In vitro modeling of hyperpigmentation associated toneurofibromatosis type 1 using melanocytes derivedfrom human embryonic stem cellsJennifer Allouchea,b, Nathalia Bellonc,d,e, Manoubia Saidanid, Laure Stanchina-Chatroussed, Yolande Massond,Anand Patwardhanf,g, Floriane Gilles-Marsensf,g, Cédric Delevoyef,g,h, Sophie Dominguesd, Xavier Nissand,Cécile Martinata,b, Gilles Lemaitrea,b, Marc Peschanskia,b, and Christine Baldeschia,b,1

aINSERM U-861, Institut des cellules Souches pour le Traitement et l’Etude des Maladies monogéniques (I-Stem), Association Française contre les Myopathies(AFM), 91030 Evry Cedex, France; bUniversité d’Evry Val d’Essonne (UEVE) U-861, I-Stem, AFM, 91030 Evry Cedex, France; cDepartment of Dermatology,Reference Center for Dermatologic Diseases, Paris Descartes–Sorbonne Paris Cité University, 75015 Paris, France; dCentre d’Etude des Cellules Souches(CECS), I-Stem, AFM, 91030 Evry Cedex, France; eInstiute Imagine, Necker-Enfants Malades Hospital, 75015 Paris, France; fInstitut Curie, Paris Sciences etLettres Research University, F-75248 Paris, France; gStructure and Membrane Compartments, CNRS UMR 144, F-75248 Paris, France; and hCell and TissueImaging Facility, CNRS UMR 144, F-75248 Paris, France

Edited by Leonard I. Zon, Howard Hughes Medical Institute, Children’s Hospital of Boston, Harvard Medical School, Boston, MA, and accepted by the EditorialBoard May 30, 2015 (received for review January 17, 2015)

“Café-au-lait” macules (CALMs) and overall skin hyperpigmentationare early hallmarks of neurofibromatosis type 1 (NF1). One of themost frequent monogenic diseases, NF1 has subsequently beencharacterized with numerous benign Schwann cell-derived tumors.It is well established that neurofibromin, the NF1 gene product, isan antioncogene that down-regulates the RAS oncogene. In con-trast, the molecular mechanisms associated with alteration of skinpigmentation have remained elusive. We have reassessed this issueby differentiating human embryonic stem cells into melanocytes. Inthe present study, we demonstrate that NF1 melanocytes repro-duce the hyperpigmentation phenotype in vitro, and further char-acterize the link between loss of heterozygosity and the typicalCALMs that appear over the general hyperpigmentation. Molecularmechanisms associated with these pathological phenotypes corre-late with an increased activity of cAMP-mediated PKA and ERK1/2signaling pathways, leading to overexpression of the transcriptionfactor MITF and of the melanogenic enzymes tyrosinase and dop-achrome tautomerase, all major players in melanogenesis. Finally,the hyperpigmentation phenotype can be rescued using specificinhibitors of these signaling pathways. These results open avenuesfor deciphering the pathological mechanisms involved in pigmenta-tion diseases, and provide a robust assay for the development ofnew strategies for treating these diseases.

neurofibromatosis type 1 | melanocytes | embryonic stem cells |hyperpigmentation | disease modeling

Neurofibromatosis type 1 (NF1) is one of the most commonmonogenic disorders, with an estimated prevalence of ap-

proximately 1 in 3,500 individuals (1). It is characterized by a widerange of clinical expression symptoms, including skin defects as-sociated with melanocytes, namely overall skin hyperpigmentation(2), skin-fold freckling, and “café-au-lait” macules (CALMs) (3),as well as numerous neurofibromas (benign tumors resulting fromSchwann cell proliferation). Hyperpigmentation and CALMs arethe initial symptoms, appearing during the first 2 years of life in allpatients (4). Although the hyperpigmentation associated withCALMs is not life-threatening, it has a strong impact on quality oflife (5, 6).NF1 is caused by mutations in a tumor suppressor gene that

encodes neurofibromin (7), a functional rat sarcoma (RAS)-gua-nosine triphosphate hydrolase (GTPase) activating protein. Neu-rofibromin down-regulates RAS signaling by accelerating theconversion of active RAS-guanosine triphosphate (GTP) to in-active RAS-guanosine diphosphate (GDP) (8, 9). The resultingdecreased expression of neurofibromin leads to activationof several important downstream signaling pathways, includingmitogen extracellular signal-regulated Kinase (MEK)/mitogen

activated protein kinase (MAPK) and cyclic adenosine mono-phosphate (cAMP)-mediated protein kinase A (PKA) pathways(10, 11). Just how these defects in multiple signaling pathwayscause the specific alterations of pigmentation originally observedin patients is not yet clear. Early histological analyses of humanmelanocytes retrieved from CALMs pointed to an overall increasein their number (12), in the size of pigment granules or melano-somes (13, 14), or in the cell content in melanogenic factors (15).Mouse models of the disease were difficult to generate, becausehomozygous Nf1−/− mice died in utero (16) and Nf1+/− micemanifested neither pigmentation abnormalities nor neurofibromas(17, 18). The hyperpigmentation phenotype has been reproducedusing a specific knock-down of Nf1 in bipotential Schwann cell-melanoblast precursors (19), but molecular mechanisms linkingneurofibromin to defective pathways in melanocytes have notbeen fully identified. The relevance of the mouse model might bequestionable in any case, given that mouse melanocytes localize inhair follicles and not in the epidermis as in human melanocytes.A significant difficulty encountered so far in the analysis of NF1

molecular mechanisms has been the lack of a reliable in vitromodel of affected human melanocytes. This has changed recentlywith the emergence of differentiation protocols of human plu-ripotent stem cells into melanocytes (20, 21). A growing number

