Isolation, Characterization, and Differentiation of Human Multipotent Dermal Stem Cells

Materials

Medium andSolution Preparation

  1. 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.
  2. 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.
  3. 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.
  4. 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×.
  5. MEF growth medium: MEF derivation medium withoutpenicillin–streptomycin.
  6. 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).
  7. Human embryonic stem cell medium 4 (HESCM4): 70% MEF conditioned HES medium and 30% HES medium, sterilize through a 0.2 mm filter.
  8. 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.
  9. Differentiation medium:

Dermal StemCell Culture

Day 1

Day 2

Day 3

Dermal Stem Cell Differentiation to Melanocytes, Neuronal Cells, Schwann Cells, Smooth Muscle Cells, Adipocytes, and Chondrocytes

DSCs Differentiation to Epidermal Melanocytes in Three-Dimensional Skin Reconstruct Culture