a, b-immnunostainings of lcmn and SSM tissue for HMB45 (A, left panel) and CD44 (B, left panel) using

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1 Figure S1 a HMB45 HMB45 + cells (% of total cells) CD44 CD44 + cells (% of total cells) In vivo characterization of. a, -Immnunostainings of and tissue for HMB45 (A, left panel) and CD44 (B, left panel) using the chromogen AEC for staining. The percentage of positive cells is represented in the histograms (right panel). Scale ar = 1 µm. Bars: SEM. **p.1 in Student s T-test.

2 Figure S2 a IgG-APC CD271-APC IgG-FITC ABCB5-FITC 5 % of laeled cells ABCB5 CD166 CD271 CD133 FACS analysis of. Flow cytometry analysis of cells laelled with different cell surface markers: ABCB5-FITC, CD271- APC, CD166-PE and CD133-PE. a-endothelial cells and leukocytes were excluded from analysis y eliminating CD31 + CD45 + cells. Isotype control dot plot is shown on the left panel. ABCB5-FITC and CD271-APC dot plot is shown on the right panel. -Histograms showing the percentage of cells expressing each marker (n=5 specimens analyzed y FACS).

3 Figure S3 a Prim mel Mel 51 Prim mel Mel 51 N2 B27 B27i DIV7 DIV13 Diameter of colonies (µm) Numer of colonies /.25 mm *** *** *** Mel51 Prim mel DIV 7 DIV 13 N2 B27 B27i ****** *** Mel51 Prim mel *** *** *** *** *** Mel51 Prim mel Mel51 Prim mel DIV 7 DIV 13 c numer of colonies /.25 mm DIV4 DIV7 DIV13 colnies diameter (µm) *** *** DIV4 DIV7 DIV13 numer of cells / colonies *** *** DIV4 DIV7 DIV13 In vitro characterization of initiating cells. a-photographs y optical microscopy of colonies were taken on DIV7 and 13 in the 3 different media tested : N2, B27 and B27+insulin. Scale ar = 1 µm. -Numer and diameter measurement of colonies for Mel51, cell suspensions of primary melanomas (Prim Mel) and in the 3 different media on DIV7 and 13 (n=5). represents the difference etween Mel51 and and * etween Prim mel and. ***, p.1 and p.1 in Student s T-test. c- numers of colonies, diameters and numer of cells per colonies at DIV4, 7 and 13. n=5 specimens. * represents the difference etween DIV in the same group, Bars: SEM. ***p.1 in Student s T-test.

4 Figure S4 a Melan-A / DAPI Nestin Melan-A + cells (% of total cells) Non sorted Sorted CD31 - CD45 - c In vitro characterization of initiating cells. a-immunocytochemistry staining of adherent cells with anti-melan-a and anti-nestin antiodies. Nestin + cells are stained in red with AEC chromogen. Melan-A + cells are stained in green y Alexa 488 conjugated secondary antiody. Scale ar = 1 µm. -Anti-Melan-A immunofluorescence staining was performed on unsorted and sorted CD31/CD45 - fresh cell suspensions. The percentage of Melan-A + cells was evaluated. c-photographs y optical microscopy of cells plated in standard 254CF melanocyte medium in soft agar at DIV7. No colonies had developed. Scale ar = 1 µm.

5 Figure S5 a 6 BrdU / Sox1 + cells (% of Sox1 + cells) 4 2 Grafted nevus Grafted neoepi. c Ki67 Melanocytes Melanocytes + HaCat In vivo xenotransplantation of cells in immunocompromised mice. Human HaCaT keratinocytes were mixed in Matrigel TM with CD31 - /CD45 - cell suspension (at a ratio of 1 melanocyte for 2 keratinocytes) and injected into the right flank of Rag2 -/- mice (n=4 injections from 4 specimens). a-3 days after BrdU injection, Brdu/Sox1 + cells were counted and reported to the numer of Sox1 + cells in order to evaluate the percentage of lael-retaining melanocytes, -Three months after xenotransplantation, a pigmented dark tumor mass developed in 2 of the 4 mice as shown in the photograph. c-immunohistochemistry of Matrigel TM containing melanocytes only or melanocytes mixed with HaCat, and stained with anti-ki67 antiody. Scale ar = 1 µm.

