Cells
Abdominal or breast-skin was obtained from healthy individuals (donor A-E) undergoing reconstructive surgery, and processed within 24 hours. When required, skin was kept in RPMI 1640 (Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin (P/S) and 0,5 μg/ml fungizone (all from Invitrogen Ltd., Paisley, UK) until processing. After removal of subcutaneous fat (if applicable), skin was cut into small fragments and incubated in phosphate buffered saline (PBS, Sigma) containing 2.4 U/mL Dispase II (Roche, Indianapolis, IL, USA) overnight at 4°C. Epidermis and dermis were separated by tweezers.
To isolate keratinocytes, up to 20 epidermal sheets were digested using 2 ml Trypsin/EDTA (Lonza, Verviers, Belgium) for 5 minutes at 37°C. Following quick neutralization by Trypsin Neutralizing solution (TNS, Lonza), suspension was vortexed after which undigested tissue was removed by subsequent filtration through 500 μm (Nedfilter, Almere, The Netherlands) and 70 μm strainers (BD Biosciences, Erembodegem, Belgium). Cells were spun down at 250 g for 10', and resuspended at approximately 2 × 105 cells/ml in Keratinocyte-SFM medium containing 1% P/S, 0.39 mM CaCl2, bovine pituitary extract (BPS) and epidermal growth factor (EGF) according to the manufacturer's descriptions (keratinocyte medium, Invitrogen). Medium was changed after 2-3 days, and islands appeared after 1-2 weeks. Keratinocytes were passed to new flasks when islands contained 30-50 cells; overall density of flasks was kept at 30-40%. For experiments, keratinocytes used were passaged 6x or less.
To isolate fibroblasts, 10-20 dermal fragments were incubated at 37°C for 1 hour in DMEM/F12 (Invitrogen) medium containing 2.5 mg/mL Trypsin, 0.2 U/mL Liberase Bz3, and 0.2 mg/ml DNAse. Following neutralization with DMEM/F12 containing 15% fetal calf serum (FCS, Greiner Bio-one, Solingen, Germany), cell-suspensions were filtrated and spun down as described above. Cells were seeded at 2 × 105 cells/ml in DMEM/F12 containing P/S, 15% FCS and 10 ng/ml EGF (fibroblast medium). Fibroblast cultures were split when density reached 100%. These cells were never split more than 8x before use in co-cultures.
Mobilized CD34+ stem cells
CD34+ cells were isolated from peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers (V1-3) treated with G-CSF (Neupogen, Amgen Inc., Thousand Oaks, CA, USA) at MUMC+ (Maastricht, The Netherlands). Isolation was performed with the Isolex 300i Magnetic cell selection system v2.5 (Baxter oncology, Brussels, Belgium) using the Isolex stem cell reagent kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The positive fraction containing > 94% CD34+ cells was frozen at a concentration of 5 × 106 cells per ml per vial. CD133+ cell preparations were obtained from Lonza.
Skin and CD34+ stem cells were obtained according to protocols approved by, and guidelines stipulated by the local medical ethical committee, and with consent from the donors.
Maturation/differentiation of keratinocytes
Keratinocytes at 30-40% density were harvested using Trypsin/EDTA and TNS (Lonza), quantified and seeded at 100% density in a 1:1 (vol/vol) mix of Keratinocyte-SFM and DF-K medium. DF-K medium is a 1:1 (vol/vol) mix of DMEM and Ham's F12 containing P/S, 0.2 ng/ml EGF, 25 μg/ml BPS and 1.5 mM L-Glutamine (Invitrogen). Medium was refreshed daily. This procedure only worked for keratinocytes isolated from breast-skin and not for abdominal skin.
The skin cell construct
Sterilized Statamatrix® (Cytomatrix, Australia) were coated by incubation in PBS containing 100 μg/ml rat tail collagen I (Roche) at 37°C, after which the coated matrices were washed twice in PBS, and maintained in PBS until use. Prior to use, matrices were transferred to non-tissue culture treated petridishes (Greiner), and PBS was aspirated. Keratinocytes and fibroblasts were harvested, resuspended in a 1:1 mixture of keratinocyte and fibroblast-medium (ker-fib medium), and quantified. Subsequently, keratinocytes and fibroblasts were combined at 2 × 106 and 1 × 106 cells/ml, respectively, and 50-100 μl of this mix was dripped onto a matrix. After 3 hours at 37°C, 5% CO2, matrices were moved to 24-well plates and 2 ml ker-fib medium was added. The skin cell constructs were cultured for 6 days and medium was changed every other day.
Seeding of CD34+/CD133+ cells
After 6 days, medium was replaced by IMDM (Invitrogen) supplemented with 10% FCS (Greiner), 20 ng/ml IL-7 and IL-15, and 100 ng/ml Flt3-L (all R & D systems, Abingdon, UK), then 1 × 104 CD34+ or CD133+ cells were dripped onto each matrix. Halve medium change was done 3 times weekly.
