1. Clonogenic Cell Subpopulations Maintain Congenital Melanocytic Nevi
Christelle Charbel, Romain H. Fontaine, Natacha Kadlub, Aurore Coulomb-L’Hermine, Thomas Rouillé, Alexandre How-Kit, Philippe Moguelet, Jorg Tost, Arnaud Picard, Selim Aractingi, Sarah Guégan 
Journal of Investigative Dermatology 
Volume 135, Issue 3, Pages (March 2015)
DOI: /jid
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Human lCMN express initiating stem cell markers. Immunohistochemical stainings of superficial spreading melanoma (SSM) and human large congenital melanocytic nevi (lCMN): MITF (a), Sox10 (b), Nestin(c), Oct4/Melan-A (d), ABCB5/Melan-A (e), and KI67(f). MITF+, ABCB5+, KI67+ cells were stained with brown/red AEC chromogen. Melan-A+ and Sox10+ cells were stained using Alexa488 secondary antibody. Oct4+ cells were stained using Cy3 secondary antibody. Scale bar=100 μm. Histograms show the number of positive cells per total cells in both the epidermis and dermis. (lCMN n=5; melanoma, n=4). *Represents differences between SSM and lCMN. Bars: SEM. *P⩽0.05, **P⩽0.001, ***P⩽ in Student’s t-test. Red dots delineate the edge of the lesions.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Large congenital melanocytic nevi (lCMN) melanocytic cells are capable of forming sphere colonies and self-renew. (a) Photomicrographs (left panel) and histograms (right panel) of Mel501 melanoma and lCMN colonies on DIV7 and DIV13 (lCMN n=5). Scale bar=100 μm. § Represents the difference between DIV7 and 13 in the same group; * represents difference between Mel 501 and lCMN. Bars: SEM. §P⩽0.05, §§§P⩽0.0001, ***P⩽ in Student’s t-test. (b) Photomicrographs (top panel) and histograms (bottom panel) for lCMN at passage 3. Scale bar=100 μm. (c) Immunocytochemistry of lCMN spheres. Nestin+ cells (middle panel) were stained with AEC chromogen. Melan-A+ (left panel) and Oct4+ cells (right panel) were stained, respectively, with Alexa 488 and Cy3 secondary antibody. Scale bar=50 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
In vitro characterization of large congenital melanocytic nevi (lCMN)-initiating cells: percentage of initiating cells and the soft agar assay. (a) lCMN cells were plated with decreasing concentrations from 500–10 cells per well. Colony numbers were counted on DIV7 and 13 (n=3 specimens). 1/302- and 1/254-initiating cells were capable of forming colonies on DIV7 and 13, respectively. (b) Soft agar assay assessed on lCMN cells (n=5). Individually seeded lCMN cells were able to form colonies. Photomicrographs taken on DIV0 and DIV13 (left panel). Scale bar=100 μm. The histogram (right panel) represents the diameter of colonies measured from DIV3 to DIV13. (c) Clinical and mutational characteristics of 5 lCMN and derived colonies. 1PAS, projected adult size; 2pyrosequencing was used to screen NRAS exon 3 mutations.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
In vivo xenotransplantation of large congenital melanocytic nevi (lCMN) tissue in immunocompromised mice. Biopsies of lCMN grafted in Rag2-/- mice were collected after 3 or 7 months (n=8 grafted specimens). (a) 4-fold expansion of the tumors after 7 months. (b) Histology of initial nevus (‘pregraft’) and of whole-graft tissue 7 months post grafting with the formation of a neoepidermis (‘grafted neoepi.’). Scale bar=100 μm, except in postgraft where scale bar=1 mm. Immunofluorescence stainings of original graft (‘grafted nevus’) and graft neoepidermis (‘graft neo-epi.’): Melan-A (c), Sox10 (d) (Alexa 488-labeled secondary antibodies), and Brdu/Melan-A (e), Brdu/Sox10 (f) co-labeling 30min or 30 days after BrdU injection, respectively (Cy3-labeled secondary antibody). Melan-A+/BrdU+ or Sox10+/BrdU+ cells are indicated with arrows. Higher magnifications are shown for each staining. Histograms show the number of double-positive cells per total cells. Scale bar=100 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
In vivo xenotransplantation of large congenital melanocytic nevi (lCMN) cells in immunocompromised mice.lCMN cell suspensions were grafted into Rag2-/- mice. (a) No tumor was formed after 7 months; Melan-A and Sox10+ cells persisted in the Matrigel (n=5 injections from five specimens, white arrows). Scale bar=100 μm. (b) lCMN cell suspensions were mixed with HaCaT keratinocytes and injected (n=4 injections from four specimens). After 3 months, hematoxylin–eosin staining revealed melanocytic theques (black arrows) and a lentiginous melanocytic hyperplasia (black arrowheads) within keratinocyte cysts (black star). Scale bar=200 μm, except in theques and lentiginous hyperplasia hematoxylin–eosin stainings where scale bar=100 μm. Melanocytic cells were visualized using anti-Melan-A and anti-Sox10 antibodies (Alexa 488 secondary antibodies; white arrows). Higher magnifications are shown for each staining. Scale bar=50 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions