypothesis confirmed that TGF-β1 regulates development of solar lentigo by reducing DKK1 expression in fibroblasts through the p38 MAP kinase pathway. This process may result in an uneven distribution of active melanocytes, with areas of hyperpigmentation in the skin.Skin as one of the largest organ of human body becomes the most important barrier between human body and external environment. The average area of the skin is to 1.8 m 2 , and its average sickness is to 1.2 mm. It is responsible of three main functions: protection (mechanical trauma, UV radiation, microorganisms, infection), regulation (temperature, fluid, immunity, synthesis of vitamin D) and sensation (pressure/pain, heat/cold)1 .

Figure 1 .

 1 Figure 1. Structure of the skin (Source from Pearson Education)

Figure 2 .

 2 Figure 2. Structure of epidermis (Source from Human Anatomy Library)

Figure 3 .

 3 Figure 3. Four stages of melanosome: stage I, premelanosome, spherical form

Figure 4 .

 4 Figure 4. Melanin synthesis in melanocytes 100

Figure 5 .

 5 Figure 5. Canonical and non-canonical Wnt signaling pathways. a. Canonical Wnt/ 𝛽-

Figure 7 .

 7 Figure 7. Schematic representation of relationships between fibroblasts and melanocytes.

Figure 8 .

 8 Figure 8. The different structure of DKKs family. DKK1, 2 and 4 have similar gene

  DKK proteins have distinct patterns of expression in adult and embryonic tissues and have a wide range of effects on tissue development and morphogenesis41 . They are involved in cancer, Alzheimer's disease, rheumatoid arthritis, keloids…41,42

Figure 9 .

 9 Figure 9. DKK1 and DKK2 interact with Wnt/β-catenin signaling pathway. (a) Wnt, Fz,

Figure 10 .

 10 Figure 10. The effects of DKK1 from palmoplantar fibroblasts on both melanocytes

Figure 11 .

 11 Figure 11. Reconstructed skin model with rhDKK1 treatment (left) showed

TGF-b superfamily

  comprises nearly 30 growth and differentiation factors including BMPs (Bone morphogenetic proteins), GDFs (Growth and differentiation factors), AMH (antimüllerian hormone), Activin, Nodal and TGF-bs (Figure12). The signaling is conducted by transmembrane receptors type I and type II. In response to ligand binding, type II receptors (BMPRII, ActRIIA, ActRIIB, TβRII (1-3)) catalyze the phosphorylation of Type I receptors (ALK1-7). Activated type I receptors phosphorylate downstream Smads (Smad1, Smad2, Smad3, Smad5, and Smad8), activating them to transduce the signal to the nucleus. It also exists non-smad signaling pathway in stem cells. TGF-b signaling pathway is involved in embryonic development, wound healing, angiogenesis by adjusting cell proliferation, differentiation, and apoptosis56,58 .

Figure 12 .

 12 Figure 12. TGF-b family signaling by type II and I receptors, and smad proteins 56 .

  signaling pathway activation by TGF-b MAPK signaling pathways are identified by four different groups including ERKs, JNK/SAPK, ERK/BMK1 and P38, which generate diverse cellular responses such as cell proliferation, differentiation, migration and apoptosis. p38-MAPK is activated by a variety of cellular stresses including inflammatory cytokines, growth factors, UV… Activated p38-MAPK phosphorylates and activates transcription factors which are involved in DNA damage response, inflammation, proliferation, apoptosis, cellular regulation. TGF-b regulates via p38/MAPK signaling pathway the expression of collagenase-3 (MMP-13)

  helping to bring thickness and firmness to the skin. They communicate with each other and neighboring cells by secreting a large number of cytokines and growth factors, playing a crucial role in skin physiology. In term of pigmentation, fibroblasts exhibit a great dynamic in the epidermal melanogenesis, and participate actively in the signal cross-talk between melanocytes and keratinocytes. Most of previous research studied the regulation of skin pigmentation by focusing on both melanocytes which synthesize melanin, and neighboring keratinocytes which receive and distribute the pigment in upper layers of the skin. Interest in fibroblasts has increased in recent years due to their ability to secrete melanogenic factors.

  addition, Salducci et al. observed an increase of melanocytes number in reconstructed epidermis cultured with conditioned media of UVA-treated fibroblasts[2]. However, fibroblasts limit the spontaneous pigmentation of melanocytes in 3D-reconstructed with cells from patient of phototype I and II, suggesting a role of fibroblasts in the control of pigmentation[3]. Murine and human fibroblasts did not secrete the same melanogenic and mitogenic melanocyte factors, resulting in a different effect on pigmentation. It has been shown that fetal fibroblasts in reconstructed skin model induce dramatic increase of pigmentation compared to adult fibroblast, resulting in the elevation of melanogenic mediators from fetal fibroblasts. Tsuchiyama et al. found that multilineage-differentiating stress-enduring cells, distinct stem cells among human fibroblasts, could be reprogrammed into melanocytes. These Muse-derived melanocytes reside in the basal layer of epidermis in 3D-skin model, and acquire melanocytic functions. This technique should permit treatment of vitiligo by autologous transplantation[4]. Moreover, Yang et al. successfully converted mouse and human fibroblasts to functional melanocytes by combination of several transcriptional factors, Microphthalmia-associated transcription factor (MITF), paired domain and homeodomain-containing transcription factor 3 (PAX3) and SRY-related

- 3 (

 3 NT-3) belongs to a family of nerve growth factors, synthesized by fibroblasts. These factors have been extensively studied for their role in the development of neurons and neural crest-derived cells such as melanocytes. NT-3 can link each Trk receptor including Trk-A, Trk-B and Trk-C, but mainly plays a biological function by binding to Trk-C. It modulates intracellular signal transduction through MAPK and PI3K-Akt pathways,

  Method I: simple enzymatic digestion (TrypLE TM ) (Figure. 1)Biopsies were incubated in TrypLE TM enzyme 1X (GIBCO Ò TrypLE TM Select TM CTS TM , ThermoFisher Scientific, France) at room temperature overnight(17 h). During the treatment, the epidermal layer was easily separated from the dermis. After incubation, all dermis were discarded, then melanocyte culture medium was added and epidermal cells were disaggregated mechanically by repeated pipetting up and down. Mixtures were filtered through 70 μm cell strainers, and centrifuged for 5 min at 1,100 rpm. Cell pellets were resuspended in melanocyte medium which consists of M254 culture medium supplemented with human melanocytes growth supplement (HMGS, Life Technologies, ThermoFisher Scientific, France), 100 IU/ ml penicillin and 100 μg/ml streptomycin. Epidermal cells were counted, placed into 25 cm2

  isolation preserves molecular markers (Xinyan et al. 2014). Prolonged passages of cultured melanocytes tend to lose their pigmentation (Virador et al. 1999). Due to the limited proliferative capacity of primary melanocytes and the variability of phenotypes generated, it's necessary to limit melanocyte passage number. In the present work, we studied the functionality of melanocytes cultured at passage 2. Several melanocytic markers can be utilized to identify typically melanocytes, including MITF. MITF is a cell-type specific regulator that is required for the development and survival of pigments cells. It acts as a master regulator of melanin production (Shi et al. 2016). We analyzed cell morphology and more precisely the dynamic cytoskeletal network by filamentous actin (F actin) labeling. Results obtained were equivalent whatever the number of 3-mm punch biopsies. Only the data for 10 x3-mm punch biopsies are shown. Similar growth rate were observed among TrypLE TM and Dispase/Trypsin methods (Figure. 5). There was also no difference in the intracellular melanin content of melanocytes (Figure. 6). Immunocytochemical staining confirmed that cells extracted by both methods positively expressed MITF and F actin (Figure.7).It was concluded that TrypLE TM and Dispase/Trypsin methods are of similar efficacy for preserving cell biological characteristics. Melanocytes extracted with our protocol and cultured with classical conditions exhibited normal functionality with respect to dendritic morphology, pigment production and growth kinetic.

- 8 - 80 - 5 -Figure. 1

 88051 Figure.1 Shematic diagram for the isolation of melanocytes from small amount of human

Figure. 2

 2 Figure.2 Epidermal-dermal separation using TrypLE TM enzyme 1X (A) and dispase II (B).

Figure. 3

 3 Figure.3 Skin epithelial cells isolated from multiple 3-mm punch biopsies. Comparison of

Figure. 4 Figure. 6

 46 Figure.4 Growth pattern of cells extracted from 10 punch biopsies of skin, by TrypLE TM (A,

Figure. 7

 7 Figure.7 Immunostaining of cells extracted from 10 punch biopsies of skin, by TrypLE TM (A)

Figure 13 .

 13 Figure 13. Hypothesis of the study: Structural damages, pro-inflammatory cytokines,

Figure 14 .

 14 Figure 14. Tense FPCL embedded by a nylon mesh ring

Figure 15 .Figure 16 .

 1516 Figure 15. Shematic representation of the irradiation protocol

  Figure 17. a) Melanin (HC, Masson-Fontana staining) and b) β-catenin (IHC, DAB

Figure 18 .Figure 19 .

 1819 Figure 18. Relative gene expression of DKK1 assessed by real-time quantitative RT-PCR.

Figure 20 .

 20 Figure 20. TGF-β1 secretion into the medium by ELISA. TGF-β1 secretion is normalized to

Figure 21 .

 21 Figure 21. Phase contrast images of melanocytes cultures in a) normal conditions, b) FL

Figure 22 .

 22 Figure 22. Immunofluorescence images of melanocytes cultures in a) normal conditions, b)

Figure 23 .

 23 Figure 23. DKK1 secretion into the medium by ELISA. TGF-β1 secretion is normalized to

  also in benign lesion development such as solar lentigo (SL). Recent studies have demonstrated that fibroblasts are activated by UV exposure to release melanogenic growth factors (HGF, KGF, SCF) which act on melanocytes both directly and indirectly through

  randomized double-blind comparative study was carried out at the CERT (Research and Study Center on Integument) located in the department of Dermatology, University Hospital of Besançon, France. The study protocol was approved by the ethics committee of the institute. This study was conducted in accordance with the ethical principles derived from the Declaration of Helsinki with Good Clinical Practices (ICH E6) and the law of local regulation clinical studies, and was considered as a non-interventional study (directive 2001/20/CE, decree dated 27 April 2006). The final protocol and the related documents were reviewed by the CPP Est2 of Franche-Comté, Besançon, as well as ANSM, Paris, France.

  1 and Graphpad Instat Version 3.06. Distribution of the data was assessed using the Shapiro-Wilk test. Statistical comparisons of the data were carried out with the Anova or Wilcoxon test (depending of the normality of the distribution). Data are presented as mean ± standard error of mean (SEM).

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. Representative images of one subject before (T0) and after the 28 days treatment (T1)

Figure 6. Melanin transfer from melanocyte to keratinocyte by melanosome. Different

	(e.g., Mc1r, NGFR, FGFR-1, FGFR-2, ETBR, Kit, gp130, LIFRα, c-Met, GMCSFR, EP1, EP2, and
	EP3) on the membrane of melanocytes and stimulate different signaling pathways, PKA, PKC,
	and MAPK.
	form between light skin and dark skin 21 .
	. Moreover, PAR-2 is
	induced by UV irradiation and inhibition of PAR-2 activation results in the prevention of
	UVB-induced tanning.
	Interactions between melanocytes and neighboring cells in the skin are important in
	regulating skin color in humans. The proliferation, differentiation, melanogenesis, and
	dendritogenesis of melanocytes in the epidermis are primarily regulated by paracrine
	factors derived from keratinocytes. Keratinocyte-derived factors such as α-MSH, ACTH, NGF,
	bFGF, ET-1, ET-2, ET-3, SCF, LIF, HGF, GMCSF, PGE2, and PGF2α bind to their specific receptors

Table 1 .

 1 . Although previously developed protocols are satisfactory in terms of cell yield and viability, they use enzymes of animal origin. In this chapter, we introduced a simple and optimized method for extraction of melanocytes from low surface of human skin sample, which should be effective for functional and molecular studies and also for medical application such cell grafting. We compare the new protocol with a classic melanocyte extraction method. This work is summarized in the following article submitted in Cytotechnology journal. Comparison of three classic melanocyte extraction methods explants is usually performed in a two-step protocol, dispase incubation to separate the dermal and epidermal layers, following by trypsin treatment to release the epidermal cells

	Protocol	Method 77	Method 78	Method 76
	Sterilization	Iodine	70% ethanol	Iodine
		70% ethanol	HBSS	Normal Saline
		DPBS Ca\Mg-free
		10μg/ml
		Gentamicin
	Size of sample 1 cm wide pieces	5×5 mm 2 pieces	0.5 mm thick pieces
		(minimum 1cm 2 )
	Solution for	10mg/ml dispase II	0.48mg/ml dispase	0.25% dispase
	separating	in Hank's solution	II(Boehringer	(Solarbio)
	dermis and		Mannheim)
	epidermis	Overnight 4℃	0.1% BSA in PBS	Overnight, 4℃
		(≥18h)	Ca/Mg-free
			18-24h, 4℃
	Solution for	Trypsin/EDTA	Trypsin/EDTA	Trypsin/EDTA
	getting cell	10-15 min, 37℃	5min, 37℃	5 min, 37℃
	suspension
	Termination RPMI Medium	HBSS+Medium	Serum
	Counting cell	Yes	No	Yes
	before culture
	Medium	RPMI (Sigma)	MCDB153 (Sigma)	MC254(Gibco)
		supplement with	supplement with	1%HMGS2
		TPA, CT, ET1, SCF	FBS, Chelated FBS, L-	100U/ml,
			Glutamine, CT, rh-	Penicillin,50μg/ml
			bFGF, rET-3, rhSCF,	streptomycin
			Heparin
	Seeding	15×10 4 cells per ml All cells in T25 cell-	5x10 5 cells / 25 cm²
	number		culture vessel	flask

pigment-related disorders, or developing in vitro pigmented skin models

(Guerra et

al. 2003; Duval et al. 2003; Yoon et al. 2003; Verma et al. 2015; Kumar et al. 2012). Although a number of cell lines originated from healthy and lesional skin are used for studying cellular and molecular biology, testing cosmetic and chemical products, primary culture remains as a more reliable model that mimics in vivo skin. Several methods for the isolation and the culture of melanocytes from human skin have been described (Hsu et al. 2005; Godwin et al. 2014; Tang et al. 2014; Nielsen et al. 1984; Gilchrest et al. 1984; Yarani et al. 2013). The first human melanocyte culture from skin was published by Hu et al. (1957). Unlike basal keratinocytes that are proliferative and capable of regenerating the epidermal layers, melanocyte cultures have low proliferation potential and short life span. To obtain high-Peacocke et al. 1988). 5-fluorouracil (5-FU) is utilized to isolate pure populations of melanocytes free from keratinocyte (Takuo et al. 1983). Isolation of melanocytes from human skin

(

  Caucasian females, 67-89 years of age) after written informed consent (University hospital of Besançon, Department of Dermatology; Pr. P Humbert).

	III.I.II.III	Masson's Fontana staining for melanin
	The sections were stained with Fontana-Masson kit following the manufacturer's
	instructions (Diapath, Italy). The stained sections were mounted with Faramount aqueous
	mounting medium, and observed using a Zeiss Axioskop 40 light microscope.
	III.I.II.IV.	Immunostaining for β-catenin
	Slides were heated at 95 °C for20 min in 10 mM citrate buffer pH 6. Samples were then
	incubated overnight at 4 °C with the primary antibodies at 1:50 (MAB 13291-SP, R&D system,
	USA). Subsequently, they were incubated for 1 h with anti-mouse HRP-DAB (CTS002, R&D
	III.I.II. system, USA) following manufacturer's procedure. The stained sections were mounted with Histology
	III.I.II.I Faramount aqueous mounting medium, and observed using a Zeiss Axioskop 40 light Fixation and sectioning
	SL and SN biopsies were fixed in PFA solution (3% in PBS), dehydrated through a graded microscope.
	series of alcohol and embedded in paraffin. Sections 7 µm in thickness were obtained using III.I.III. Cell culture
	a rotary microtome (Leitz 1512; Leica). They were cleared in xylene rehydrated in solutions III.I.III.I. Extraction of fibroblasts
	of alcohol and briefly washed in distilled water. All cell culture products were purchased from PAN Biotech, Germany. Primary human
	III.I.II.II fibroblasts from SL (FL) and from SN (FN) were obtained from the biopsies by explant Masson's trichrome staining for collagen fibers
	The sections were stained in Harris hematoxylin solution, rinsed in distilled water, stained culture, and cell cultures were maintained in Dulbecco's Modified Eagle's Medium (DMEM)
	with 0.1% eosin solution, treated with 1% acetic acid-water, washed again and differentiated supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S, 100
	in 1% phosphomolybdic acid. Finally, the sections were stained with 0.5% light green and units/mL penicillin, 100 µg/mL streptomycin) in a humidified 5% CO2 atmosphere at 37℃.
	rinsed in distilled water before 1% acetic acid-water. The stained sections were mounted III.I.III.II. Establishment of skin fibroblast cultures
	with Faramount aqueous mounting medium, and observed using a Zeiss Axioskop 40 light FL and FN were maintained until the monolayer cultures reached 80% confluency and were
	microscope. passaged using 0.25% trypsin-EDTA solution. Cells were used for experiments at passages
	2-7. FL and FS were seeded at 400,000 cells in 25 cm 2 culture flasks (F25) with 3 mL full
	DMEM medium.

Table 2 .

 2 Primers and Taqman probes used for qRT-PCRIII.I.VII. Conditioned medium from FL and FN in cultured human melanocytes

Table 3 .

 3 Preparation of different concentrations of fibroblast-conditioned medium

	III.I.VIII.	MTT assay
	Melanocytes were cultured in 96-well plates at an initial density of 5×10 3 cells per well for
	24 h, and then treated with several concentrations of fibroblast-conditioned medium. After
	48 h, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0,5 mg/mL,
	Sigma-Aldrich, France) was added to each well and the cells were incubated at 37°C for 4 h.
	The medium was removed and dimethyl sulfoxide (DMSO) was added to dissolve the

Table 4 .

 4 Optical density measurements for MTT assay of viability of melanocytes cultured

		0.0025	**	0.0015
		0.002
	TGF-β1 pg /cell	0.0005 0.001 0.0015	0.0009
		0
			FN	FL

Table 1 .

 1 List of ingredients of test cream

Test cream: Anti-sensitive moisturizing tolerance-extreme cream Aqua, Glycerin, Pentylene glycol, butyrospermum parkII (shea butter), tridecyl, trimellitate, dimethicone, glyceryl stearate, prinsepia utilis oil, sodium hyaluronate, portulaca oleracea extract, beta-glucan, acrylates/c10-30 alkyl acrylate crosspolumer, xanthan gum, butylene glycol, aminomethyl propano Control cream: Tolerance extreme cream Thermal spring water, glycerin, mineral oil (paraffinum liquidum), squalene, carthamus tinctorius (Safflower), seed oil (carthamus tinctorius seed oil), cyclomethicone, glyceryl stearate, sodium carbomer, titanium dioxide

Acknowledgments

This work was financially supported by Program for Innovative Research Team in University of Ministry of Education of China (Grand No. IRT13067) and The Fund of Yunnan Province Chinese Academy of Sciences Cooperation (Grande No.2014IB008) (China) and Greentech SA (France). Acknowledgments: this work was financially supported by Program for Innovative Research Team in University of Ministry of Education of China (Grand No. IRT13067), the Fund of Yunnan Province Chinese Academy of Sciences Cooperation (Grand No.2014IB008) and Greentech SA (France). Acknowledgments This work was financially supported by Program for Innovative Research Team in University of Ministry of Education of China (Grand No. IRT13067) and the Fund of Yunnan Province Chinese Academy of Sciences Cooperation (Grande No.2014IB008) (China).

We would like to thank Céline Thiebaut, Aurélie Durai and Vanessa Ecarnot for their technical help.

Conflicts of interest

The authors have no conflict of interest to declare.

Dermatol. 19 (2010) 865-872.

Author Biography

Yinjuan Wang is a Ph.D. candidate from the Laboratory of Engineering and Cutaneous Biology, UMR 1098, Bourgogne Franche-Comte University, Besancon, France. Her thesis, supervised by Professors Philippe Humbert and He Li, is concerned with skin pigmentation.

She received Medical Doctor on 2011 and Dermatological Master on 2014, from Sichuan

University and Kunming Medical University of China, respectively. Her research interests involve melanin synthesis by melanocytes and its pathways, in particular the epidemiology and physiopathology of hyperpigmented skin lesions including melasma and lentigo.

Chapter II Development and validation of a simple method for the extraction of human skin melanocytes

Abstract

Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products.

Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as After 3 days of culture, supernatants were collected with 10% (v/v) anti-protease solution (P1860, Sigma-Aldrich, France) and stored at -80°C. ELISA analyzes for DKK1 (kit EHDKK1, Thermo Scientific, USA) and TGF-b1 (kit BMS2065, Invitrogen, Australia) were performed according to the manufacturer's instructions. Duplicate analyses were performed for each sample. Dkk1 concentration in the supernatants was normalized to the concentration of intracellular protein (which corresponds directly to the cell number) determined by the Biuret method. TGF-b1concentration in the supernatants was normalized to the cell number.

