Skin, as the body’s largest organ, has been extensively used to study adult stem cells. Most previous skinrelatedstudies have focused on stem cells isolated from hair follicles and from keratinocytes. Here we presenta protocol to isolate multipotent neural crest stem-like dermis-derived stem cells (termed dermal stemcells or DSCs) from human neonatal foreskins. DSCs grow like neural spheres in human embryonic stemcell medium and gain the ability to self-renew and differentiate into several cell lineages including melanocytes,neuronal cells, Schwann cells, smooth muscle cells, adipocytes, and chondrocytes. These cells expressneural crest stem cell markers (NGFRp75 and nestin) as well as an embryonic stem cell marker (OCT4).Key words Skin , Stem cells , 3-D skin reconstruct , Cell differentiation1 IntroductionAdult stem cells are well known for their potential therapeuticvalue and their accessibility from patient-derived samples to avoidethical problems. Skin contains somatic stem cells that generatekeratinocyte, melanocyte, and mesenchymal cell lineages. Thesesomatic stem cells have traditionally been thought to be restrictedin their differentiation and regeneration potential to the tissues inwhich they reside ( 1 ) . The advantage of skin stem cell research isthat skin is a readily accessible organ from which to obtain a biopsy.Hair follicle stem cells and keratinocyte stem cells from humanepidermis have been well characterized. Human induced pluripotentstem (iPS) cells can be generated from human dermalfi broblasts following upregulation of different transcription factors,including Oct3/4, Sox2, c-Myc, Klf4, Lin28, and Nanog,using retroviral and/or lentiviral vectors ( 2– 4 ) . However, someissues of iPS cells have been recently observed such as low ef fi ciencyof the reprogramming process, high number of retroviral insertions,and tumorigenesis by reactivation of the c-Myc transgene ( 5 ) .

Isolation, Characterization, and Differentiation of Human Multipotent Dermal Stem CellsMaterialsMedium andSolution PreparationForeskin transporting medium: Dulbecco’s modification of Eagle’s medium (DMEM; Cellgro #10-017-CM) supplemented with gentamycin (100 mg/mL; Cellgro #30-005-CR). After sterilization through a 0.2 mm filter, the medium is transferred into sterile containers in 20 mL aliquots and stored at 4°C for up to 1 month.Dispase solution ( 0.48% ): Dispase (grade II, 0.5 U/mg; Boehringer Mannheim #165859) 0.48 g is dissolved in 100 mL phosphate-buffered saline (PBS) without Ca 2+ and Mg 2+ (Cellgro #MT21-031-CM). Sterilize the enzyme solution through a 0.2 mm filter, aliquot into 5 mL/tubes, and store at −20°C for up to 3 months.Collagenase solution ( 1 mg/mL ): Collagenase type IV (Invitrogen #17104-019) 100 mg is dissolved in 100 mL DMEM to yield a final concentration of 1 mg/mL. Sterilize the enzyme solution through a 0.2 mm filter, aliquot into 5 mL/tubes, and store at −20°C for up to 3 months.Mouse embryonic fi broblast ( MEF ) derivation medium : The medium contains 87% DMEM (Invitrogen #11965-092), 10% defined FBS (Invitrogen #16000-044; heat inactivate for 30 min at 57°C), 1% 200 mM L -glutamine (Invitrogen #21051024), 1% nonessential amino acids 100× (Invitrogen #11140) and 1% penicillin-streptomycin 100×.MEF growth medium: MEF derivation medium withoutpenicillin–streptomycin.Human embryonic stem cell medium (HES): 78% DMEM/F-12 (Invitrogen #11330-032), 20% Knockout-Serum Replacer (Invitrogen #10828-028), 1% 100 mM L -glutamine + b -mercaptoethanol (Invitrogen #21051-024—add 7 mL b -mercaptoethanol to 10 mL L -glutamine), 1% nonessential amino acids 100× (Invitrogen #11140), 4 ng/mL basic fi broblast growth factor (bFGF; Fitzgerald Industries #30R-AF015).Human embryonic stem cell medium 4 (HESCM4): 70% MEF conditioned HES medium and 30% HES medium, sterilize through a 0.2 mm filter.L-Wnt3a cell growth medium and conditioning medium: 90% DMEM (Cellgro #10-017-CM), add 10% FBS and 0.4 mg/ mL G418 (Sigma #G-8168). Conditioning medium including 99% DMEM and 