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 reservoir in glabrous has not yet been found. Human 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 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 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 , where they contact with surrounding ( 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 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 ( 7, 8 ). Glabrous 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 , 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 (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 -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 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 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. Shaker (Taitec, microincubator M-36).

11. CO2 incubator (CO2 at 5% (vol/vol); humidifi ed, T = 37°C). 12. Centrifuge (Eppendorf Centrifuge 5810). 13. Inverted light microscope. 14. 4-Well chamber slides (Fisher, Pittsburgh, PA). 15. Tissue culture 6-well trays with inserts (Organogenesis, Canton, MA). 16. Multi cassettes (Surgipath, Richmond, IL). 17. TBS biopsy papers (Triangle Biomedical Sciences, Durham, NC). 18. Plastic boats (Fisher, Pittsburgh, PA).

2.4. Cells and Sources 1. CF-1 female mice (days 13–14 gestation) for MEF derivation (Jackson Laboratory). 2. L-Wnt-3A cells (ATCC #CRL-2647). 3. Neonatal human foreskin. 4. Human fi broblasts. 5. Human keratinocytes.

3. Methods

3.1. MEF-Conditioned 1. Sacrifi ce a female mouse (CF-1, WiCell) at day 13 or 14 of HES Medium pregnancy. 2. Soak the abdomen with 70% ethanol. 3. Using a forceps, pull up the skin separating the hide from the peritoneum and cut a nick in the skin with scissors. 4. Using a new set of forceps and scissors, cut the peritoneum to expose the abdominal cavity. 5. Grab hold of the uterine horns with a blunt-point forceps and using a scissors cut them from the abdominal cavity. Place the uterine horns in a 100-mm cell culture dish that contains 10 mL of DPBS without Ca++ and Mg ++ . 6. Wash the uterine horns three times with 10 mL of DPBS with- out Ca++ and Mg ++ . 7. Using two fi ne-pointed forceps, tease open the uterine walls or cut using a scissor to release the embryos into the culture dish. 8. Separate the embryos from the placenta and fetal membranes. 9. Transfer the embryos to a new culture dish and wash them three times using 10 mL of DPBS without Ca++ and Mg ++ . 20 L. Li et al.

10. Using a fine-tipped forceps, individually, dissect out and discard the viscera, liver, and , which appear as red spots, from each embryo. 11. Wash the embryos three times with 10 mL of DPBS without Ca++ and Mg ++ . 12. Remove the DPBS and add 2 mL of trypsin/EDTA solution to the washed embryos. 13. Using curved Iris scissors, fi nely mince each embryo. Add 5 mL trypsin/EDTA solution. 14. Incubate at 37°C for 20–30 min on shaker until individual cells are visible (using an inverted culture microscope). 15. Add 20 mL of MEF derivation media to the plate after the incubation. Transfer the individualized cells to a 50-mL tube. 16. Rinse remaining tissue in the plate with a few milliliters of MEF derivation medium. Transfer to the 50-mL tube. Mix by pipetting a few times. Allow the debris in the suspension to settle to the bottom of the tube for 1 min. 17. Remove the top 12 mL of the suspension containing the indi- vidualized cells and spin down to remove all the trypsin, and then resuspend the pellet and divide into T75 fl ask (three embryos/fl ask). 18. Mix the remaining 8 mL with the debris, spin, resuspend, and add into one fl ask. 19. Add additional MEF derivation medium to each fl ask; bring the fi nal volume to 20 mL/fl ask.

20. Incubate fl asks in a 37°C, humidifi ed incubator at 5% CO2 for 2–3 days, until 80–90% confl uent. At this time, the MEFs are ready to be harvested and passaged. 21. Split MEF cells at 1:4 into gelatin-coated T75 fl asks with MEF growth medium and grow to reach 80% confl uence. 22. Remove MEF growth medium. Add 20 mL HES medium to each fl ask and incubate for 24 h. 23. Collect conditioned medium in a bottle and store at −70°C. This is the HES conditional medium.

