Human dermal fibroblast cell lines GM18436 and GM05659 (Coriell Institute for Medical Research, Camden, USA) were obtained by skin biopsies from one-year old male Caucasian donors. GM18436 exhibits compound heterozygous mutations in the NPC1 gene (c.1628delC and GLU612ASP), representing a frameshift mutation and a missense mutation, respectively. The mutations lead to a non-functional protein as demonstrated by cholesterol esterification assay . Fibroblast cell line GM05659 is obtained from a healthy donor. In the following cells of the cell line GM18436 will be referred to as mutNPC1 and cells of the cell line GM05659 will be referred to as wtNPC1. Cells were cultivated in fibroblast medium containing DMEM high glucose, 10% FBS and 1% Penicillin/ Streptomycin. Mitotically inactivated mouse embryonic fibroblasts (GlobalStem, Rockville, USA) were used as the feeder cell layer for hiPSCs. Cells were plated in fibroblast medium at a density of 33.000 cells/ cm2 onto 0.1% gelatine coated wells in fibroblast medium 24 h before hiPS cell split. HiPS cells were cultured on a feeder cell layer in iPS medium containing DMEM/ F12, 20% knockout serum replacement, 1% Penicillin/ Streptomycin, 1% GlutaMAX, 1% MEM non essential amino acids, 0.2% 2-mercaptoethanol, and 10 to 15 ng/ ml hFGF-2 (Globalstem, Rockeville, USA). hiPS cells on matrigel (BD Biosciences, Heidelberg, Germany) were cultured in mTESR1 medium (Stemcell Technologies, Grenoble, France). Medium was changed daily and cells were passaged weekly using 10 μM ROCK inhibitor Y-27632 (Stemgent, Cambridge, USA) for increased plating efficiency. HiPS cells growing on a feeder cell layer were split mechanically weekly using pulled glass hooks by performing the cut and paste technique. Cells growing on matrigel were harvested enzymatically using 1 mg/ ml dispase (Stemcell Technologies, Grenoble, France) for 7 min and large bore tips to break down large clumps according to manufacturer’s recommendations. HEK293FT cells (Invitrogen, Darmstadt, Gemany) used to obtain the viral vectors were cultivated in fibroblast medium without Penicillin/ Streptomycin. All cells were cultivated at 37°C in a saturated humidity atmosphere containing 5% CO2.
Generation of retroviruses
Retroviral pMIG vectors containing the cDNA of the human genes Oct4, Sox2, Klf4, and c-Myc were used as described recently . Briefly, 3×106 HEK293FT cells per 10 cm-dish were seeded onto ten dishes and incubated overnight. A solution containing 2.5 μg retroviral vector encoding for GFP and one of the transcription factors (Sox2, Klf4, Oct4, or c-Myc) was incubated with 0.25 μg VSV-G and 2.25 μg Gag-Pol in X-tremeGENE9 (Roche, Mannheim, Germany)/ DMEM High Glucose mixture (1:4) which was added to each of the dishes. Medium was renewed after 18 h and cells were incubated further for 48 h. Subsequently, the virus-containing medium was collected and passed through a 0.45 μm filter. To concentrate the virus, the medium was centrifuged at 70.000 × g at 4°C for 90 min, resuspended in 0.1 to 1 ml DMEM medium, and stored at −80°C. All four vectors contained a GFP sequence thus enabling titering by determining the percentage of GFP positive HEK293FT cells using FACS. Therefore, 1×105 HEK293FT cells were seeded per 12-well in Penicillin/ Streptomycin free fibroblast medium containing 5 μg/ ml protamine sulfate and concentrated virus in the following volumes: 6.25 μl, 12.5 μl, 25 μl, and 50 μl. After 48 h cells were washed with PBS containing Ca2+/ Mg2+, trypsinized and centrifuged for 5 min at 500 × g. Pellet was resuspended in 100 μl PBS without Ca2+/ Mg2+ and fixed by adding 100 μl of 4% paraformaldehyde for 15 min. Afterwards, the percentage of GFP positive cells was determined via FACS analysis.
