dc-hiOL A
Homo sapiens
Protocols: Newborn skin (FibA, B) and fetal lung (FibC) fibroblasts were acquired from ATCC (PCS-201-010, CRL-2097 and CCL-15, respectively). Fibroblasts derived from three PPMS diseased individuals were kindly provided by Gianvito Martino. Fibroblasts derived from 20 year old male PMD (643C>T) patient were acquired from Coriell Institute (ID:GM0954). Fibroblasts derived from healthy adult male individual were used as control for PMD cells. The study was approved by the local ethical committees in Münster and Milan (AZ 2018-040-f-S). Fibroblasts were cultured in fibroblast medium (FM): Dulbecco's Modified Eagle Medium high glucose (Sigma) containing 10% FBS (Gibco), 1% nonessential amino acids (NEAA) (Sigma), 1% GlutaMAX (Gibco), and 1% penicillin/streptomycin solution (Sigma). All cell lines were negative for HIV, HCV, HBV and mycoplasma contamination. Fibroblasts were seeded at a density of 12x103 cells/cm2 on a Poly-L-lysine (Sigma) / mouse Laminin (Sigma) coated plate in FM. The next day cells were tranduced with the SF-SON lentivirus in fresh FM and 16h later the medium was changed to Glial Induction Medium (GIM). GIM consisted of DMEM-F12 (Gibco), 1:200 N2 supplement (Gibco), 1:100 B27 supplement lacking vitamin A (Gibco) and 1% penicillin/streptomycin/glutamine (Sigma) supplemented with 0,5 μM SAG (Cayman Chemical), 10 ng/mL PDGF (Peprotech), 200 μM AA (Sigma) and 20 ng/mL T3 (Sigma). GIM was changed every other day and the concentration of T3 was increased to 60ng/ml at day 4. