Dataset in support of the generation of Niemann-Pick disease Type C1 patient-specific iPS cell lines carrying the novel NPC1 mutation c.1180T>C or the prevalent c.3182T>C mutation – Analysis of pluripotency and neuronal differentiation

Data presented in this article demonstrate the generation and characterization of two novel Niemann-Pick disease Type C1 (NPC1) patient-specific induced pluripotent stem cell (iPSC) lines, related to the research article Trilck et al. (Diversity of Glycosphingolipid GM2 and Cholesterol Accumulation in NPC1 Patient-Specific iPSC-Derived Neurons; Brain Res.; 2017; 1657:52-61. doi: 10.1016/j.brainres.2016.11.031). For reprogramming fibroblasts, carrying the novel homozygous mutation c.1180T>C and the prevalent homozygous mutation c.3182T>C, were used. Reprogramming into patient-specific iPSCs was induced by retroviral transduction of the transcription factors Sox2, Klf4, Oct4 and c-Myc, and confirmed according to their pluripotency. The iPSCs were subsequently differentiated into neural progenitor cells, which were terminally differentiated into functional neurons and glial cells. The generation of these cell lines provides further valuable tools to investigate pathogenic mechanism of NPC1 in human neuronal cells carrying different NPC1 mutations.


Value of the data
Data in support of the generation of patient-specific iPSCs carrying novel NPC1 mutation. Data demonstrating the pluripotency of generated iPSCs and derived neural progenitors. Data showing differentiation of neural progenitors into neurons and glial cells. Data describing the establishment of human cell model applicable in NPC1 disease modeling and drug discovery.

Data
These data show the generation of NPC1 patient-specific induced pluripotent stem cells (iPSCs) using a standardized protocol [1]. Data presented in this article are related to the research article Trilck et al. [2]. Fibroblasts of NPC1 patients, carrying the homozygous NPC1 mutation c.1180T 4C or c.3182T4C, were used for reprograming. The c.1180T 4C is a novel unpublished mutation while mutation c.3182T 4 C is the most prevalent mutation of the NPC1 gene found in patients [3]. Data of the new cell lines were compared with a control cell line for examination of differences in reprogramming and pluripotency which might occur due to NPC1 mutation. Data demonstrate the pluripotency of the generated iPSCs by immunocytochemical detection of a panel of specific pluripotency Fig. 1. Examination of pluripotency of iPSCs and karyotype. Alkaline phosphatase (AP) staining (upper row). Photographs of immunocytochemistry, taken with a Keyence Biozero 800 microscope, show iPSC colonies being positive for pluripotency specific marker Nanog (red), Tra-1-60, Tra-1-81, SSEA3 and SSEA4 (all green). Scale bars in lower row are representative for all photographs and indicate 100 mm. Phase contrast picture of karyotype by Giemsa trypsin banding (last row) indicate no abnormalities due to reprogramming. No scale is given for karyotypes. marker ( Fig. 1). Pluripotency was further shown by induction of embryoid bodies and the immunocytochemical detection of nestin, muscle actin and α-feto protein (αFP), specific markers for the three germ layers (Fig. 2). Data present the differentiation of iPSCs into neural progenitor cells (Fig. 3). Terminal neuronal differentiation in neurons and in glial cells is shown by immunocytochemical detection of the neuron specific marker βIII-tubulin (βIII-tub) and synaptophysin (Fig. 5) and the glial cell specific marker GFAP (Fig. 4). Data obtained by patch clamp recordings show differentiation of neural progenitor cells into functional neurons (Fig. 5).

Reprogramming of fibroblasts into iPSCs
Human iPSCs were generated as described earlier [1]. In brief, retroviruses were produced in HEK293FT cells using a retroviral vector encoding for GFP and one of the transcription factors (Sox2, Klf4, Oct4, or c-Myc) as well as VSV-G and Gag-Pol by XtremeGene 9 (Roche, Germany). Virus titering was performed through FACS analysis of the percentage of GFP-positive HEK293FT cells. The human fibroblasts were transduced with Sox2, Oct4, Klf4 (70-80% infection efficiency), and c-Myc (40-50% infection efficiency) in the presence of 5 mg/ml protamine sulfate (in fibroblast medium). After 48 h, cells were reseeded onto a gelatin-coated 6 cm-dish. The following day, medium was replaced with iPSC 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. Initial iPSC colonies were picked and further cultivated on 45,000 feeder cells/cm 2 until a stable cell line was expanded.

Karyotyping
Karyotyping was performed by Giemsa Trypsin banding. Colonies were incubated with colcemid solution (10 mg/ml in HBSS) overnight for metaphase arrest. IPSCs were harvested by trypsin treatment (0.25%), wherein the reaction was stopped with Amniomax solution (Invitrogen, Germany), centrifuged at 300g for 10 min and the pellet was resuspended in 4 ml KCl solution (5.62%). Cells were incubated for 5 min at 37°C and centrifuged at 300g for 10 min. After resuspension cells were fixed in 5 ml glacial acetic acid and methanol (1:3) and subsequently centrifuged for 7 min at 350g. This step was repeated once. Next, 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.

Generation of embryoid bodies
Embroid body (EB) formation was used for proving pluripotency of iPSCs by using marker for the three germ layers. Whole iPSC colonies were mechanically lifted off the feeder cell layer and transferred into the cavity of a low attachment 6-well plate in 5 ml of differentiation medium (knockout DMEM, 20% FBS, 1% MEM non-essential amino acids, 2 mM GlutaMAX, and 0.1 mM beta-mercaptoethanol). EBs were formed after five to seven days and reseeded onto gelatin-coated glass cover slips. After 10 days of random differentiation, spread cells were fixed with 4% PFA for 15 min and immunocytochemically stained for muscle actin (MA, 1:50, mouse IgG, Dako, Denmark), alpha fetoprotein (aFP, 1:500, mouse monoclonal IgG, Sigma-Aldrich, Germany) and nestin (1:100, mouse IgG, R&D, Germany). Alexa Fluor 488 (1:500, goat anti-mouse IgG (Invitrogen, Germany) was used as secondary antibody, incubated 1 h at room temperature in PBS. Cells were washed three times with PBS and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system (Keyence, Germany). Teratoma formation assay in immune deprived mice was not performed any more, following the 3Rs principle, to replace, refine or reduce, the use of animals, mandated by the German Protection of Animal Act. In regards of the teratoma formation assay in stem cell research the applicability of the in vitro embryoid body formation assay is discussed by Sheridan and colleagues [4].

Patch clamp recordings
Patch clamp recordings were performed using an EPC-10 amplifier (Heka, Germany). Patch pipettes were pulled from borosilicate glass tubing (Harvard Apparatus, USA). The internal solution contained (mM): KCl 130, NaCl 10, HEPES 10, EGTA 11, MgCl 2 x6H 2 O 1, CaCl 2 xH 2 O 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, CaCl 2 xH 2 O 2, MgCl 2 x6H 2 O 1, NaHCO 3 26, NaH 2 PO 4 xH 2 O 1.25, glucosexH 2 O 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 (V H ) of À 60 or À80 mV. Current voltage responses were evoked by applying 100 ms voltage steps from À60 mV to þ50 mV in 10 mV increments. 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 V H of À 60 mV. Mini Analysis 6 (Synaptosoft, USA) was used to analyze recordings of post-synaptic currents.