3.2. L-Wnt3a- 1. The L-wnt3a cells are cultured in L-Wnt3a medium (DMEM Conditioned Medium containing 10% FBS and 0.4 mg/mL G418). for Melanocyte 2. When cells reach confl uence, split 1:10 into 100-mm culture Differentiation dishes and add 10 mL L-Wnt3a conditioning medium (1% Medium (Mel-1) FBS without G418) in each dish.

3. Incubate cells for 4 days at 37°C 5% CO2 . 4. Collect the medium and fi lter sterilize. This is Batch 1. 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional… 21

5. Add 10 mL fresh L-Wnt3a conditioning medium and culture for another 3 days. 6. Collect the medium and sterile fi lter. This is Batch 2. Discard the cells. 7. Mix Batch 1 and Batch 2 medium at 1:1 ratio. This is the L-Wnt3a-conditioned medium.

3.3. Dermal Stem Day 1 Cell Culture 1. Prepare the following in a laminar fl ow hood: One pair each of sterile forceps, curved scissors, and surgical scalpel blade; 5 mL of epidermal isolation solution (see Subheading 2.1) in a sterile centrifuge tube; 10 mL of Ca++ - and Mg++ -free HBSS in a 100-mm culture dish; and 10 mL of 70% ethanol in a sepa- rate sterile100-mm culture dish. 2. Soak the neonatal human foreskin specimens in 70% ethanol for 1 min. Transfer skin to the dish containing HBSS to rinse off ethanol (see Notes 1 and 2 ). 3. Cut skin ring open, and trim off fat and with scissors (see Note 3 ). 4. Cut skin into pieces (approximately 5 × 5 mm2 ) using the surgi- cal scalpel blade with one-motion cuts (see Note 4 ). 5. Transfer the skin pieces into the tube containing epidermal iso- lation solution. Cap, invert, and incubate the tube in the refrig- erator at 4°C for 18–24 h (see Note 5 ). Day 2 1. Remove the tube containing the sample from the refrigerator and incubate at 37°C for 5 min. 2. Prepare the following in a laminar fl ow hood: Two pairs of sterile forceps and a surgical scalpel blade; two empty sterile Petri dishes; 2 mL of dermal isolation solution. 3. Pour tissue in epidermal isolation solution into one of the empty 100-mm culture dishes. Separate the epidermis (thin, brownish, translucent layer) from the dermis (thick, white, opaque layer) using the forceps. Hold the dermal part of the skin piece with one pair of forceps, and the epidermal side with another. Gently tease them apart. Discard the epidermis immediately (see Note 5). Transfer the harvested dermis to an empty culture dish. Repeat the above-described proce- dure for each piece of tissue and then mince them as small as possible with a surgical scalpel blade (see Note 5 ). Transfer the minced tissue to a 50-mL tube with 2 mL of dermal isolation solution and leave the tube for 24 h at room temperature. 22 L. Li et al.

Day 3

1. Add 25 mL of HBSS without Ca++ and Mg ++ in the tube of dermal tissue with dermal isolation solution. Mix well and serially fi lter through 100-, 70-, and 40-m m cell strainers (see Note 6). 2. Spin down at 200 ´ g for 5 min. 3. Resuspend pellets in 5 mL of HESCM4 medium at 4 × 106 per T25 fl ask.

4. Incubate in 37°C and 5% CO2 . 5. Change ½ medium with fresh HESCM4 medium twice a week.

3.4. Melanocyte 1. Coat T25 fl ask or tissue culture-grade 4-well chamber slides Differentiation with 10 ng/mL fi bronectin (3 mL for T25 fl ask, 0.5 mL/well for chamber slides) overnight at 37°C. 3.4.1. Monolayer Differentiation Culture 2. Collect dermal spheres into a 50-mL tube and let spheres settle to the bottom (see Note 7 ). 3. Remove as much medium without disturbing settled spheres. 4. Add 5-mL melanocyte differentiation medium (Mel-1) to spheres. Pipette up and down fi ve times. Aspirate fi bronectin from chamber slide wells or T25 fl ask. 5. Gently remove as much medium from spheres as possible with- out disturbing. 6. Add 5 mL Mel-1 and transfer spheres to fi bronectin-coated chamber slide well or T25 fl ask.