Significance

There are few suitable laboratory models for human pigmenta-tion disease. Neurofibromatosis type 1 (NF1) is a common neu-rocutaneous disease whose initial symptoms in all patients are“café-au-lait” macules and overall skin hyperpigmentation. Toanalyze the molecular mechanisms associated with this pheno-type, we have developed an in vitro model of NF1 based onhuman embryonic stem cells (hESCs). Melanocytes derived fromNF1 hESCs reproduced the hyperpigmentation phenotype in vitroand were characterized by deregulation of melanogenesis fac-tors. The model allowed us to identify the cellular pathways in-volved in this phenotype. The hyperpigmentation phenotypecould be rescued by small molecules, demonstrating the potentialof pluripotent stem cells as models for pigmentation disorders.

Author contributions: J.A. and C.B. designed research; J.A., N.B., M.S., L.S.-C., Y.M, A.P.,F.G.-M., C.D., S.D., X.N., and G.L. performed research; J.A. and C.B. analyzed data; and J.A.,C.M., M.P., and C.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. L.I.Z. is a Guest Editor invited by the EditorialBoard.1To whom correspondence should be addressed. Email: cbaldeschi@istem.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1501032112/-/DCSupplemental.

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of examples illustrate how such cells, retrieved from genetically se-lected donors carrying the causal mutation of a monogenic disorder,may reproduce disease-associated phenotypes (22–26). Thus, we usedtwo human embryonic stem cell (hESC) lines derived from embryoscharacterized as mutant gene carriers for NF1 during a pre-implantation diagnosis procedure, to explore mechanisms associatedwith hyperpigmentation in melanocytes and potential treatments forthe pathological phenotype. In this study, we demonstrate the use-fulness of human pluripotent stem cells in deciphering the mecha-nisms underlying the hyperpigmentation phenotype of NF1. At themolecular level, our results indicate that neurofibromin controlsmelanogenesis via cAMP-mediated PKA and extracellular signal-regulated kinase (ERK) pathways. Consequently, the decreasedexpression of neurofibromin in a pathological context leads to dys-regulation of these pathways, resulting in hyperpigmentation. In-terestingly, our cellular model has allowed us to identify smallmolecules capable of restoring the pathological phenotype to normal.

ResultsNF1 hESCs-Derived Melanocytes Reproduced the Decreased Expressionof Neurofibromin and Exhibited a Hyperproliferative Phenotype. TwohESC lines, NF1-1 and NF1-2, which carry a heterozygous de-letion of four nucleotides leading to a stop codon in the NF1 gene,and two control cell lines were successfully differentiated towardhomogenous populations of melanocytes as described previously(20). Melanocytes derived from NF1 and WT hESCs, termed mel-NF1 (mel-NF1-1 and mel-NF1-2) and mel-WT (mel-WT-1 andmel-WT-2), showed morphology typical of melanocytes associatedwith expression of tyrosinase-related protein 1 (TYRP1), a mel-anogenic enzyme (Fig. 1A and Table S3). Flow cytometry analysisconfirmed that more than 97% of mel-WT and mel-NF1 cellsexpressed microphthalmia-associated transcription factor (MITF),

a key marker of melanocytes (Fig. S1A), suggesting that this mu-tation does not prevent differentiation of melanocytes.To determine whether the deletion of the four nucleotides in the

NF1 gene affected neurofibromin expression, we analyzed mRNAand protein levels for this gene. Quantitative RT-PCR (qRT-PCR)analyses showed similar levels of NF1 mRNA (Fig. S1B and TableS1) and the presence of a mutated transcript in mel-NF1 comparedwith mel-WT (Fig. S1C and Table S2); however, as expected,Western blot analysis demonstrated a twofold decrease in neuro-fibromin protein levels relative to control (Fig. 1B).Because neurofibromin has a normal role in cell cycle pro-

gression (27), we investigated whether reduced expression ofneurofibromin results in an alteration of cell proliferation capacity.

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Fig. 1. Characterization of mel-NF1 cells derived from hESCs carrying anNF1 mutation. (A) Microscopy analysis of mel-WT and mel-NF1 cells, andimmunofluorescence analysis of the melanocyte marker TYRP1 in mel-WTand mel-NF1 cells. (Scale bar: 100 μm.) (B) Western blot analysis of neuro-fibromin expression in mel-WT and mel-NF1 cells. β-tubulin served as aloading control. Densitometry measurement of protein levels was relative tocontrol mel-WT-1 cells. Data are presented as mean ± SD (n = 3) normalizedto the expression in WT-1 cells. (C) Automated quantification at differenttime points after plating of DAPI nuclear staining in mel-WT and mel-NF1cells. Results are expressed as mean ± SD (n = 3). ***P < 0.001, ANOVAfollowed by Dunnett’s multiple-comparison test with WT-1.

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Fig. 2. Phenotypic changes in melanocytes associated with NF1 mutation.(A) Representative cell lysates from mel-WT and mel-NF1 cells. (B) Quanti-fication of melanin cell content by spectrophotometry in mel-WT and mel-NF1 cells. Measurements were performed using 105 cells of each cell type.Results are expressed as mean ± SD (n = 3). (C) Western blot analysis of MITF,tyrosinase, and DCT expression in mel-WT and mel-NF1 cells. β-actin served asa loading control. Densitometry measurement of protein levels is presentedas mean ± SD (n = 3) normalized to the expression in WT-1 cells. (D) Rep-resentative EM images of melanosome stages in mel-WT and mel-NF1 cells.Characteristic immature unpigmented (stage I/II) and mature pigmented(stage III/IV) melanosomes are observed in the soma of melanocytes. (Scalebar: 1 μm.) (E) Quantification of melanosome maturation in mel-WT-2 andmel-NF1-2 cell lines. Data are presented as mean ± SD (n = 100 stages ofmelanosomes). *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA followed byDunnett’s multiple-comparison test with WT-1 cells.

Allouche et al. PNAS | July 21, 2015 | vol. 112 | no. 29 | 9035

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For this purpose, we performed automated quantification ofDAPI nuclei stained at different time points after plating of mel-WT and mel-NF1 cells. Mel-NF1 cells proliferated more activelyby day 4 compared with mel-WT cells (Fig. 1C). Flow cytometryanalysis of cell cycle progression at day 4 after EdU incorporationconfirmed a greater number of replicative mel-NF1 cells at theS/G2M stage than in mel-WT cell lines (Fig. S2). Taken together,our results indicate that down-expression of neurofibromin doesnot interfere with the capacity of hESCs to differentiate intomelanocytes; however, the protein might be involved in regulatingthe proliferative properties of these melanocytes.

NF1 hESCs-Derived Melanocytes Phenocopied the HyperpigmentationPhenotype Associated with NF1. We next sought to analyze theconsequences of neurofibromin down-expression on melano-genesis in hESCs-derived melanocytes. Interestingly, cell lysatesfrom mel-NF1 were dark brown in color, whereas mel-WT lysateswere much lighter, suggesting a hyperpigmentation defect analo-gous to that observed in patients with NF1 (Fig. 2A). To confirmthis observation, we quantified melanin content by spectropho-tometry. A twofold increase in intracellular melanin content wasdetected in mel-NF1 cells relative to control cells (Fig. 2B). Con-sistent with these results, Western blot analysis showed greater ex-pression in mel-NF1 cells than in control mel-WT cells of the keytranscription factor MITF and of melanogenic enzymes such astyrosinase and dopachrome tautomerase (DCT) (Fig. 2C).Given the increased melanin content observed in mel-NF1 cells,

we used electron microscopy (EM) to analyze the biogenesis andmaturation of melanosomes (the pigment granules of melanocytes)at the ultrastructural level. Melanosome biogenesis occurred in twosteps, corresponding to four morphologically distinct melanosomalstages. Unpigmented immature melanosomes (stages I and II) weregenerated, and then acquired melanin pigment in their lumensto form dark (stage III) and fully mature pigmented (stage IV)melanosomes (28). Mel-NF1 cells contained more numerous darkpigmented granules (stages III and IV) compared with mel-WTcells, suggesting that the maturation of melanosomes is increased bytheNF1mutation. Indeed, more mature melanosomes accumulatedin mel-NF1 cells than in mel-WT cells (Fig. 2D). The proportion ofstage III/IV melanosomes reached 60% in NF1 mutant cells,compared with 10% in WT cells (Fig. 2E). These results confirmethe contribution of neurofibromin to melanogenesis in human cells,and also highlight a possible role for this protein in melanin bio-synthesis and melanosome maturation.To confirm a direct involvement of neurofibromin down-expres-

sion in the hyperpigmentation phenotype, we transfected mel-WTcells with three distinct siRNAs targeting the NF1 gene. Decreasedexpression of NF1 was confirmed at both the RNA and protein levels(Fig. 3 A and B and Fig. S3 A and B). At the functional level, down-expression of neurofibromin in mel-WT cells resulted in increasedmelanin content (Fig. 3C and Fig. S3C), as well as greater tyrosinaseexpression (Fig. 3D and Fig. S3D). EM analysis revealed a qualitativeand quantitative increase in dark-pigmented granules (stages III andIV) in siRNA targeting NF1 (siNF1)-mel-WT cells compared withcontrol siRNA (siCtrl)-mel-WT cells (Fig. S3 E and F). Automatedquantification of DAPI nuclei stained at 8 days after plating ofsiNF1-mel-WT and siCtrl-mel-WT cells revealed that siNF1-mel-WTcells proliferated more actively by day 8 (Fig. S4A). Flow cytometryanalysis of cell cycle progression at day 4 after 5-ethynyl-2′-deoxy-uridine (EdU) incorporation confirmed an increase of replicativemel-WT cells transfected with siNF1 at the S stage compared withcells treated with control siRNAs (Fig. S4B). These results suggestthat down-regulation of neurofibromin expression in mel-WT cells issufficient to result in pathological phenotypes analogous to thoseobserved in mel-NF1 cells.We next asked whether complete depletion in neurofibromin

might be associated with pigmentation defects such as thoseobserved in CALMs from patients with NF1. For this purpose,mel-NF1 cells were transiently transfected with specific siRNAstargeting neurofibromin expression still present in these mutantcells. qRT-PCR and Western blot analysis confirmed the reduction

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Fig. 3. Neurofibromin depletion with siRNA in mel-WT and mel-NF1 cells.(A) qRT-PCR analysis of NF1 transcript expression in mel-WT-1 and mel-WT-2 cells transfected with either siRNA targeting NF1 (siNF1) or control siRNA(siCtrl). qRT-PCR levels are presented as fold change relative to siCtrl-mel-WT cells. Results are expressed as mean ± SD (n = 3). (B) Western blotanalysis of neurofibromin expression in mel-WT-1 and mel-WT-2 cellstransfected with siNF1 or siCtrl. β-tubulin served as a loading control (n = 1).(C) Melanin content in siCtrl-mel-WT-1 and WT-2 and siNF1-mel-WT-1 andWT-2 cells. Measurements were performed using 105 cells of each cell type.Data are presented as mean ± SD (n = 3) normalized to the expression insiCtrl-mel-WT-1 cells. (D) Western blot analysis of tyrosinase expression insiNF1-mel-WT-1 and WT-2 and siCtrl-mel-WT-1 and WT-2 cells. β actin servedas a loading control. Densitometry measurement of protein levels was rel-ative to siCtrl-mel-WT-1 cells. Results are expressed as mean ± SD (n = 3).(E) qRT-PCR analysis of NF1 transcript expression in mel-NF1-1 and NF1-2 cellstransfected with siNF1 and siCtrl. qRT-PCR values are presented as foldchange relative to siCtrl-mel-NF1 cells. Results are expressed as mean ± SD(n = 3). (F) Western blot analysis of neurofibromin expression in mel-NF1-1and mel-NF1-2 cells transfected with siCtrl or siNF1. β-tubulin served as aloading control (n = 1). (G) Melanin content in siCtrl-mel-NF1-1 and NF1-2and siNF1-mel-NF1-1 and NF1-2 cells. Measurements were performed using105 cells of each cell type. The results are expressed as a relative level nor-malized to siCtrl-mel-NF1-1 cells. Data are presented as mean ± SD (n = 3).(H) Western blot analysis of tyrosinase expression in siCtrl-mel-NF1and siNF1-mel-NF1 cells. β-actin served as a loading control. Results are expressed asmean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA followed byDunnett’s multiple-comparison test with siCtrl-mel-WT cells (A, C, and D) orsiCtrl-mel-NF1-1 cells (E, G, and H).

9036 | www.pnas.org/cgi/doi/10.1073/pnas.1501032112 Allouche et al.

of neurofibromin in siRNA-treated mel-NF1 cells compared withcontrol mel-NF1 cells (Fig. 3 E and F). Interestingly, NF1 de-pletion led to increases in intracellular melanin content as wellas tyrosinase expression (Fig. 3 G and H), confirming the in-volvement of neurofibromin in the regulation of melanogenesisin human melanocytes. No significant effect of NF1-targetedsiRNA transfection was observed in mel-NF1 cells on cell prolife-ration (Fig. S4C). We then performed a comparative analysis of theintracellular melanin content in mel-WT, siNF1-mel-WT, mel-NF1,and siNF1-mel-NF1 cells. As expected, the down-expression ofneurofibromin in mel-WT cells led to a similar level of melanincontent as was observed in mel-NF1 cells (Fig. S5). In addition,the loss of neurofibromin induced by the NF1 siRNA treatment inmel-NF1 cells exacerbated the increased melanin content (Fig. S5).Our results also suggest the possibility of using hESCs-derivedmelanocytes to reproduce the effect of gradient neurofibrominexpression on the hyperpigmentation phenotype.

NF1-Related Alteration of ERK1/2 and cAMP Signaling Pathways. Todecipher the molecular mechanisms linking down-expression ofneurofibromin with the hyperpigmentation phenotype, we focusedour attention on a possible role of cAMP-mediated PKA and ERKsignaling pathways, which have been shown to be misregulated invarious NF1 models (10, 29, 30). ELISA revealed a fourfold in-crease in cAMP levels in mel-NF1 cells compared with mel-WTcells (Fig. 4A). Analysis of posttranslational activation of MAPK byphosphorylation disclosed major differences between mel-NF1 andmel-WT cells. Mel-NF1 cells showed a twofold increase in pERKlevel compared with mel-WT cell lines (Fig. 4B). To correlate thesemodifications with deregulation of neurofibromin expression, wefurther down-expressed neurofibromin in mel-WT cells. This led toan increase in cAMP activity and activation of the ERK signalingpathway (Fig. 4 C and D and Fig. S3 G and H). Consistent withthese results, NF1 depletion in mel-NF1 cells also resulted in afourfold increase in cAMP intracellular content compared withcontrol cells (Fig. 4E).We then investigated whether the defective melanogenesis ob-

served in NF1 hESCs-derived melanocytes could be rescued bypharmacologic modification of cAMP-mediated PKA and ERKsignaling pathways. For this, we treated mel-NF1 cells with specificinhibitors of cAMP-mediated PKA, MEK, and tyrosinase activity.The MEK inhibitor PD032059 induced an obvious decrease in theexpression of phospho-ERK (Fig. 5A), correlated with decreases intyrosinase and DCT cell protein content (Fig. 5B). We obtained asimilar result using the PKA-cAMP pathway inhibitor HA1004.Treating mel-NF1 cells with HA1004 resulted in decreased cAMPcontent (Fig. 5C), which in turn led to a reduction in melanincontent (Fig. 5D), as well as down-regulated expression of key en-zymes involved in melanogenesis, such as tyrosinase and DCT (Fig.5E). We also evaluated the effect of kojic acid, a specific inhibitorof tyrosinase activity. As expected, treatment with kojic acid led toa reduction in melanin content (Fig. 5F). Consistent with thesefindings, Western blot analysis showed that the decrease in tyrosi-nase and DCT expression was correlated with the concentration ofkojic acid (Fig. 5G). All of the foregoing results demonstrate thatdecreased expression of neurofibromin affects melanogenesis,probably via its action on ERK and cAMP signaling pathways. In-terestingly, our results also demonstrate that it is possible to blockthese cascades of events pharmacologically.

DiscussionThe main finding of this study is the demonstration that skindefects associated with NF1 can be reliably modeled in vitro usingmelanocytes derived from hESCs. This has allowed us to definethe molecular mechanisms responsible for hyperpigmentation andCALMs. These mechanisms involve activation of the cAMP-mediated PKA and ERK1/2 signaling pathways, which affects theexpression of major proteins involved in melanin biosynthesis andpigmentation of melanosomes. Our results also suggest that tar-geting these proteins may have clinical significance in diminishingthe hyperpigmentation associated with CALMs.

Melanocytes Derived from Mutant NF1-Carrying hESCs Replicate thePathological Phenotypes in Vitro. Few previous studies have ana-lyzed the effects of the NF1 mutation on melanogenesis. Untilnow, only Kaufman et al. (15) had demonstrated a higher mel-anin content in human melanocytes obtained from biopsy spec-imens of CALMs and other areas of skin from patients with NF1compared with melanocytes from healthy donors. Interestingly,those authors correlated this phenotype with increased tyrosinehydroxylase activity (15). Owing to the limited accessibility ofhuman NF1 melanocytes, these results have not been confirmed,however. Although pigment abnormalities could not be dem-onstrated in Nf1+/− mice, cultured melanocytes from these micehave shown increased expression of Mitf, Tyrosinase (Tyr),Tyrp1, and Dct at the RNA level (31). The experimental setup

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Fig. 4. Impact of neurofibromin expression in cAMP and ERK1/2 signal-ing pathways in melanocytes. (A) Direct ELISA analysis of cAMP contentin mel-WT and mel-NF1 cells. Results are expressed as mean ± SD (n = 3).Measurements were performed using 105 cells of each cell type. The re-sults were normalized to siCtrl-mel-WT-1 cells. (B) (Upper) Western blotanalysis of Phospho-p44/42 MAPK (ERK1/2) in mel-NF1 cells comparedwith mel-WT cells. Total ERK served as a control. (Lower) Densitometrymeasurement of protein levels relative to control mel-WT-1 cells. Resultsare expressed as mean ± SD (n = 3). (C ) Direct ELISA analysis of cAMPcontent in siCtrl-mel WT-1 and WT-2 and siNF1-mel WT-1 and WT-2 cells.Measurements were performed using 105 cells of each cell type. Results werenormalized to siCtrl-mel WT-1 cells and are presented as mean ± SD (n = 3).(D) Western blot analysis of Phospho-p44/42 ERK1/2 in siCtrl-mel WT-1 andWT-2 and siNF1-mel WT-1 and WT-2 cells. Total ERK served as a control (n = 1).(E) Direct ELISA analysis of cAMP content in siCtrl-mel NF1-1 and NF1-2 andsiNF1-mel NF1-1 and NF1-2 cells. Measurements were performed using 105

cells of each cell type. The results were normalized to siCtrl-mel NF1-1. Resultsare expressed as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ANOVA followedby Dunnett’s multiple-comparison test compared with WT-1 (A and B), siCtrl-mel WT-1 (C), or siCtrl-mel NF1-1 (E).

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used in the present study allowed us to overcome some of thetechnical problems encountered by others, making it possible toexplore homogeneous proliferative populations of human me-lanocytes in amounts as large as required by each analysis.Our first and most important result is that melanocytes derived

from hESCs carrying a heterozygous NF1 mutation repro-duced the generalized hyperpigmentation phenotype observed inpatients with NF1. The increased melanin content associated withup-regulation of MITF, TYR, and DCT in mel-NF1 cells is con-sistent with the observations reported in Nf1+/− mice. Confirmingthe direct implication of neurofibromin in the development ofthese phenotypes, NF1 knockdown in mel-WT cells led to asimilar phenotype. Moreover, it was possible to demonstrate agene dosage effect; the knockdown neurofibromin expression inmel-NF1 cells led to significant exacerbation of the already hy-perpigmented cell phenotype, confirming that CALMs are directly

related to the spontaneous loss of heterozygosity in discrete pop-ulations of melanocytes in patients with NF1.To date, only a few studies have described the consequences of

the NF1 gene defect on the size and distribution of melanosomes(32, 33). It has been shown that melanocytes from some patientswith NF1 contain giant pigment granules known as macro-melanosomes in both CALMs and non–CALM-derived melano-cytes (34). More recently, it was reported that a loss ofneurofibromin expression induced by shRNA in human primarycultures of melanocytes derived from healthy patients led to aber-rant subcellular localization of melanosomes (32). Our study high-lights the functional consequences of decreased neurofibrominexpression on melanosome maturation. Hyperpigmented NF1-hESCs–derived melanocytes were characterized by a very significantshift in the four different stages of maturation, involving repartitionof melanosomes toward the most mature stages (III and IV). Takentogether, these results provide important insights into the possiblefunction of NF1 during the maturation, transport, and distributionof melanosomes; however, further studies are needed to determinethe exact contribution of NF1 to these processes.

