SUPPORTING INFORMATION

3.4.6 MATERIAL AND METHODS mESC culture

mESC maintenance culture was performed as described in Kuegler et al. (Kuegler et al. 2011). Briefly, the mESC lines CGR8 and E14.1 were maintained on 0.1% gelatine coated-cell culture plastic (Nunc) in GMEM containing high glucose (4.5 g/L), 10%

fetal bovine serum (FBS; PAA, Pasching, Austria), 2 mM GlutaMax, 2 mM sodium pyruvate, 2 mM non-essential amino acids, 50 µM β-mercaptoethanol and 1000 U/ml LIF (1000 U/ml, Millipore, Billerica, MA, USA) and passaged 1/10 every other day.

Medium with fresh LIF was fed every day.

Differentiation to NPC

For induction of neuralisation, a protocol supplied by Ying et al. (Ying and Smith 2003) was modified. In brief, mESC were seeded at 10.000 cells/cm² into 0.01%

gelatine coated 10 cm² dishes (Nunclon, Nunc, Roskilde, Denmark) in N2B27 medium (50% DMEM/F12, 50% Neurobasal medium, 2 mM Glutamax, 0.5x B27 supplement, 0.5x N2 supplement, 20 µg/ml insulin, 50 µg/ml BSA) under adherent conditions for 7 days. Medium was changed every other day. At day 7, the cells were washed with PBS and replated onto Nunclon cell culture flasks which had been coated with 0.1%

gelatine over night into N2B27 medium supplemented with 20 ng/ml EGF and 20 ng/ml bFGF (both R&D Systems, Minneapolis, MN, USA). The cells were fed every other day and passaged at 50-70% confluency. Areas of unwanted differentiation were manually removed using a pipette tip before feeding/replating.

Differentiation to astrocytes

For astroglial differentiation, the cells were trypsinised and replated onto polyornithin/laminin coated cell culture plates at densities of 30.000-50.000 cells/cm² in N2B27 medium supplemented with CNTF (20 ng/ml) and BMP4 (20 ng/ml) (both R&D Systems) for up to five days. The cells were fed every three days.

LUHMES cell culture

Culture conditions for the fetal mesencephalic neuronal cell line LUHMES (Lund human mesencephalic) were modified from Scholz et al. (Scholz et al. 2011).

LUHMES were maintained in proliferation medium (Advanced Dulbecco’s modified Eagle’s medium/F12, 1x N2-supplement (Invitrogen, KA, Germany), 2 mM GlutaMax (Gibco, Rockville, MD, USA) and 40 ng/ml recombinant basic fibroblast growth factor (R&D Systems) on poly-L-ornithine (50 µg/ml) and fibronectin (10 µg/ml, Sigma-Aldrich, St. Louis, MO, USA) coated cell culture plastic (Nunclon). For maturation, the cells were pre-differentiated by medium switch to differentiation medium (Advanced Dulbecco’s modified Eagle’s medium/F12, 1x N2-supplement, 2 mM GlutaMax, 1 mM dibutyryl cAMP (Sigma Aldrich), 10 µg/ml tetracycline (Sigma-Aldrich) and 2 ng/ml recombinant recombinant human GDNF (R&D Systems). After 2 days, the predifferentiated LUHMES were trypsinised and seeded into the desired format. Unless stated otherwise, co-culture medium (Advanced Dulbecco’s modified Eagle’s medium/F12, 1x N2-supplement, 2 mM GlutaMax, 10 µg/ml tetracycline) was used for further differentiation and in co-cultures.

Co-culture of LUHMES and astrocytes

For co-cultures, NPC were differentiated to astrocytes as described above. On day 5, LUHMES cells were seeded in co-culture medium onto the differentiated astrocytes. If astrocytes thawed from cryopreservation were used, they were thawed on poly-L-ornithine/laminin coated plates or glass bottom chamber slides and grown in N2B27 medium for 2 days before LUHMES cells were added. Astrocyte-conditioned medium (N2B27) was conditioned in pure, confluent astrocyte cultures for 24-48 h and added in equal volumes to co-culture LUHMES medium (50:50). Non-conditioned N2B27 served as control.

Immunofluorescence analysis

Cultures were washed once with PBS, fixed with ice-cold MeOH for 20 min at -20 °C

33342 (Hoechst dye). The cells were imaged using an Olympus IX81 inverted microscope equipped with CellP Software.

Calcein staining of live cells was performed according to Lotharius et al.

(Lotharius et al. 2005). Calcein and H-33342 were added to live cells and imaged after 30 min.

Lentiviral transduction of NPC

mESC-derived NPC were transduced with an eGFP expression construct phsCEUemptyW (Figure 3.4-5), expressing a GFP gene under a cytomegalovirus (CMV) immediate early promoter. Lentivirus production was perfomed as described previously (Scholz et al. 2011). 70% confluent NPC cultures were incubated over night with 40 µl virus, concentrated 500-fold from HEK293 FT cell supernatant using the PEG-it^TM (System Biosciences). The medium was changed the following day.

Viral transduction was performed in 48 well format. The experiments were performed under biosafety level 2 conditions until passage 4.

HEK293 FT culture and virus production

The virus was constructed by Christiaan Karreman and produced and purified from HEK293 FT culture supernatant by Dominik Pöltl. HEK293 FT cells were grown in DMEM medium containing 10% FCS and 1 mM Glutamax and were split 1/10 twice a week. The experiments were performed under biosafety level 2 conditions.

Flow cytometry and FACS analysis

For flow cytometry or FACS analysis, 90% confluent NPC were detached from surface using trypsin. Trypsin was removed by pelleting at 500 x g for 5 min and the pellet was resuspended in N2B27 medium. For FACS analysis, penicillin and streptomycin (both 10 µg/ml) was added to the medium for at least 7 days after sorting.

Non-transduced cultures served as control. On day 3 after lentiviral transduction, flow cytometry analysis revealed 13.8% of the cells were GFP positive. After 4 passages the cells were sorted for GFP expression using a fluorescent activated cell sorter (FACS Aria II, BD).

MPTP conversion and HPLC analysis

Confluent day 5 cultures were used for 1-methyl-4-phenyl-tetrahydropyridine (MPTP)-conversion experiments. The cells were cultured over night in medium containing DMEM/F12, 2 mM Glutamax and 2% serum. MPTP was added to the medium directly. Supernatants were cleared by centrifugation, acidified with formic acid and filtered using a 22 µM filter. They were frozen in liquid nitrogen and stored at -80°C until further use. HPLC analysis was performed using a Bio-Tek Instruments HPLC equipped with a tuneable UV-detector (BIO-TEK instruments, GOEBEL Instrumentelle Analytik GmbH, Au/Hallertau, Germany) and a C18 reverse phase column. 12.5% acetonitril in water titrated to a pH of 2.3 using TEA was used as solvent. MPTP elution was detected at 245 nm and the metabolites 1-methyl-4-phenyl-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenyl-pyridinium (MPP⁺) were detected at 345 nm and 295 nm respectively (Di Monte et al. 1991). The amounts were quantified according to standard curves obtained from serial dilutions of MPTP, MPP⁺ (both Sigma Aldrich) or MPDP⁺ (mCAT, Konstanz, Germany).

Analysis of cell division

DNA of dividing cells was labelled using the Click-iT EdU Imaging Kit (Invitrogen).

In brief, cells were primed for 24-48 h using 5-ethynyl-2-deoxyuridine (EdU; 10 µM).

Afterwards, the cells were fixed using -20°C cold MeOH for 10 min and permeabilised with 0.5% Triton X-100 in PBS for 15 min. After washing in PBS, the wells were blocked using 3% BSA in PBS for 30 min and subsequently Click-iT reaction mix (azide-coupled fluorescein fluorophore) was clicked to the alkyne-sidechain of EdU for 30 min at RT in the dark. Nuclei were counterstained with H-33342. The cells were imaged on an Olympus IX81 inverted microscope equipped with CellP software and nuclei and EdU-positive cells were counted manually.

3.4.7 ACKNOWLEDGEMENTS

We are grateful for technical support by Heidrun Leisner for FACS analysis and Regina Pape for HPLC measurements. This work was supported by the DFG, the Doerenkamp-Zbinden Foundation, the Konstanz Research School Chemical Biology (PBK) and the RTG1331 (BZ).

4 General Discussion