7. Incubate at 37°C and in a 5% CO2 tissue culture incubator for 3 weeks, changing ½ of Mel-1 medium twice a week (see Note 7 ). 8. After a 3-week culture, cells can be passaged in T25 fl ask at a ratio of 1:3 and grown in melanocyte differentiation medium without TPA. Cells in chamber slide wells are ready for staining.

3.4.2. Three-Dimensional 1. Acellular layer: Mix the following reagents in a 50-mL tube: Skin Reconstruct Culture 0.59 mL 10× minimal essential medium (EMEM), 50 m L 200 mM L -glutamine (keep frozen; thaw immediately before use), 0.6 mL FBS, 120 m L 7.5% sodium bicarbonate, 4.6 mL bovine collagen I. Add 1 mL of mixture into one insert of tis- sue culture trays. Incubate for 30 min at room temperature. 2. Detach human fi broblasts from culture fl asks with 0.25% trypsin/EDTA, and add DMEM containing 10% FBS to neu- tralize. Collect cells by centrifugation and resuspend 0.45 × 106 cells in 0.75-mL skin-reconstruct medium. 3. Collect 6600 dermal spheres in 0.75-mL HESCM4 medium ( see Note 8 ). 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional… 23

4. Cellular layer: Mix the following in a 50-mL tube: 1.65 mL 10× MEM, 150 m L 200 mM L -glutamine, 1.85 mL FBS, 350 m L 7.5% sodium bicarbonate, 14 mL bovine collagen I, 0.75 mL fi broblasts suspension from step 2 , and 0.75 mL der- mal spheres from step 3 and mix well. Add 3 mL to each acel- lular layer-coated insert. Incubate for 45 min at 37°C in a 5%

CO2 tissue culture incubator. Add skin-reconstruct medium I (2 mL inside and 10 mL outside of insert). Incubate for 4 days. 5. Detach human keratinocytes from culture fl asks with 0.05% trypsin/EDTA, neutralize trypsin with soy bean trypsin inhibitor, spin down, and resuspend 3 × 10 6 cells in 600 m L skin-reconstruct medium I. 6. Remove skin-reconstruct tray from incubator, and aspirate medium from both inside and outside of insert. 7. Add skin-reconstruct medium I (1.5 mL inside and 10 mL outside of insert). 8. Drop 100 m L keratinocyte suspension to inside insert. Incubate for 2 days. 9. Aspirate skin-reconstruct medium I from both inside and outside of insert. Add skin-reconstruct medium II (2 mL inside and 10 mL) outside. Incubate for another 2 days. 10. Aspirate skin-reconstruct medium II both inside and outside, and add 7.5 mL skin-reconstruct medium III to only outside of insert (see Note 9). Change medium III every other day until day 18. 11. Harvest skin reconstruct at day 18. (a) Aspirate media from insert both inside and outside. (b) Remove insert from tray with forceps. (c) Cut out the reconstruct (including the polycarbonate fi lter) by tracing a circle close to the edge with a scalpel blade. (d) Cut the reconstruct in half on a hard surface. 12. For paraffi n sections: Place a half of the reconstruct in a histol- ogy cassette between two black TBS biopsy papers and soak the whole cassette in 10% formalin for 4–6 h. Then, place the cassette in 70% ethanol and store it at 4°C until you are ready to have it processed for paraffi n embedding. 13. For frozen sections: (a) Place the other half of reconstruct in 50% sucrose at 4°C. After 1–2 h, change the sucrose to 2 M and store it at 4°C for another 1–2 h. (b) Dispense OCT freezing media into a plastic boat so that it is about ½ full. Let the OCT polymerize at room tempera- ture for about 15 min. Avoid any bubbles in the OCT. 24 L. Li et al.

(c) Remove the reconstruct from the sucrose using forceps to grab the edge and a spatula underneath the polycarbonate fi lter, and place it on 4 Kimwipes. Dispense enough OCT on top to cover the surface and let it sit for about 10 min (the Kimwipes absorb the sucrose and the OCT starts to bind to the reconstruct). (d) Transfer the reconstruct into the boat on top of the OCT using forceps and a spatula. Dispense more OCT over the top of the reconstruct until the boat is completely full (make sure that you do not have any bubbles in the OCT which make cutting diffi cult). (e) Place the boat evenly on crushed dry ice, pit a lid on the bucket for faster freezing, and allow the OCT to freeze completely (see Note 10 ). (f) Wrap the boat in tin foil and store it at −70°C until you are ready to cut it using a cryostat.

3.4.3. Immunostaining For monolayer differentiated cells: 1. Fix differentiate cells in chamber slides with 4% paraformalde- hyde for 20 min. 2. Rinse slides three times with 1× PBS. 3. Unless extracellular are being targeted, permeabilize cells using 0.5% Triton X-100 (Sigma #T9284) for 5 min. 4. Rinse slides twice with 1× PBS. 5. Incubate slides in blocking solution (3% BSA) for 30 min. 6. Removing blocking solution and add primary antibodies diluted in 1× PBS without Ca++ and Mg++ . Incubate overnight at 4°C. 7. Wash off primary antibody solution three times with 1× PBS without Ca++ and Mg ++ . 8. Incubate slides in fl uorescence-conjugated secondary antibod- ies for 45 min at room temperature in the dark. 9. Rinse twice with 1× PBS without Ca++ and Mg ++ . 10. Dispense one drop of VECTASHIELD mounting medium onto the slide. Use VECTASHIELD mounting medium with DAPI if nuclear counterstaining is desired. Then coverslip and allow the mounting media to disperse over the entire slide. For skin reconstructs: 1. Deparaffi nization in xylene twice for 10 min. 2. Redehydrate in 100, 100, 95, 70, and 50% ethanol for 2 min. 3. Rinse three times with 1× PBS without Ca++ and Mg ++ . 4. Retrieve with trypsin. 5. Repeat steps 4– 10 for monolayer cells. 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional… 25

Fig. 1. Isolating and culturing dermal spheres in HESCM4 medium. (a ) Day 0: Single cells were isolated from neonatal foreskin dermis and grown in HESCM4 medium. ( b) Day 5: Small cell clusters were formed. ( c) Day 14: Cell clusters became large 3-dimensional spheres which are loosely adherent or nonadherent to culture fl asks. (d ). Mature melanocytes isolated from epidermis died in HESCM4 medium (day 7 picture).

3.5. Results HESCM4 medium is suffi cient to maintain human embryonic stem cells in an undifferentiated state in the absence of feeder cells 3.5.1. Dermal Sphere ( 10 ). After enzymatic digestion, dermis-derived single cells were Formation and grown in HESCM4 (Fig. 1a ). Although most of the single cells Characterization gradually died, some cells started forming small cell clusters within 5–7 days of culture (Fig. 1b ). After 10–14 days, a majority of theses cell clusters became loosely adherent to culture plates and formed characteristic 3D (Fig. 1c ). In contrast to dermal spheres, mature melanocytes isolated from foreskins did not survive in HESCM4 medium more than 1 week (Fig. 1d ). The dermal spheres expressed a neural crest cell marker NGFRp75 and an embryonic stem cell marker OCT4, and were capable of self- renewal and differentiated into multiple neural crest-derived cell lineages, including melanocytes ( 9 ) .

3.5.2. Differentiated When the dermal spheres were cultured for 2–3 weeks in melanocyte Melanocytes Express differentiation medium ( 11 ) , most of the cells died and fl oated, Melanocytic Markers whereas some of the attached cells developed dendritic processes 26 L. Li et al.

Fig. 2. Dermal stem cell-derived melanocytes express different melanocytic markers. (a ) When the dermal spheres were cultured for 2–3 weeks in melanocyte differentiation medium, cells migrated from dermal spheres and became adherent single cells. Some of the attached cells developed dendritic processes. (b –f ) After 3 weeks cultured in melanocyte differ- entiation medium, immunofl uoresent staining was performed. The differentiated cells were positive for melanocytic mark- ers MITF (b ), DCT ( c ), TYRP1 (d ), HMB45 ( e ), and S100 (f ). Nuclei were counterstained with DAPI (blue ) .

(Fig. 2a ). These adherent cells expressed the melanocyte markers MITF, DCT, TYRP1, HMB45, and S100 (Fig. 2b–f ). Undiffer- entiated dermal spheres were embedded into the dermis of skin reconstructs. At day 14, after seeding keratinocytes, single cells which migrated out from dermal spheres were observed at the 2 Isolation and Cultivation of Dermal Stem Cells that Differentiate into Functional… 27

Fig. 3. Melanocyte differentiation in three-dimensional skin-reconstruct model. Undifferentiated dermal spheres were embedded in the reconstructed dermis consisting of collagen with fi broblasts, and after 4 days keratinocytes were added on top of the dermis. At day 14, after seeding keratinocytes, single cells which migrated out from dermal spheres were observed at the basement membrane at the epidermis–dermis junction. (a ) These cells derived from dermal spheres expressed a melanocytic marker S100 (red ). They expressed E-cadherin (green ) when they resided within the basal layer of epidermis. Surrounding keratinocytes are also positive for E-cadherin. (b ) These single cells were positive for another melanocytic marker HMB45 (red ). White dotted lines indicate the epidermis–dermis junction. Nuclei were counterstained with DAPI (blue ) .

basement membrane at the epidermis–dermis junction. These cells expressed only the melanocytic markers HMB45, S100, and E-cadherin (Fig. 3a, b ) and not neural crest marker NGFRp75 ( 9 ) .

4. Notes

1. Tissue source and collection: The source of tissue for dermal stem cell cultures is human neonatal foreskins obtained from routine circumcision. At the time of excision, the skin is placed into a sterile container with 20 mL of normal skin-transporting medium supplied in advance and kept near the surgical area at 4°C. Specimens are delivered immediately to the tissue culture laboratory or stored at 4°C. Neonatal foreskins can be kept for up to 72 h. However, the fresher the specimens, the higher the yield of live cells upon isolation. 2. Sterilization of skin specimens: Reduce contamination by a short treatment (1 min) of intact skin with 70% ethanol in a laminar fl ow hood. After sterilization, rinse samples thoroughly with Ca++ - and Mg++ -free HBSS. 3. Preliminary tissue preparation: Place tissue on a 100-mm dish, and remove most of the subcutaneous fat and membranous material with curved scissors. 4. Adjustment of tissue size for enzymatic digestion: To improve reagent penetration, cut the skin samples into small pieces (approximately 5 × 5 mm2 ) rinsed in Ca++ - and Mg++ -free HBSS. 28 L. Li et al.

5. Separate epidermis from dermis: Pieces of skin are incubated in epidermal isolation solution for up to 24 h at 4°C to allow detachment of epidermis from dermis. Each piece of skin is secured with two pairs of forceps; one holds the epidermis and the other the dermis. The epidermal sheet is then peeled apart from the dermis and discarded immediately. Since melanocytes are located just above the basement membrane in the epidermis, scrape the basement membrane with a scalpel blade to try to get rid of as many epidermal melanocytes as possible even though epidermal melanocytes cannot survive in stem cell medium. Dermis is transferred to a 100-mm dish and minced with a scalpel blade as small as possible (approximately 1 × 1 mm 2 ). 6. Cell dispersal techniques: A single-cell suspension is generated from clumps of dermal tissue by enzymatic treatment with dermal isolation solution for 24 h at room temperature. The suspensions are diluted in HBSS and are serially fi ltered through 100-, 70-, and 40-m m cell strainers. Cells are collected by cen- trifugation, resuspended with stem cell medium, and seeded in a T25 culture fl ask. 7. Change ½ medium: Remove half volume of medium from fl ask or chamber slide wells, and add same amount of fresh medium. 8. Harvest dermal spheres: The majority of derma spheres adhere to plastic, and only a small number of spheres fl oated. The dermal spheres are easily detached by tapping the fl asks as they separate from the monolayer cells, which are strongly attached to the culture fl asks. 9. Air-lift epidermis: Add skin-reconstruct medium III only out- side of insert to expose epidermis in air. This step induces kera- tinocyte differentiation to form thick epidermis. 10. OCT embedding: Make sure that you do not have any bubbles in the OCT, which makes cutting diffi cult. The OCT becomes white when frozen.

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