NF1-Induced Hyperactivity of cAMP-Mediated PKA and ERK1/2Signaling Pathways Is Involved in Overexpression of MelanogenicEnzymes. So far, the functional consequences of the down-regu-lation of neurofibromin expression have been explored mainly inSchwann cells and central nervous system neurons. Those studiespointed to misregulation of several important downstream sig-naling pathways, including the MEK/MAPK and cAMP-medi-ated PKA pathways; however, depending on the cell typeanalyzed, either up- or down-regulation of cAMP content wascorrelated with decreased expression of neurofibromin. Thus,Schwann cells retrieved from neurofibromas exhibited increasedcAMP levels (30, 35). Activation of MEK/MAPK was also ob-served in the peripheral nerve sheath in one mouse model (36).Conversely, Nf1+/− heterozygous mice showed decreased cAMPlevels in astrocytes (37, 38) and central nervous system neurons(39). Recently, Anastasaki and Gutmann (40) analyzed neuronsderived from induced pluripotent stem cells from patients withNF1 and proposed a way to reconcile those conflicting results byshowing that in neurons, decreased neurofibromin expression ledto lower rather than higher cAMP levels. Our results indicatethat in melanocytes, decreased expression of neurofibromin onsignaling pathways is characterized by an increase in the MEK/MAPK and cAMP-mediated PKA pathways, as observed inSchwann cells. Therefore, our results strongly suggest that NF1mutation might affect melanocytes and Schwann cells throughsimilar metabolic pathways, raising the possibility that a commontherapeutic strategy could be developed to address the dys-function of these two cell types in patients with NF1.Activation of P38-MAPK and cAMP enhances MITF expres-

sion and melanin production via phosphorylation of the cAMP-responsive element-binding protein (CREB) (41). As observedwith the cAMP pathway, activation of the MAPK signalingpathways may lead to either up- or down-regulation of melano-genesis, depending on the cell type analyzed (42, 43). Diwakaret al. (31) reported opposite effects of MEK inhibition, leading todecreased melanogenic gene expression in primary murine mela-nocytes and increased gene expression in their immortalizedcounterparts, melan-a cells. In the present study, we have dem-onstrated that in mel-NF1 cells, the MEK inhibitor PD032059 andthe PKA-cAMP pathway inhibitor HA1004 induced marked de-creases in levels of phospho-ERK and cAMP, respectively, inparallel with the down-regulation of tyrosinase and DCT expres-sion expected in human primary melanocytes. Identification ofthese molecular cascades responsible for the phenotypic alter-ations that mimic those observed in patients with NF1 (Fig. S6)enabled us to target them with specific inhibitors, resulting in theexpected functional recovery. We obtained similar results usingkojic acid, a specific inhibitor of tyrosinase. Taken together,our results highlight the therapeutic potential of human plu-ripotent stem cell-derived melanocytes to identify or validate new

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Fig. 5. Effects in mel-NF1 cells of specific inhibitors targeting downstreamsignaling pathways of neurofibromin on melanogenesis. (A) Western blotanalysis of phospho-p44/42 MAPK (ERK1/2) after PD0325901 treatment inmel-NF1-1 and NF1-2 cells. Total ERK served as a control (n = 2). (B) Western blotanalysis of tyrosinase and DCT expression after PD0325901 treatment in mel-NF1-1 and NF1-2 cells. β-actin served as a loading control (n = 2). (C) Direct ELISAanalysis of cAMP content in mel-NF1-1 and NF1-2 cells after HA1004 treatment.Measurements were performed using 105 cells of each cell type. Results werenormalized to mel-NF1 control and are expressed as mean ± SD (n = 3).(D) Melanin content in mel-NF1-1 and NF1-2 cells after HA1004 treatment. Mea-surements were performed using 105 cells of each cell type. Results were nor-malized to mel-NF1 control and are expressed as mean ± SD (n = 3). (E) Westernblot analysis of tyrosinase and DCT expression after HA1004 treatment in mel-NF1-1 and NF1-2 cells. β actin served as a loading control. (F) Melanin contentafter treatment of mel-NF1-1 and mel-NF1-2 cells with various concentrations ofkojic acid. Results are expressed as mean ± SD (n = 3). (G) Western blot analysis oftyrosinase and DCT expression after treatment with various concentrationsof kojic acid in mel-NF1-1 and mel-NF1-2 cells. β-actin served as a loading control(n = 1). *P < 0.05, ***P < 0.001, ANOVA followed by Dunnett’s multiple-com-parison test with NF1-1 control or NF1-2 control cells (C, D, and F).

9038 | www.pnas.org/cgi/doi/10.1073/pnas.1501032112 Allouche et al.

pharmacologic approaches targeting hyperpigmentation pheno-types. Although the hyperpigmentation associated with CALMs isnot life-threatening, it does impair the quality of life of affectedpatients (5, 6); thus, further studies involving topical application ofa specific tyrosinase inhibitor may be clinically relevant.

MethodsMelanocyte Differentiation. hESCs were differentiated into melanocytes asdescribed previously (20).

Measurement of Melanin Content. Melanocytes were treated for 7 d with 1 μMalpha-Melanocyte Stimulating Hormone αMSH and 100 μM 3-Isobutyl-1-meth-ylxanthine (IBMX) after seeding. Then 100,000 cells were lysed in 1 M NaOH(Sigma-Aldrich) and centrifuged. Absorbance of supernatants was measured at405 nm in 96-well microplates. A standard synthetic melanin curve (0–50 μg/mL;Sigma-Aldrich) was performed in triplicate for each experiment.

Determination of Intracellular cAMP Content. cAMP content was measured byELISA (ADI-901-066 direct cAMP ELISA kit; Enzolife). Melanocytes were treated

with 1 μM αMSH and 100 μM IBMX for 60 min at 37 °C. cAMP was quantifiedon 100,000 cells and on the basis of a standard curve.

The methodology is described in more detail in SI Methods.

ACKNOWLEDGMENTS. We thank S. Viville and Dr. P. Tropel for providinghESCs; Dr. Y. Laabi for hESCs banking; D. Vidaud and Dr. B. Parfait for hESCssequencing; Dr. A. Benchoua for providing HA1004; Dr. L. Larribere forparticipating in preliminary experiments; and S. Julie, A. Alquezar, andB. Champon for technical assistance. I-Stem is part of the BiotherapiesInstitute for Rare Diseases, supported by the Association Française contre lesMyopathies. This work was supported by the Institut National de la Santé etde la Recherche Medicale, University Evry Val d’Essonne, Genopole, KokcineloAssociation for Neurofibromatosis Type 1, Domaine d’Intérêt Majeur (DIM)Biothérapies, Institut Curie, Laboratoire d’Excellence “Labex Revive” (Inves-tissement d’Avenir; ANR-10-LABX-73), and the program INGESTEM (inves-tissement d’avenir ANR-11-INBS-009-01). We acknowledge the Plateformed’Imagerie Cellulaire et Tissulaire-Infrastructure en Biologie Santé et Agronomie,BioImaging Cell and Tissue Core Facility of the Institut Curie (PICT-IBiSA), mem-ber of the France BioImaging national research infrastructure, supportedby the CelTisPhyBio Labex (ANR-10-LBX-0038) part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL).

1. Trovó-Marqui AB, Tajara EH (2006) Neurofibromin: A general outlook. Clin Genet70(1):1–13.

2. Maertens O, et al. (2007) Molecular dissection of isolated disease features in mosaicneurofibromatosis type 1. Am J Hum Genet 81(2):243–251.

3. De Schepper S, Boucneau J, Lambert J, Messiaen L, Naeyaert J-M (2005) Pigment cell-related manifestations in neurofibromatosis type 1: An overview. Pigment Cell Res18(1):13–24.

4. Gutmann DH, et al. (1997) The diagnostic evaluation and multidisciplinary manage-ment of neurofibromatosis 1 and neurofibromatosis 2. JAMA 278(1):51–57.

5. Wolkenstein P, Zeller J, Revuz J, Ecosse E, Leplège A (2001) Quality-of-life impairmentin neurofibromatosis type 1: A cross-sectional study of 128 cases. Arch Dermatol137(11):1421–1425.

6. Ablon J (1996) Gender response to neurofibromatosis 1. Soc Sci Med 42(1):99–109.7. Abramowicz A, Gos M (2014) Neurofibromin in neurofibromatosis type 1 mutations in

NF1gene as a cause of disease. Dev Period Med 18(3):297–306.8. Fountain JW, et al. (1989) Physical mapping of the von Recklinghausen neurofibro-

matosis region on chromosome 17. Am J Hum Genet 44(1):58–67.9. Marchuk DA, et al. (1991) cDNA cloning of the type 1 neurofibromatosis gene:

Complete sequence of the NF1 gene product. Genomics 11(4):931–940.10. Lau N, et al. (2000) Loss of neurofibromin is associated with activation of RAS/MAPK

and PI3-K/AKT signaling in a neurofibromatosis 1 astrocytoma. J Neuropathol ExpNeurol 59(9):759–767.

11. Tong J, Hannan F, Zhu Y, Bernards A, Zhong Y (2002) Neurofibromin regulates Gprotein-stimulated adenylyl cyclase activity. Nat Neurosci 5(2):95–96.

12. Johnson BL, Charneco DR (1970) Café au lait spot in neurofibromatosis and in normalindividuals. Arch Dermatol 102(4):442–446.

13. Jimbow K, Szabo G, Fitzpatrick TB (1973) Ultrastructure of giant pigment granules(macromelanosomes) in the cutaneous pigmented macules of neurofibromatosis.J Invest Dermatol 61(5):300–309.

14. Jimbow K, Horikoshi T (1982) The nature and significance of macromelanosomes inpigmented skin lesions: Their morphological characteristics, specificity for their oc-currence, and possible mechanisms for their formation. Am J Dermatopathol 4(5):413–420.

15. Kaufmann D, Wiandt S, Veser J, Krone W (1991) Increased melanogenesis in culturedepidermal melanocytes from patients with neurofibromatosis 1 (NF 1). Hum Genet87(2):144–150.

16. Lakkis MM, Epstein JA (1998) Neurofibromin modulation of ras activity is required fornormal endocardial-mesenchymal transformation in the developing heart. De-velopment 125(22):4359–4367.

17. Brannan CI, et al. (1994) Targeted disruption of the neurofibromatosis type-1 geneleads to developmental abnormalities in heart and various neural crest-derived tis-sues. Genes Dev 8(9):1019–1029.

18. Jacks T, et al. (1994) Tumour predisposition in mice heterozygous for a targetedmutation in Nf1. Nat Genet 7(3):353–361.

19. Deo M, Huang JL-Y, Fuchs H, de Angelis MH, Van Raamsdonk CD (2013) Differentialeffects of neurofibromin gene dosage on melanocyte development. J Invest Dermatol133(1):49–58.

