Chapter 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional Epidermal Melanocytes Ling Li , Mizuho Fukunaga-Kalabis , and Meenhard Herlyn Abstract Human melanocytes have been extensively studied, but a melanocyte stem cell reservoir in glabrous skin has not yet been found. Human dermis contains cells that are nonpigmented but can differentiate to sev- eral different cell types. We have recently shown that multipotent dermal stem cells isolated from human neonatal foreskins are able to differentiate to multiple cell lineages, including pigmented melanocytes. The dermal stem cells grow as three-dimensional spheres in human embryonic stem cell medium and express some neural crest stem cell and embryonic stem cell markers. Melanocytes derived from dermal stem cells express melanocytic markers and act the same way as mature epidermal melanocytes. Dermal spheres, embedded in the reconstructed dermis consisting of collagen with fi broblasts, can migrate to the basement membrane, where they become pigmented in the same way as epidermal melanocytes suggesting that dermal stem cells can give rise to epidermal melanocytes. Key words: Melanocyte , Stem cells , Dermal reservoir , 3D skin reconstruct 1. Introduction Melanocytes originate from the neural crest in vertebrate development, and undergo a complex process of fate specifi cation, proliferation, survival, and differentiation, before fi nally homing in the epidermis, where they contact with surrounding keratinocytes ( 1 ) . Different techniques of the isolation and subsequent culture of human epidermal melanocytes had been attempted ( 2– 5 ) until Eisinger and Marko established the method to grow homogeneous human melanocytes to yield cells in suffi cient quantity for biological, biochemical, and molecular analyses ( 6 ) . Since then, epidermal melanocytes have been extensively studied. Recently, the presence of a stem cell niche for melanocytes has been shown in hair follicles Ragai R. Mitry and Robin D. Hughes (eds.), Human Cell Culture Protocols, Methods in Molecular Biology, vol. 806, DOI 10.1007/978-1-61779-367-7_2, © Springer Science+Business Media, LLC 2012 15 16 L. Li et al. of mouse and human skin ( 7, 8 ) . Glabrous skins are also abundant in melanocytes; however, no obvious, spatially restricted stem cell niche for melanocytes has been found in such areas. We have recently shown that multipotent dermal stem cells isolated from human foreskins lacking hair follicles are able to home to the epi- dermis to differentiate into melanocytes ( 9 ) . These dermal stem cells grow as three-dimensional spheres in human embryonic stem cell medium, self-renew, and express a neural crest marker NGFRp75 and an embryonic stem cell marker OCT4, but not melanocytic markers, such as HMB45 or S100. In addition, these cells are able to differentiate into multiple neural crest-derived cell types, including melanocytes. In a three-dimensional skin recon- struct model, the dermal stem cells are differentiated into HMB45/ E-cadherin-positive melanocytes, which migrated from the dermis to the epidermis and reside singly among the basal-layer keratino- cytes. In this chapter, we outline our procedures for the isolation of human dermal stem cells and differentiation toward mature functional melanocytes that home to the epidermis in skin recon- structs, produce pigment, and express the appropriate melanocytic markers MITF, DCT, TYRP1, S100, HMB45, and E-cadherin. 2. Materials 2.1. Reagent Setup 1. Normal skin-transporting medium: The medium for collecting normal skin is composed of Hanks’ balanced salt solution with- out Ca ++ and Mg ++ (HBSS; Gibco-BRL Grand Island, NY) supplemented with penicillin (100 U/mL; USB Cleveland, OH), streptomycin (100 m g/mL; USB), gentamicin (100 m g/mL; Bio Wittaker Walkersville, MD), and Fungizone (0.25 m g/mL; JRH Biosciences, Lenexa, KS). After sterilization through a 0.2- m m fi lter, the skin-transporting medium is transferred into sterile containers in 20-mL aliquots and stored at 4°C for up to 1 month. 2. Epidermal isolation solution: Dissolve 0.48 g of dispase (grade II, 0.5 U/mg; Boehringer Mannheim, Indianapolis, NJ) in 100 mL of phosphate-buffered saline (PBS) without Ca ++ and Mg ++ (Cellgro by Mediatech, Herndon, VA) containing 0.1% bovine serum albumin (BSA) (fraction V; Sigma, St. Louis, MO) to yield a fi nal dispase activity of 2.4 U/mL. Sterilize the enzyme solution through a 0.2- m m fi lter, aliquot into a 5-mL tube, and store at −20°C for up to 3 months. 3. Dermal isolation solution: Collagenase type IV (Invitrogen, Grand Island, NY) 100 mg is dissolved in 100 mL of Dulbecco’s modifi ed Eagle’s medium (DMEM) (Cellgro) to yield a fi nal concentration 1 mg/mL. Sterilize the enzyme 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional… 17 solution through a 0.2- m m fi lter, aliquot into a 5-mL tube, and store at −20°C for up to 3 months. 4. Mouse embryonic fi broblast (MEF) derivation medium: Combine 870 mL DMEM (Invitrogen), 100 mL defi ned FBS (Invitrogen; heat inactivate for 30 min at 57°C), 10 mL 200 mM L -glutamine (Invitrogen – keep frozen, thaw immedi- ately before use), 10 mL nonessential amino acids 100× (Invitrogen), and10 mL penicillin–streptomycin 100×. 5. MEF growth medium: Combine 880 mL DMEM (Invitrogen), 100 mL defi ned FBS (Invitrogen; heat inactive for 30 min at 57°C), 10 mL 200 mM L -glutamine (Invitrogen – keep frozen, thaw immediately before use), and 10 mL nonessential amino acids 100× (Invitrogen). 6. Human embryonic stem cell medium (HES): Combine 800 mL DMEM/F-12 (Invitrogen), 200 mL Knockout- Serum Replacer (Invitrogen), 10 mL 100 mM L -glutamine + b -mercaptoethanol (Invitrogen – add 7 m L b -mercaptoethanol to 10 mL), 10 mL nonessential amino acids 100× (Invitrogen), and 1 mL basic fi broblast growth factor [bFGF; Fitzgerald Industries – dissolve in 0.1% BSA/1× Dulbecco’s PBS (DPBS; Cellgro, Manassas, VA) at a concentration of 4 m g/mL and store at −70°C]. 7. Human embryonic stem cell medium 4 (HESCM4): Mix 700 mL MEF-conditioned HES medium and 300 mL HES medium, sterilize through a 0.2- m m fi lter. 8. L-Wnt3a cell medium: Add 100 mL FBS and 8 m L of 50 mg/mL G418 (Sigma) to 900 mL DMEM (Cellgro). 9. L-Wnt3a conditioning medium: Add 10 mL FBS to 990 mL DMEM (Cellgro). 10. Melanocyte differentiation medium (Mel-1): Combine 30 mL DMEM-Low Glucose (Invitrogen), 20 mL MCDB201, and 50 mL Wnt3a conditioned medium, add 20 m L Dexamethasone (Sigma – dissolve in ddH2 O at a concentration of 0.25 M and store at −20°C), 1 mL ITS Liquid Medium Supplement (Sigma), 1 mL Linoleic Acid-BSA (Sigma), 1 mL L -ascorbic acid (Sigma), 1-mL stem cell factor (SCF; Fitzgerald Industries, Concord, MA – dissolve in 0.1% BSA/1× DPBS at a concen- tration of 10 m g/mL and store at −70°C), 100 m L 4 m g/mL bFGF (Fitzgerald Industries), 100 m L endothelin-3 (ET-3; American Peptide Co., Sunnyvale, CA – dissolve in 0.1% BSA/1× DPBS at a concentration of 264 m g/mL and store at −70°C), 150 m L Cholera toxin (Sigma – dissolve in 0.1% BSA/1× DPBS at a concentration of 3.32 m g/mL and store at 4°C), and 12.5 m L TPA [Sigma – dissolve in dimethyl sulfoxide (DMSO); Fisher Scientifi c, Fair Lawn, NJ] at a concentration of 250 m g/mL and store at −20°C. 18 L. Li et al. 11. Skin reconstruct medium preparation: Basic medium (500 mL): Add the following reagents to 490 mL keratinocyte serum-free medium (Invitrogen), 1.8 mL bovine pituitary extract (BPE; Invitrogen 1×), 10 mL dialyzed fetal bovine serum (dFBS) (Hyclone, Logan, UT), 500 m L of 10 m g/mL SCF (Fitzgerald Industries), 125 m L 4 m g/mL bFGF (Fitzgerald Industries), and 500 m L of 264 m g/mL ET-3 (American Peptide Co.). Medium I: To 100 mL basic medium, add 10 m L Epidermal Growth Factor (EGF; Invitrogen, Camarillo, CA – dilute to 100 m g/mL in 0.1% BSA in 1× DPBS and store at −70°C). Medium II: To 100 mL basic medium, add 2 m L EGF. Medium III: To 300 mL basic medium, add 720 m L CaCl2 (Sigma – dissolve in ddH2 O at 1 M and store in room temperature) . 12. 0.1% gelatin (500 mL): Dissolve 500 mg gelatin powder (Sigma), 500 mL endotoxin-free, reagent-grade water. Autoclave gelatin solution for 45 min on a liquid cycle and store at room temperature. 13. Fibronectin (Advanced Biomatrix, Sandiego, CA): Dissolve 1 mg in 2 mL 1× DPBS, store in −20°C. For coating fl asks, dilute to 10 ng/mL in DPBS. 14. Soy bean trypsin inhibitor (Invitrogen): Dissolve 250 mg in 1,000 mL of 1× DPBS. 2.2. Additional 1. 0.25% Trypsin/EDTA (Cellgro, Manassas, VA). Reagents 2. Minimal essential medium with Earle’s salts (10× EMEM) (Lonza, Walkersville, MD). 3. FBS (Hyclone, Logan, UT). 4. Bovine tendon acid-extracted collagen I (Organogenesis, Canton, MA). 5. Optimal cutting temperature (OCT) freezing media (Sakura, Torrance, CA). 2.3. Equipment 1. Forceps (Roboz, Gaithersburg, MD). 2. Scissors (Roboz, Gaithersburg, MD). 3. Iris scissors (Roboz, Gaithersburg, MD). 4. Surgical blades (Feather, Japan). 5. 100-mm culture dish (Corning Incorporated, Corning, NY). 6. 100, 70, and 40 m m cell strainers (Becton Dickinson, Franklin Lakes, NJ). 7. 50-mL sterile centrifuge tubes (Becton Dickinson, Franklin Lakes, NJ). 8. 15-mL sterile centrifuge tubes (Becton Dickinson, Franklin Lakes, NJ). 9. T25 and T75 fl asks (Becton Dickinson, Franklin Lakes, NJ). 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional… 19 10.