III.I.V. Biuret protein assay

After 3 days of culture, cells were scrapped, lysed with NaOH 0.1N and mixed with Biuret reagent (the Biuret reaction (Cu 2+ + e -→ Cu + ) uses bicinchoninic acid and it is based on the measurement of the formation of purple-colored complex of bicinchoninic acid and Cu + ) 73 .

Mixtures were centrifugated, and absorbance of supernatants was determined at 620 nm.

Bovine serum albumin was used as standard. Duplicate analyses were performed for each sample.

III.I.VI. Real-time quantitative reverse transcription-PCR

After 3 days of culture, cell pellets were collected with 0.25% trypsin-EDTA solution, lysed in RLT buffer (Qiagen, France) with 1% b-mercaptoethanol and stored at -80°C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, France) according to the manufacturer's protocol. Next, cDNA was performed using the High Capacity RNA-to-cDNA kit (Thermofisher, Applied Biosystems, France). Taqman Ò Gene Expression Assay kits (Thermofisher, Applied Biosystems, France) were used to assess DKK1 (Hs00183740_m1)

and Abl (Abelson, Dm01843160_g1) gene expressions. Abl was used as housekeeping gene.

Up-regulation of melanocyte functions caused by aging and UV irradiation is involved in the development of solar lentigo lesions or other age-related hyperpigmentation disorders.

Cosmetic companies usually develop anti-photoaging products by focusing on melanocyte and keratinocyte functions. This study brings new approach to prevent and treat solar lentigo lesions, by controlling the expression and secretion of several fibroblast-derived soluble factors.

List of Publications & Communications

Test product

The test product is an anti-sensitive moisturizing tolerance-extreme cream which is manufactured by Beitaini Bio-technological Co.,Ltd. (China). A tolerance extreme cream was used as control. Both products were provided by Beitaini Bio-technological Co.,Ltd.

Ingredients of test and control creams were listed in Table 1.

Treatment

Volunteers served as their own controls with one side of the face receiving the test cream and the other side of the face receiving the control cream. Evaluations were performed at

Bioinstrumentation

Transepidermal water loss (TEWL)

The skin barrier function of the stratum corneum was measured by Vapometer® (Delfin Technologies). The sensor monitor in closed cylindrical chamber measures the increasing relative humidity (RH) of evaporated water from the skin covered by chamber. The evaporation rate value was expressed in g /m²/ h. One assessment was performed on external side of each cheekbone.

Skin hydration

Skin hydration was determined on each cheekbone by the measurement of the electric capacitance (i.e.the capacity of the intercellular water to conduct electrons) of the stratum corneum using a Corneometerâ CM825 (Courage-Khazaka Electronic). The mean value (hydrate index, HI) of three assessments was calculated.

Lipid index

Quantitative analysis of the sebum was measured on forehead by a Sebumeter® SM 815 (Courage-Khazaka Electronic). Values were expressed in mg/cm 2 . The mean value of three assessments was calculated.

Skin texture

Visioscanâ VC98 (Courage-Khazaka Electronic) was used for assessing skin texture and roughness. 15 It takes pictures of the skin surface under standardized homogenous ringshaped UVA illumination. Evaluation of images was carried out according texture parameters: energy reflecting overall skin condition (NRJ), skin smoothness (Sesm), skin roughness (Ser), and arithmetic mean roughness (R5). The mean value of three acquisitions on each temple was calculated.

Skin surface wettability

The measurement of the contact angle formed between the skin surface and water is an indicator of its lipophilic or hydrophilic property, thus the content in lipids. The system is composed of a surgical microscope (Wild M650 Heerbrugg®, Leica) with a mirror oriented at 45° to the skin surface. A drop of distilled water was deposited on the skin surface and the contact angle was measured using software based on the geometrical dimensions of the drop. 16 The mean value of three measures on forehead was calculated.

Skin color

The Minolta 400 Chroma Meter is a colorimeter that combines a source-filter-photodetector and a unit that directly computes the chromatic dimensions of the colour. The mean value of three measures on each cheekbone was calculated.

complex cream could give further assistance to the continuous treatment of sensitive skin.

Conflicts of interest

The authors have no conflict of interest to declare.