Isolation, Characterization, and Differentiation of Human Multipotent Dermal Stem Cells
Materials
Medium andSolution Preparation
- Foreskin 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 Culture
Day 1
- Take out a foreskin from the transfer tube; rinse it with 70% ethanol for 1 min
- Transfer the foreskin to a sterile 100 mm culture dish, add 20 mLHBSS without Ca 2+ and Mg 2+, wait for 2 min
- Open the foreskin ring with scissors and cut the foreskin into several pieces of approximately 5 × 5 mm 2 using a surgical scalpel blade
- Transfer the skin pieces into a 50 mL Falcon tube with 5 mL0.48% dispase II, and incubate at 4°C overnight
Day 2
- Remove the Falcon tube containing the skin sample from 4°Cand incubate it at 37°C for 5 min
- Pour the skin pieces with the disease into a sterile 10 cm culture-dish, transfer all skin pieces to a new dish using forceps
- Separate 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 skin
- Transfer 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 3
- Add 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 strainers
- Centrifuge the cell suspension at 200 × g for 5 min
- Resuspend the cell pellet in 10 mL HESCM4 medium and seed the cells in two T25 flasks
- Put the flasks into an incubator with 5% CO 2 at 37°C
- After 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 days
Dermal Stem Cell Differentiation to Melanocytes, Neuronal Cells, Schwann Cells, Smooth Muscle Cells, Adipocytes, and Chondrocytes
- Tissue 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 possible
- Add 0.5 mL 1 mg/mL collagenase IV and 0.5 mL 0.25%trypsin/EDTA. Incubate at 37°C for 5 min
- Pipette 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 slides
- For neuronal and Schwann cell differentiation, use the medium-I during the first week and switch to medium-II during the second week
- Incubate for 2–3 weeks, replace 1/2 fresh medium twice a week. Cells are ready to fix for staining using differentiation markers
DSCs Differentiation to Epidermal Melanocytes in Three-Dimensional Skin Reconstruct Culture
- Coat 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 trays
- Collect dermal spheres by centrifugation and resuspend 6,600dermal spheres in 0.75 mL HESCM4 medium
- Trypsinize fibroblasts and collect cells by centrifugation and resuspend 0.45 × 10 6 cells in 0.75 mL skin reconstruct medium-I
- Mix 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 day
- Harvest human keratinocytes, resuspend 3 × 10 6 cells in 600 m Lskin reconstruct medium-I
- Remove the skin reconstruct tray from the incubator, aspiratemedium from both inside and outside of transwells
- Add 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 days
- Remove 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 days
- Aspirate 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 18
- Harvest 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