All cells were grown in a humidified atmosphere at 37°C and 5% CO2. Individual culture
conditions are listed below.
VII.3.1.1. Cell culture of tumor cells and murine embryonic fibroblasts
Tumor cells and murine embryonic fibroblasts were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) with 4500 mg/l glucose and 4 mM L-alanyl-L-glutamine supplemented with 10% (v/v) heat inactivated fetal bovine serum (FCS), 1% (v/v) 100x non-essential amino acids (NEAA), 100 units/ml penicillin, 100 µg/ml streptomycin and 0.1 mM β-mercapthoethanol, from here on referred as complete medium. Every 3-5 days when 80% confluence was reached the cells were subcultured using a 21 mM trypsin solution.
VII.3.1.2. Melanocyte cell culture
Human melanocytes were obtained from foreskins, kindly provided by Dr. Uysal, Mannheim. Donor age ranged from new born to three years old patients. Excised foreskins were incubated for 15 min at room temperature in 10% Braunol solution, were washed with PBS and separated from subcutaneous fat followed by cutting into 10 x 4 mm pieces. Specimens were digested in dispase (1 mg/ml) at 4°C overnight followed by 15 min digestion of the epidermis at 37°C in trypsin/EDTA. Primary melanocytes were washed and transferred to 10 cm dishes in medium 254 supplemented with 1% (v/v) 100x human melanocyte growth supplement (HMGS) resulting in a final concentration of 0.2% (v/v) bovine pituitary extract, 0.5% v/v fetal bovine serum, 1 µg/ml recombinant human insulin-like growth factor-I, 5 µg/ml bovine transferrin, 3 ng/ml basic fibroblast growth factor (bFGF), 0.18 µg/ml hydrocortisone, 3 µg/ml heparin and 10 ng/ml phorbol 12-myristate 13-acetate. Cells were subcultured before reaching 90% confluence.
VII.3.1.3. Generation of iPS and iPC cells
For reprogramming of human tumor cells, fibroblasts and melanocytes 105 cells per cm2 were
seeded on gelatin-coated plates and transduced with a reverse tetracycline-controlled transactivator (FUdeltaGW-rtTA-zeocin) containing a zeocin resistance gene. Cells were selected with 100 µg/ml zeocin in complete medium generating resistant clones that were manually picked and expanded. The clones were co-infected with a doxycycline-inducible vector expressing a stem cell cassette (STEMCCA) encoding for the transcription factors Oct4, Sox2, Klf4 and a puromycin resistance or alternatively for Oct4, Sox2, Klf4 and c-Myc. The next day, superinfection was performed to reach higher efficiencies. All transductions were
conducted by incubation of cells with virus for 24 h at 37°C in DMEM medium supplemented with 10 µg/ml polybrene. 24 h after the last infection 105 cells in complete medium were plated
onto six well tissue culture plates coated with gelatin. After cell attachment doxycycline was added to the medium to induce transgene expression. From here on, medium was changed every second day. After 30-40 days first colony-forming cells originated. In order to create reprogrammed clones derived from single cells individual colonies were manually transferred onto fresh feeder cells in DMEM/F12 with 20% (v/v) knockout serum replacement (KOSR), 2 mM L-glutamine, 1% (v/v) NEAA, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM ß- mercaptoethanol, 1 µg/ml doxycycline and supplemented with 10 ng/ml human LIF, from here on referred to as naïve hES medium, until homogenous colonies were established.
VII.3.1.4. Human induced pluripotent stem cell culture
Stable clones of human iPSCs were cultivated under xeno-free cell culture conditions using a synthetic surface matrix. Therefore, one day prior use six well tissue culture plates were coated with Matrigel for one hour at room temperature and stored at 4°C. Human iPSCs were washed and undifferentiated parts were manually dissociated into cell clusters of 50-100 cells. These small cell aggregates were transferred to Matrigel-coated plates in mTeSR1 medium containing 20% (v/v) mTeSR1 supplements of bovine serum albumin, recombinant human bFGF, recombinant human TGF-β, lithium chloride, pipecolic acid and γ-aminobutyric acid. Every other day medium was changed and differentiated parts manually removed. Alternatively, human iPSCs were cultivated on feeder cells in DMEM/F12 with 20% (v/v) knockout serum replacement, 2 mM L-glutamine, 1% (v/v) NEAA, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM β-mercaptoethanol and 10 ng/ml bFGF, from here referred as human ES medium.