6 Supplementary Materials and methods Immunohistochemistry Formalin-fixed paraffin emedded CMN tissue sections (5 µm) were otained from the department of Pathology at Trousseau Hospital. Tissue sections were deparaffinized with xylene, rehydrated with grade ethanol concentrations and laeled with the following antiodies: Rait anti-human MITF (dilution 1:25, a2663, Acam, Camridge, UK), mouse anti-human Nestin (dilution 1:1, a2235, Acam), mouse anti-human HMB45 (prediluted, a51935, Acam), mouse anti-human KI67 (dilution 1:1, NCL-L-Ki67-MM1, Novocastra, Wetzlar, Germany), rait anti-human CD44 (dilution 1:1, a5137, Acam). Antigen retrieval was performed y incuating the slides with citrate uffer (ph=6) or EDTA uffer (ph=9) at 98 C for 2 min. Endogenous peroxidase activity was locked with 3 % H 2 O 2 for 1 min. Non specific antigen sites were locked in PBS with 3% BSA with 1 or 2 % serum goat. The sections were incuated with the different antiodies for 2h at 37 C or overnight. Staining was achieved using appropriate iotinylated goat anti-mouse or anti-rait secondary antiodies (BA92, BA1, Vector Laoratories, Burlingame, CA, USA) and Avidine Biotine complex (PK-61, Vector Laoratories). Bound antiodies were visualized using aminoethylcarazol (SK-425, Vector Laoratories) as chromogen. Nuclei were counterstained with Mayer s haematoxylin (TA-125-MH, Thermoscientific, Waltham, MA, USA). Negative controls were performed in parallel with the samples sustituting the primary antiody with the equivalent isotype. For immunofluorescence, slides were incuated with primary antiodies diluted in PBS with 3% BSA with or without serum goat (for Sox1) and.1% triton for intracellular staining for 2 hours. The following antiodies were used: rait anti-human Oct4 (dilution 1:1, a19857, Acam), mouse anti-human Melan-A (dilution 1:1, a3168, Acam), rait antihuman Sox1 (dilution 1:1, sc-17342, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rait anti-human ABCB5 (dilution 1:5, HPA26975, Sigma-Aldrich, Saint-Louis, MO, USA). Sections were incuated with primary antiodies diluted with locking solution overnight at 4 C, washed with PBS followed y secondary antiody goat anti-mouse Alexa 488 (dilution 1:1, A168, Life Technologies, Carlsad, CA, USA), or goat anti-rait Cy3 (dilution 1:1, A1522, Life Technologies) or chicken anti-goat Alexa 488 (dilution 1:1, A1155, Life Technologies) for 45 min at room temperature. Slides were also incuated with DAPI (D9542, Sigma-Aldrich) for 5 min for nuclei staining and coverslipped using Fluoromount G (1-1, Southern Biotech, Birmingham, AL, USA).

7 Stainings were analyzed on Nikon Eclipse 9i microscope (Nikon, Tokyo, Japan). Pictures were taken under x2 ojective. Using Image J software, positive cells were counted in 1 µm 2 randomized surface squares in oth epidermis and dermis of each specimen and reported to the numer of total cells. Quantitations were done on various parts of the and and always in areas within the lesions: these lesions were dissected in a gridded fashion and 3 different sectors were analyzed; for each different sector of the lesions, 3 different areas of 1 µm 2 were counted. In vitro assays For sphere formation assay, fresh nevocytic cell suspension deriving from CMN were seeded at a density of 5 cells per well in a 12-well plate in DMEM/F12 ( , Life Technologies) supplemented with either N2 supplement 1X ( , Life Technologies), with EGF (2ng/mL, PHG311, Life Technologies), FGF (2 ng/ml, PHG26, Life Technologies) or with B27 supplement ( , Life Technologies) with or without insulin (5 ug/ml, I , Sigma-Aldrich) plus EGF (2 ng/ml) and FGF (4 ng/ml). Primary melanoma cells or Mel51 melanoma cells were seeded at the same density in DMEM supplemented with 1% fetal ovine serum. The numer of colonies and the numer of cells per colony were counted. The diameter was also measured on day in vitro DIV4, DIV7 and DIV13. For serial passages and clonogenic assay, the culture medium was collected at DIV13, centrifuged at 5 rpm for 15 min, and then spheres were dissociated with pro-accutase (A1115, Life Technologies) 1 min at 37 C, then centrifuged at 7 rpm for 5 min. The pellet was mixed with 2 ml of B27+insulin and single cells were seeded again. For the clonogenic assay, cells were plated at a density of 1 cells per well in 24-well plates. For each passage, the diameter of spheres was measured on DIV7 and DIV13. For colony formation assay, the colony formation capaility of CMN cells in soft agar media was investigated. 5 single cell suspension of cells were re-suspended in 1,5mL B27+insulin media containing.35% low melting temperature agarose and were plated on 6- well plate over a asal layer of 3mL of B27+insulin media containing,6% low melting temperature agarose. The cells were cultured for 13 days, and colony formation was monitored and the diameters of colonies were measured on DIV3, 5, 7, 9 and 13 using Image J software. The numer and frequency of initiating cells in a given test cell population can e determined using limiting dilution analysis ased on Poisson statistics and the method of maximum likelihood if the numer of sphere colony produced per initiating cell is unknown for a given