1% FBS.Differentiation medium:For melanocyte differentiation (100 mL): 50 mL Wnt3a conditioned medium, 30 mL DMEM-Low Glucose (Invitrogen #11885), 20 mL MCDB201 (Sigma #M6770), 1× ITS Liquid Medium Supplement (Sigma #I-3146), 1 mg/mL linoleic acid-BSA (LA-BSA; Sigma #L-9530), 10 −4 M L -ascorbic acid (Sigma #A-4403), 100 ng/mL stem cell factor (SCF; Fitzgerald Industries, #RDI-307- 255X), 0.05 m M dexamethasone (Sigma #D-2915), 20 pM Cholera toxin (Sigma #C-3012), 50 nM TPA (Sigma #P-1583), 4 ng/mL bFGF (Fitzgerald Industries #30RAF015), 100 nM endothelin-3 (ET-3; American Peptide Co. #88-5-10).For neuronal cell differentiation (100 mL): (1) 60 mL DMEM, 30 mL F12 (GIBCO #11765), 10 mL FBS, 40 ng/mL bFGF. (2) 60 mL DMEM, 30 mL F12, 10 mL FBS, 10 ng/mL nerve growth factor (Millipore #GF028), 10 ng/mL brain-derived neurotrophic factor (Peprotech #450-02-10), 10ng/mL NT-3 (Stem Cell Technologies #02508).For smooth muscle cell differentiation (100 mL): 90 mL DMEM-F12 (GIBCO m#11330), 10 mL FBS, 0.1 M nonessential amino acids solution and 60 pM transforming growth factor- b 1 (TGF- b 1; R&D Systems #240B).For adipocyte differentiation (100 mL): 90 mL lowglucose DMEM (Sigma #D6046), 10 mL horse serum (Invitrogen #26050-070), 1× ITS, 1 mg/mL LA-BSA, 1 m M hydrocortisone (Sigma #H4001), 60 m M indomethacin (Sigma #I7378), 0.5 mM isobutylmethylxanthine (Sigma #I5879).For chondrocyte differentiation (100 mL): 90 mL highglucose DMEM, 10 mL FBS, 1× ITS, 1 mg/mL LA-BSA, 50 nM dexamethasone, 60 pM TGF- b 1.For Schwann cell differentiation (100 mL): (1) 60 mL DMEM, 30 mL F12 (GIBCO #11765), 10 mL FBS. (2) Medium I plus 4 m M foskolin (Sigma #F3917).Skin reconstruct medium: Basic medium: 400 mL Keratinocyte-SFM (Invitrogen #10724), 2% dialyzed FCS (Gibco LTI #16440-034), 60 ng/mL bovine pituitary extract (BPE; Invitrogen #13028-014), 4.5 ng/mL bFGF, 100 nM ET3 (American Peptide Co. #88-5-10), 10 m g/mL SCF (Fitzgerald Industries #RDI-307-255X). (1) Add 1 ng/mL EGF (Invitrogen #10450-013) to 100 mL basic medium; (2) Add 0.2 ng/mL EGF (Invitrogen #10450-013) to 100 mL basic medium; (3) Add 2.4 mM CaCl 2 (Sigma; #C-7902) to 200 mL basic medium.Dermal StemCell CultureDay 1Take out a foreskin from the transfer tube; rinse it with 70% ethanol for 1 minTransfer the foreskin to a sterile 100 mm culture dish, add 20 mLHBSS without Ca 2+ and Mg 2+, wait for 2 minOpen the foreskin ring with scissors and cut the foreskin into several pieces of approximately 5 × 5 mm 2 using a surgical scalpel bladeTransfer the skin pieces into a 50 mL Falcon tube with 5 mL0.48% dispase II, and incubate at 4°C overnightDay 2Remove the Falcon tube containing the skin sample from 4°Cand incubate it at 37°C for 5 minPour the skin pieces with the disease into a sterile 10 cm culture-dish, transfer all skin pieces to a new dish using forcepsSeparate the epidermis from the dermis by holding the dermal part of each skin piece with one pair of forceps and gently remove the epidermal part with a surgical scalpel blade. Discard the epidermis. Repeat the procedure for each piece of skinTransfer all dermal pieces to a new dish and mince them as small as possible. Collect the minced dermal pieces using a pipette and transfer to a 50 mL Falcon tube containing 2 mL 1 mg/mL collagenase IV. Incubate at room temperature for 24 Day 3Add 25 mL HBSS without Ca 2+ and Mg 2+ to the tube containing the dermis with collagenase IV. Mix well by pipetting up and down; serially filter through 100, 70, and 40 cell strainersCentrifuge the cell suspension at 200 × g for 5 minResuspend the cell pellet in 10 mL HESCM4 medium and seed the cells in two T25 flasksPut the flasks into an incubator with 5% CO 2 at 37°CAfter 48 h, aspirate 2.5 mL medium from the flask, and replace with 2.5 mL fresh HESCM4 medium. Change half the volume of the medium every 3–4 daysDermal Stem Cell Differentiation to Melanocytes, Neuronal Cells, Schwann Cells, Smooth Muscle Cells, Adipocytes, and ChondrocytesTissue culture-grade 4-well chamber slides (Becton Dickinson) are precoated with: 0.5 mL/well 10 ng/mL fibronectin (Advanced Biomatrix #5050; for melanocyte, chondrocyte, and adipocyte differentiation), 0.1% Matrigel (BD Biosciences #354234; for neuronal and smooth muscle cell differentiation), and a mixture of 20 mg/mL laminin (BD Biosciences #354232) with 200 mg/mL poly- D -lysine (BD Biosciences #354210) (for Schwann cell differentiation)Collect dermal spheres and transfer into a 50 mL tube, spindown, remove the supernatant as much as possibleAdd 0.5 mL 1 mg/mL collagenase IV and 0.5 mL 0.25%trypsin/EDTA. Incubate at 37°C for 5 minPipette up and down for 1 min, then add 9 mL soybean trypsin inhibitor. Spin down. Resuspend the sphere cell pellet in the various differentiation media and seed the cells in coated chamber slidesFor neuronal and Schwann cell differentiation, use the medium-I during the first week and switch to medium-II during the second weekIncubate for 2–3 weeks, replace 1/2 fresh medium twice a week. Cells are ready to fix for staining using differentiation markersDSCs Differentiation to Epidermal Melanocytes in Three-Dimensional Skin Reconstruct CultureCoat the transwell (Organogenesis) with the collagen mixture: 0.59 mL 10× minimal essential medium (EMEM), 50 m L 200 mM L -glutamine, 0.6 mL FBS, 120 m L 7.5% sodium bicarbonate, 4.6 mL bovine collagen I and mix well. Add 1 mL of the mixture into one transwell of tissue culture traysCollect dermal spheres by centrifugation and resuspend 6,600dermal spheres in 0.75 mL HESCM4 mediumTrypsinize fibroblasts and collect cells by centrifugation and resuspend 0.45 × 10 6 cells in 0.75 mL skin reconstruct medium-IMix the following reagents 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 dermal spheres from step 2 , 0.75 mL fi broblasts suspension from step 3 and mix well. Add 3 mL to each coated transwell. Incubate for 45 min at 37°C in a 5% CO 2 tissue culture incubator. Add skin reconstruct medium I (2 mL inside and 10 mL outside of the transwell). Incubate for 4 dayHarvest human keratinocytes, resuspend 3 × 10 6 cells in 600 m Lskin reconstruct medium-IRemove the skin reconstruct tray from the incubator, aspiratemedium from both inside and outside of transwellsAdd skin reconstruct medium I (1.5 mL inside and 10 mL outside of insert). Drop 100 m L keratinocyte suspension to each inside transwell. Incubate for 2 daysRemove skin reconstruct medium I from both inside and outside of transwells. Add skin reconstruct medium II (2 mL inside and 10 mL outside). Incubate for another 2 daysAspirate skin reconstruct medium II both inside and outside of transwells, add 7.5 mL skin reconstruct medium III to only the outside of the transwells ( see Note 12 ). Change medium-III every other day until day 18Harvest the skin reconstruct at day 18: Remove the transwellfrom the tray with forceps. Cut out the reconstruct (including the polycarbonate fi lter) by tracing a circle close to the edge with a scalpel blade. Place the reconstruct in a histology cassette (Surgipath #02275-BX) between two black TBS biopsy papers (Triangle Biomedical Sciences, #BP-B) and soak the whole cassette in 10% formalin (Fisher Healthcare #245-685) for 4–6 h. Then place the cassette in 70% for paraffin embedding

Day 1 1. Prepare the following in a laminar flow hood: one pair of sterile forceps, curved scissors, and surgical scalpel blade; 5 ml of dispase II (Boehringer Mannheim) in a sterile centrifuge tube; 20 ml of Ca2+, Mg2+ -free HBSS in a sterile non-tissue culture Petri dish; and 10 ml of 70% ethanol in a separate Petri dish. 2. Soak the skin specimens in 70% ethanol for 30 seconds. Transfer skin to another Petri dish containing HBSS to rinse off ethanol (see notes 1 and 2). 3. Cut skin-ring open and trim off fat and subcutaneous tissue with scissors (see note 3). 4. Cut skin into pieces (approximately 5 x 5 mm2) using the surgical scalpel blade with one-motion cuts (see note 4). 5. Transfer the pieces into the tube containing dispase II. Cap, invert, and incubate the tube in the refrigerator at 4°C for 15-18 hours.