VII.3.1.5. Culture of human iPS and iPC cells in the alternative pluripotent state
Mitotic inactivated feeder cells were plated on gelatin-coated six well tissue culture plates in complete medium and incubated for two days to ensure proper attachment and spread. Then iPS cells were transferred onto the feeder cells and medium was changed to naïve hES medium. For passaging cells were harvested every 4-7 days using trypsin and replated at 1:30 to 1:100 ratios in naïve hES medium containing 10 µM ROCK inhibitor (Y27632). In order to separate iPS or iPC cells from feeder cells by preplating, cells were harvested using trypsin, dissociated into single cells, washed and resuspended in naïve hES medium containing ROCK inhibitor. Then, the cell suspension was transferred onto gelatin-coated tissue culture plates and incubated for 2 hours at 37°C. Afterwards undifferentiated cells floating in the supernatant were collected and prepared for further experiments.VII.3.1.6. Preparation of murine embryonic fibroblast
Day 12.5 to 13.5 embryos postcoitum of C57BL/6 mice were dissected from the uterus and incubated for five minutes in 10% Braunol. Embryos from one mouse were rinsed in PBS followed by the mechanical removal of internal organs and the head. The carcass was manually minced in trypsin/EDTA solution using scalpels and incubated at 37°C for ten minutes. Afterwards the cell suspension was neutralized in complete medium, washed and resuspended in complete medium. Subsequently, the cells of one embryo were transferred to one 150 mm tissue culture dish and cultivated until confluence.
VII.3.1.7. Mitotic inactivation of feeder cells
Murine embryonic fibroblasts were expanded until passage three in either T175 cell culture flasks or 150 mm cell culture dishes. Dense fibroblasts were incubated with 10 µg/ml mitomycin C for 4 hours and washed with Ca2+ and Mg2+ containing PBS for three times followed by
rinsing the cells with Ca2+ and Mg2+ free PBS. The postmitotic cells were trypsinized for 5-7
min at 37°C until the cell detached followed by neutralization of the enzymatic digestion with complete medium. After centrifugation the cells were resuspended in 80% FCS with 20% (v/v) dimethyl sulfoxide (DMSO) and aliquoted at a concentration of 1x106 cells per vial. Until
thawing the vials were stored in liquid nitrogen.
VII.3.1.8. Fibroblast differentiation
For the differentiation into fibroblast-like cells HT-144-iPCCs were seeded onto 80% confluent mitotic-inactivated feeder cells in naïve hES medium with 10 µM ROCK inhibitor and 1 µg/ml doxycycline and cultivated for two to five days until small colonies were formed. In order to establish the clones A-C, different protocols were followed. For clone A medium was switched to complete medium until stably expandable colonies emerged. Clone B was generated by changing the medium to DMEM/F12 1:1 with Neurobasal medium containing 1% B27 and 0.5% (v/v) N2 supplement (Gibco) for 3 days. Then the medium was also switched to complete medium with 20% (v/v) FCS. For clone C iPCC colonies were cultivated in DMEM/F12 3:1 supplemented with 10% (v/v) FCS, 0.18 mM adenine, 0.5 µg/ml hydrocortisone, 100 pM cholera toxin, 10 ng/ml EGF, 5 µg/ml insulin for 10 days and supplemented on days four to ten with 0.5 nM BMP-4. Afterwards fibroblast-like cells were split and maintained in T75 cell culture flasks with complete medium.
VII.3.1.9. Neuronal differentiation
For neuronal induction, 2x104 cells per cm2 were seeded on Matrigel-coated dishes in human
naïve ES medium supplemented with 10 µM ROCK inhibitor. When small colonies of 5-10 cells appeared medium was changed to DMEM/F12 and Neurobasal mixed at a 1:1 ratio with 1% (v/v) B27 and 0.5% (v/v) N2, 100 ng/ml noggin, 0.5 µM LDN-193189, 10 µM SB-491542, 2 µM
CHIR-99021, 10 µM forskolin and 10 ng/ml bFGF for 3-10 days. Subsequently, the cells were cultivated for additional 5-10 days without small compound inhibitors but in the presence of 10 ng/ml bFGF.
VII.3.1.10. Small molecule inhibitors
All small molecule inhibitors were obtained from Selleck Chemicals as powder. Vemurafenib (PLX4032), trametinib (GSK1120212), LDN193189 and SB431542 were dissolved in DMSO to a stock concentration of 10 mM, CHIR99021 was dissolved in DMSO to a 30 mM stock solution. Aliquots of the inhibitors solutions were stored at -20°C and applied at the indicated concentrations.
Inhibitor Target Targeted pathway Resolvent
PLX4032 BRAFV600E MAPK DMSO
GSK1120212 MEK1/MEK2 MAPK DMSO
CHIR99021 GSK-3α/β GSK-3β DMSO
LDN193189 ALK2, ALK3 BMP4 DMSO
SB431542 ALK5, ALK4, ALK7 TGF-β DMSO