8 cell sample (37). Here, CMN cells were seeded at ratios of 5, 1, 2, 5, 25, 1, 5 and 1 cells per wells. Using optical microscope (Olympus ULWCD.3, Tokyo, Japan), wells containing spheres were counted on DIV7 and 13. The frequency of initiating cells corresponding to 37% of negative wells according to the Poisson statistics was calculated with GraphPad Prism software starting from 5 cells. For immunocytochemistry, sphere colonies from CMN in B27+insuline were seeded on 12- well plate coated y Poly-D-Lysine (1ug/mL, P64-7, Sigma-Aldrich) for 2 hours, then fixed with paraformaldehyde for 15min. The following primary antiodies were used for staining: anti-melan-a (mouse anti-human, dilution 1:1, Acam), anti-oct4 (rait antihuman, dilution 1:5, Acam) and anti-nestin (mouse anti-human, dilution 1:1, Acam). Primary antiodies were applied overnight at 4 C. The following secondary antiodies were used: anti-mouse Alexa 488 and anti- rait Cy3. Nestin expression was visualized with Expose Mouse and Rait Specific HRP/AEC Detection Kit (Vector Laoratories). Nuclei were stained with DAPI and coverslips were mounted with Fluoromount G. Adherent cells were also stained using anti-melan-a and anti-nestin antiodies as previously descried. Cell preparation Fresh tissues were manually dissected into small pieces, efore sequential enzymatic digestion in Dispase II (2%, , Roche, Basel, Switzerland) overnight at 4 C then in Collagenase I (3.5%, , Life Technologies) for 1 hour at 37 C. Cells were filtered (using 1 µm and 4 µm cell strainers) to otain a single-cell suspension. Cell laeling and sorting All antiodies laelings were performed for 15 min at 4 C followed y washing and centrifugation. Secondary antiody was conjugated to Streptavidin PercP (55464, BD Pharmingen, San Diego, CA, USA). Primary isotype controls were used to set ackground. Cells were susequently stained with primary iotinylated antiodies to human CD31 (13-319, Eiosciences, San Diego, CA, USA) and CD45 (13-459, Eiosciences) for xenograft suspensions to exclude endothelial and hematopoietic cells. In Fig. 5, cells were also stained with primary CD271 Alexa 647 (56326, BD Pharmingen) and unconjugated ABCB5 (AP6122a, Agent, San Diego, CA, USA) antiodies and goat anti-rait Alexa 488 (A117, Life Technologies) as a secondary antiody. Cells were sorted using a LSRII (Life Technologies).

9 Flow cytometric analysis Analysis of ABCB5, CD133, CD166, CD271 surface markers in clinical patient-derived nevus cell suspension was performed y four color flow cytometry. Cells were susequently incuated with PE conjugated CD133/1 (AC133, , Miltenyi Biotec, Bergisch Gladach, Germany) or PE conjugated CD166 (559263, BD Pharmingen), Alexa 647 conjugated CD271 (56326, BD Pharmingen) and unconjugated ABCB5 (AP6122a, Agent) followed y counterstaining with Alexa 488 goat anti-rait secondary antiody (A117, Life Technologies). Primary isotype controls were used to set ackground. In vivo assays For transplantation of tissue, standardized specimens (3mm punch iopsies) were xenografted in rown adipose tissue in Rag2 -/- mice. Xenografts were collected after 3 or 7 months. Xenograft diameters were measured with a caliper, the grafted volume tumor was otained as V= Width (w) x Length (l) x Height (h). For transplantation of human nevocytic cells, Rag2 -/- mice were maintained under defined conditions. After sorting, CD31 - CD45 - cells were resuspended in Matrigel TM (354234, BD Pharmingen) either alone or mixed with human keratinocyte cell line HaCat at a ratio of 1:2, then injected sucutaneously into the flank of recipient Rag2 -/- mice. The xenografts were collected after 3 months or 7 months respectively. For the in vivo cell proliferation assay, xenografted mices were given a single i.p injection of.5mg BrdU (B52, Sigma-Aldrich) 3 min efore sacrifice. For the in vivo lael retaining (LR) assay, mices were given 1 ip injection of,5mg BrdU (2 injection /day) 1 month efore the sacrifice. BrdU laeling was detected y immunofluorescence on paraffin xenograft sections using anti-brdu antiody (dilution 1:5, a6326, Acam) and goat anti-rat Cy3 (dilution 1:1, A117, Life Technologies) as secondary antiody. The percentages of Brdu/Melan-A and BrdU/Sox1 positive cells were counted in 1 µm2 randomized surface squares in oth grafted nevus and grafted neo-epidermis using Image J software. Statistical analysis The data were analyzed using Unpaired Student s t-test. For all results, n indicates the numer of independent experiments performed. In all histograms, asterisks correspond to: *p.5, **p.5, ***p.1.