Transduction of human fibroblasts
For transduction, 1×105 fibroblasts were seeded per cavity of a 6-well plate and cultured for 18 h in fibroblast medium without Penicillin/ Streptomycin. Afterwards, fibroblast medium without Penicillin/ Streptomycin was supplemented with a volume of retrovirus of Sox2, Oct4, Klf4 (corresponding to 70 – 80% infection efficiency), and c-Myc (corresponding to 40–50% infection efficiency) in the presence of 5 μg/ ml protamine sulfate. Cells were incubated for 48 h. Subsequently, medium was aspirated and cells were washed twice with PBS containing Ca2+/ Mg2+. Transduced cells were trypsinized and reseeded onto a gelatin coated 6 cm-dish. The next day, medium was replaced with iPS medium supplemented with 0.5 mM valproic acid to further increase the efficiency of reprogramming. Medium was changed daily and valproic acid was omitted after seven days.
Generation of hiPS cell lines
Initial hiPS colonies were routinely observed after three to four weeks. For further cultivation, they were picked using a 100 μl pipette tip and a pulled glass hook. Single colonies were transferred to the cavities of a 24-well plate, coated with 0.1% gelatin and 45.000 feeder cells/ cm2. After 4 to 7 days of proliferation hiPS colonies were mechanically divided into two to four pieces and further expanded. Within six weeks each single hiPS colony was expanded to obtain different clones.
Karyotyping was performed by Giemsa Trypsin banding. In short, colonies were incubated with a colcemid solution (10 μg/ ml in HBSS) for three hours to arrest cells in metaphase. Cells were treated with trypsin (0.25%) and the enzymatic reaction was stopped with Amniomax solution (Invitrogen, Darmstadt, Germany). Cells were centrifuged at 300 × g for 10 min and the pellet was resuspended in 4 ml hypotonic potassium chloride solution (5.62%). Cells were incubated for 5 min at 37°C and centrifuged at 300 × g for 10 min. The cells were resuspended and fixed in 5 ml glacial acetic acid and methanol (1:3) and subsequently centrifuged for 7 min at 350 × g. This step was repeated once. Finally, most of the supernatant was removed and cells were resuspended. Cell suspension was dropped onto cold slides and dried at 100°C for 1 h. Giemsa solution (5%) was added and incubated for 5 min. Slides were washed in distilled water two times, dried at room temperature and sealed with cover slips.
Genomic DNA of fibroblasts, iPS cells grown on matrigel or neural progenitor cells were isolated using AllPrep Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Exon regions were amplified using HotStart Taq (Qiagen, Hilden, Germany) as follows: 95°C for 15 min followed by 13 cycles of 94°C for 30 s, 66°C for 30 s with 1.5°C decrease/ cycle and 72°C for 20 s; followed by 8 cycles of 94°C for 30 s, 46.5°C for 30 s with 1°C increase/ cycle and 72°C for 20 s; followed by 13 cycles of 94°C for 30 s, 66°C for 30 s with 1.5°C decrease/ cycle and 72°C for 20 s; followed by 11 cycles of 94°C for 30 s, 54.5°C for 30 s with 1°C increase/ cycle and 72°C for 20 s. Products were purified using ExoSAP Kit (USB Europe GmbH, Staufen, Germany) according to manufacturer’s recommendations. Sequence analysis was performed on a 3130XL Genetic Analyzer (Applied Biosystems, Carlsbad, USA).
Alkaline phosphatase staining
HiPSCs were cultivated on a feeder cell layer for five days. Medium was removed, cells were washed with PBS and fixed with ice-cold methanol (100%) for 10 min at −20°C. Methanol was removed and cells were washed with PBS. Subsequently, cells were incubated at room temperature for 15 min with the staining solution: 75% distilled water, 10% sodium chloride solution (1 M), 10% Tris solution (1 M, pH 9.8), 5% magnesium chloride solution (1 M), and NBT/ BCIP solution (1:50, Roche, Mannheim, Germany). Staining solution was removed and cells were washed with distilled water. Microphotographs were taken using a Nikon Eclipse TS100 (Nikon, Düsseldorf, Germany).
Cells were fixed at room temperature for 15 minutes in 4% paraformaldehyde, washed with PBS and stored in 0.02% NaN3 at 4°C. Immunocytochemistry was performed for Nanog (1:100, rabbit IgG polyclonal), Oct4 (1:100, rabbit IgG polyclonal), SSEA3 (1:100, rat IgM), SSEA4 (1:100, mouse IgG3), Tra-1-60 (1:100, mouse IgM), Tra-1-81 (1:100, mouse IgM, all Stemgent, Cambridge, USA), Smooth muscle actin (SMA, 1:50, mouse monoclonal, Dako, Glostrup, Denmark), alpha fetoprotein (alpha FP, 1:500, mouse monoclonal IgG, Sigma-Aldrich, Hamburg, Germany), Nestin (1:100, mouse monoclonal, R&D, Wiesbaden, Germany), MAP2ab (1:100, mouse monoclonal, Chemicon, Schwalbach, Gemany), Tuj1 (1:100, mouse monoclonal Tu-20, Santa Cruz biotechnology, Heidelberg, Germany) and Sox-2 (1:200, rabbit monoclonal, Abcam, Cambridge, UK). Blocking and permeabilization was carried out using 0.3% Triton X-100 and 5% normal goat serum (Dako, Glostrup, Denmark) for 30 min at room temperature. Cells were incubated with primary antibodies for 3 hours at room temperature in 1% normal goat serum, followed by three washing steps with PBS. Alexa Fluor 568 (1:1000, goat anti-mouse IgG or goat anti-rabbit IgG, Invitrogen, Darmstadt, Germany), Alexa Fluor 488 (1:1000, goat anti-mouse IgG or goat anti-rabbit IgG, Invitrogen, Darmstadt, Germany), or Alexa Fluor 488 (1:1000, goat anti-mouse IgM or goat anti-rat IgM, Invitrogen, Darmstadt, Germany) were used as secondary antibodies, incubated 1 h at room temperature with 1% normal goat serum in PBS. After washing with PBS, cells were stained with DAPI (5 minutes, 250 ng/ ml), washed three times and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system (Keyence, Hamburg, Germany).
Generation of embryoid bodies
To generate embryoid bodies (EBs), whole hiPS colonies were mechanically lifted off the feeder cell layer and transferred into a 15 ml conical tube. Once the colonies settled at the bottom of the conical tube, the medium was removed and 5 ml of differentiation medium, containing knockout DMEM, 20% FBS, 1% MEM non-essential amino acids, 2 mM GlutaMAX, and 0.1 mM beta-mercaptoethanol, was added. Afterwards, colonies were transferred into the cavity of a low attachment 6-well plate and incubated at 37°C/ 5% CO2. Medium was changed every second day until EBs were formed. After five to seven days EBs were transferred onto gelatin coated glass cover slips and supplied with differentiation medium. Once EBs were attached, medium was changed every second or third day. After 10 days of random differentiation, spread cells were washed with PBS and fixed with 4% PFA for 15 min. Fixed cells were washed with PBS and immunocytochemical stainings for nestin (ectoderm), smooth muscle actin (mesoderm), and alpha-fetoprotein (endoderm) were performed.
Teratoma formation assay
Immunodeficient (SCID) hairless mice (Charles River Laboratories, Sulzfeld, Germany) were used for the teratoma formation assay. HiPSCs for injection were cultured on feeder cells in 6-well culture plates. For each injection the amount of 3 cavities of a 6-well culture plate were collected mechanically and centrifuged for 2 min at 200 × g. The pellet was resuspended in 1 ml of 0.25% trypsin/ EDTA. After 1 min, the reaction was stopped by adding 2 ml of fibroblast medium and centrifuged again for 2 min at 200 × g. Cells were resuspended in 140 μl of cold DMEM/ F12 and stored on ice. Directly before injection, cell suspension was mixed with 60 μl matrigel. Cells were injected subcutaneously into the flank of the hind limb. After 8–12 weeks, when tumors were clearly visible, the animals were sacrificed and tumors were removed. Tumor tissue was fixed in 4% formalin for 12 to 18 hours and embedded in paraffin for subsequent staining.
H&E staining of tumor sections
4 μm thick tumor tissue sections were deparaffinized in xylol for 10 min and a descending ethanol concentration for 5 min each. Afterwards, the sections were washed in distilled water and stained with Mayers hematoxylin (Merck, Darmstadt, Germany) for 1 min. Next, the tissue was washed two times in distilled water and stained with eosin Y (Sigma-Aldrich, Hamburg, Germany) for 2 min. The slides were washed again twice and then dehydrated using an ascending ethanol concentrattion and xylol. Slides were mounted in Mowiol-DABCO. Microphotographs were taken with a Biozero 8000 microscope system (Keyence, Hamburg, Germany).
To differentiate hiPS colonies into neural direction, the colonies were cut, transferred to Poly-L-ornithine (15 μg/ ml)/ laminin (10 μg/ ml) coated dishes, and cultivated for 10 days in medium consisting of Neurobasal, DMEM/ F12, 1xN2 supplement, 1xB27 supplement, GlutaMAX (2 mM) complemented with mouse recombinant noggin Fc-chimera (500 ng/ ml, R&D, Wiesbaden, Germany), SB431542 (20 μM, Sigma-Aldrich, Hamburg, Germany) and hFGF-2 (5 ng/ ml, GlobalStem, Rockeville, USA). Neural rosettes were manually isolated using pulled glass hooks, gently trypsinized, and seeded as single cells on Poly-L-ornithine/ laminin coated dishes in medium consisting of Neurobasal, DMEM/ F12, 1×N2, 1×B27, and GlutaMAX (2 mM) supplemented with hFGF-2 (10 ng/ ml) and hEGF (10 ng/ ml, Peprotech, Hamburg, Germany). Neural progenitor cells were seeded at high densities (100–150.000 cells/ cm2) and passaged one day after reaching confluence using Trypsin/ Benzonase. Differentiation was induced by seeding the cells at a density of 50.000 cells/ cm2 and withdrawal of growth factors in the presence of BDNF (20 ng/ ml, Peprotech, Hamburg, Germany).
Patch clamp recordings
Patch clamp recordings were performed using an EPC-10 amplifier (Heka, Lambrecht, Germany). Patch pipettes were pulled from borosilicate glass tubing (Harvard Apparatus, Holliston, USA). The internal solution contained (mM): KCl 130, NaCl 10, HEPES 10, EGTA 11, MgCl2×6H2O 1, CaCl2×H2O 2, Mg-ATP 2. pH was adjusted to 7.2. When filled, electrodes had a resistance of 6–8 MΩ. Cell cultures were continuously superfused with an extracellular solution, consisting of (mM): NaCl 125, KCl 2.5, CaCl2×H2O 2, MgCl2×6H2O 1, NaHCO3 26, NaH2PO4×H2O 1.25, glucose×H2O 25. Solution was continuously bubbled with carbogen to maintain a pH of 7.4. Recordings were made in the whole cell configuration with holding potentials (VH) of −60 or −80 mV. Current voltage responses were evoked by applying 100 ms voltage steps from −60 mV to +50 mV in 10 mV increments. Data were filtered at 3 kHz, digitized and stored on-line using Pulse (Heka, Lambrecht, Germany). Na+ and K+ currents were identified via their I-V relationship. Na+ currents were antagonized in some experiments by TTX (1 μM). Current clamp mode was used to apply current steps to induce action potentials or to measure spontaneous action potentials. Postsynaptic currents were measured in the voltage clamp mode at a VH of −60 mV. Mini Analysis 6 (Synaptosoft, USA) was used to analyse recordings of post-synaptic currents. Data are given as mean ± SEM.
Filipin is a polyene antibiotic which binds to free cholesterol and is widely used to analyze the sequestration of unesterified cholesterol in NPC1-deficient cells. Therefore, cells were fixed with 4% paraformaldehyde (in PBS) for 15 min, washed with PBS and incubated at room temperature for 45 min in the dark with a staining solution containing 100 μg/ ml Filipin (Polysciences, Eppelheim, Germany) in PBS. Cells were washed twice with PBS for 5 min. Slides were mounted using Mowiol-DABCO and sealed with cover slips. After 12 h of drying in the dark at room temperature, fluorescence pictures were taken with a Biozero 8000 microscope (Keyence, Hamburg, Germany).
Amplex red assay
To quantify the amount of cholesterol we used the Amplex Red cholesterol assay . Fibroblasts, iPS cells grown on matrigel and neural progenitor cells were harvested in PBS/ SDS (0.1%) at room temperature, sheared through a 27 G needle and cholesterol levels were determined using the Amplex Red cholesterol assay kit (Molecular Probes, Darmstadt, Gemany) according to the manufacturer’s instructions. Protein concentrations in lysates were measured using the bicinchoninic acid assay (BCA, Pierce, USA).
Analysis of the data was carried out with GraphPad Prism 5 (GraphPad Software Inc., USA). Data are given as mean ± SEM. Unless otherwise stated, unpaired t-tests were used to test for significance, with * = p<0.05 and ** = p<0.01, *** = p<0.001.