TSt-4
Thymic stromal cell lines TSt-4 and TSt-4 transduced with hDLL1 or hDLL4 were kindly supplied by Dr. H. Kawamoto (RCAI-RIKEN, Yokohama, Japan) and maintained in RPMI 1640 containing 5% fetal bovine serum (FBS), 1% PS, 1 mM sodium pyruvate, 0.1 mM MEM non essential amino acids (NEAA), and 5 × 10-5 M 2-mercaptoethanol (bME)(all from Invitrogen Ltd., Paisley, UK).
Flowcytometry
All antibodies, materials and equipment were obtained from BD Biosciences unless stated otherwise, and were used according to the manufacturer's instructions. At different time points, cells were analyzed using different combinations of fluorescein isothiocyanate (FITC), R-phycoerithrin (PE), peridinin chlorophyll-a protein (PerCP)-, and allophycocyanin (APC) conjugated monoclonal antibodies (mAbs). The following antibodies (clones) were used: CD1a (HI149), CD3 (UCHT1, SK7), CD4 (SK3), CD5 (UCHT2, L17F12, MEM32 - Immunotools, Friesoythe, Germany), CD7 (M-T701, 7F3 - Sanquin), CD8a (HIT8a, RPA T8), CD14 (M5E2), CD19 (HIB19), CD34 (8G12), CD38 (HB7), CD45 (2D1, HI30), CD45RA (HI100), CD46 (E4.3), CD56 (B159), CD90 (AS02, Dianova, Hamburg, Germany), CD271 (C40-1457), IFN-γ (25723.11), NKG2A (131411, R&D Systems, Minneapolis, MN, USA), NKp46 (9E2), TCRαβ (IP26 - eBioscience), and TCRγδ (B1.1 - eBioscience). Unconjugated goat and rabbit antibodies used were DLL1 (#H20 and #H265), Nitric Oxide Synthase 2 (NOS2), IFN-γ Responsive Factor 1 (IRF1), Angiotensin-converting enzyme (ACE) (all Santa Cruz Biotechnology, Santa Cruz, CA, USA), for which the following conjugates were used according to the manufacturer's instructions: donkey anti-goat FITC and donkey anti-rabbit FITC or APC (all Jackson ImmunoResearch, West Grove, PA, USA). 7-amino-actinomycin D (7AAD) was used to differentiate between viable and dead cells. For intracellular stainings, cells were permeabilized using perm/wash. Cells were analyzed on a FACSCalibur or FACScan with WinMDI (Joe Trotter,
http://facs.scripps.edu/) software.
Immunofluorescence
Upon aspiration of media, monolayers or cells were washed with PBS and fixed with cold methanol: acetone (Merck) 1:1 for 10 minutes on ice. Following fixation, preparations were washed 3 times with PBS at RT, with each wash for 4 minutes on an orbital shaker. Blocking was done with for 30' with 1% normal donkey serum (JIR) in PBS, after which preparations were washed twice with PBS. The following primary antibodies were used, each at the proper, predetermined dilution: CD46 (BD Biosciences), CD90 (Dianova), DLL1, Jagged-1 (both Santa Cruz), pan-cytokeratin (Acris, Hiddenhausen, Germany), Keratin-10 (RKSE60), -14 (RCK107) and -16 (LL025)(all MuBio, Maastricht, The Netherlands). Preparations were incubated with primary antibodies for 1 hour at RT in the dark, after which they were washed 5 times with PBS, followed by incubation with secondary antibody for 1 hr at RT in the dark. Appropriate Texas Red or FITC-labeled donkey anti-rabbit anti-goat, or anti-mouse antibodies were obtained from Jackson Immunoresearch. Following another washing procedure, preparations were post-fixed for 15' with 2% paraformaldehyde in PBS. Then, preparations were washed twice, and covered with mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Preparations were analyzed using an Axioplan 2 microscope and Axiovision software (Zeiss, Jena, Germany).
PCR analysis
RNA was extracted from immature and matured keratinocytes using Trizol according to the manufacturer's instructions (Invitrogen). Following quantification and DNAse treatment, cDNA was synthesized using Superscript III according to the manufacturer's instructions (Invitrogen). PCR was carried out in 20 μl reaction volumes containing ≤ 80 ng or 40 ng RNA equivalent from keratinocytes or TSt-4 cells, respectively. All PCR components were used according to the manufacturer's instructions (iTaq, BioRad, Hercules, CA, USA) 100 nM of each primer (Eurogentec, Liege, Belgium). Primers used were (anneal temperature, optimal MgCl2): Keratin-14 f-CACCTCTCCTCCTCCCAGTT r-CATCGTGCACATCCATGAC (63°C, 3 mM), DLL-1 f-CGTCGACTCCTTCAGTCTGC r-TTCTGTTGCGAGGTCATCAG (60.5°C, 3 mM), DLL-4 f-TCCAACTGCCCTTCAATTTC r-ACTGCAGATGACCCGGTAAG (57°C, 5 mM), and Jagged-1 f-CGGCCTCTGAAGAACAGAAC r-CCTCAGAGGCTGAGTGTGTG (62°C, 3 mM). All PCR products were validated by TA-cloning and sequencing according to standard procedures.