20. Nissan X, et al. (2011) Functional melanocytes derived from human pluripotent stemcells engraft into pluristratified epidermis. Proc Natl Acad Sci USA 108(36):14861–14866.

21. Mica Y, Lee G, Chambers SM, Tomishima MJ, Studer L (2013) Modeling neural crestinduction, melanocyte specification, and disease-related pigmentation defects inhESCs and patient-specific iPSCs. Cell Reports 3(4):1140–1152.

22. Carvajal-Vergara X, et al. (2010) Patient-specific induced pluripotent stem cell-derivedmodels of LEOPARD syndrome. Nature 465(7299):808–812.

23. Zhang J, et al. (2011) A human iPSC model of Hutchinson Gilford progeria revealsvascular smooth muscle and mesenchymal stem cell defects. Cell Stem Cell 8(1):31–45.

24. Marteyn A, et al. (2011) Mutant human embryonic stem cells reveal neurite andsynapse formation defects in type 1 myotonic dystrophy. Cell Stem Cell 8(4):434–444.

25. Feyeux M, et al. (2012) Early transcriptional changes linked to naturally occurringHuntington’s disease mutations in neural derivatives of human embryonic stem cells.Hum Mol Genet 21(17):3883–3895.

26. Telias M, Ben-Yosef D (2014) Modeling neurodevelopmental disorders using humanpluripotent stem cells. Stem Cell Rev 10(4):494–511.

27. Luo G, Kim J, Song K (2014) The C-terminal domains of human neurofibromin and itsbudding yeast homologs Ira1 and Ira2 regulate the metaphase to anaphase transi-tion. Cell Cycle 13(17):2780–2789.

28. Raposo G, Marks MS (2007) Melanosomes—dark organelles enlighten endosomalmembrane transport. Nat Rev Mol Cell Biol 8(10):786–797.

29. Sharma R, et al. (2013) Hyperactive Ras/MAPK signaling is critical for tibial nonunionfracture in neurofibromin-deficient mice. Hum Mol Genet 22(23):4818–4828.

30. Dang I, De Vries GH (2011) Aberrant cAMP metabolism in NF1 malignant peripheralnerve sheath tumor cells. Neurochem Res 36(9):1697–1705.

31. Diwakar G, Zhang D, Jiang S, Hornyak TJ (2008) Neurofibromin as a regulator ofmelanocyte development and differentiation. J Cell Sci 121(Pt 2):167–177.

32. De Schepper S, et al. (2006) Neurofibromatosis type 1 protein and amyloid precursorprotein interact in normal human melanocytes and colocalize with melanosomes.J Invest Dermatol 126(3):653–659.

33. Arun V, Worrell L, Wiley JC, Kaplan DR, Guha A (2013) Neurofibromin interacts withthe cytoplasmic Dynein Heavy Chain 1 in melanosomes of human melanocytes. FEBSLett 587(10):1466–1473.

34. Martuza RL, et al. (1985) Melanin macroglobules as a cellular marker of neurofibro-matosis: A quantitative study. J Invest Dermatol 85(4):347–350.

35. Kim HA, Ratner N, Roberts TM, Stiles CD (2001) Schwann cell proliferative responsesto cAMP and Nf1 are mediated by cyclin D1. J Neurosci 21(4):1110–1116.

36. Jessen WJ, et al. (2013) MEK inhibition exhibits efficacy in human and mouse neu-rofibromatosis tumors. J Clin Invest 123(1):340–347.

37. Dasgupta B, Dugan LL, Gutmann DH (2003) The neurofibromatosis 1 gene productneurofibromin regulates pituitary adenylate cyclase-activating polypeptide-mediatedsignaling in astrocytes. J Neurosci 23(26):8949–8954.

38. Warrington NM, et al. (2007) Spatiotemporal differences in CXCL12 expression andcyclic AMP underlie the unique pattern of optic glioma growth in neurofibromatosistype 1. Cancer Res 67(18):8588–8595.

39. Brown JA, Diggs-Andrews KA, Gianino SM, Gutmann DH (2012) Neurofibromatosis-1heterozygosity impairs CNS neuronal morphology in a cAMP/PKA/ROCK-dependentmanner. Mol Cell Neurosci 49(1):13–22.

40. Anastasaki C, Gutmann DH (2014) Neuronal NF1/RAS regulation of cyclic AMP re-quires atypical PKC activation. Hum Mol Genet 23(25):6712–6721.

41. Ahn JH, Jin SH, Kang HY (2008) LPS induces melanogenesis through p38 MAPK acti-vation in human melanocytes. Arch Dermatol Res 300(6):325–329.

42. Park H-Y, et al. (2009) Role of BMP-4 and its signaling pathways in cultured humanmelanocytes. Int J Cell Biol 2009:750482.

43. Englaro W, et al. (1998) Inhibition of the mitogen-activated protein kinase pathwaytriggers B16 melanoma cell differentiation. J Biol Chem 273(16):9966–9970.

44. Tropel P, et al. (2010) High-efficiency derivation of human embryonic stem cell linesfollowing pre-implantation genetic diagnosis. In Vitro Cell Dev Biol Anim 46(3-4):376–385.

45. Delevoye C, et al. (2009) AP-1 and KIF13A coordinate endosomal sorting and posi-tioning during melanosome biogenesis. J Cell Biol 187(2):247–64.

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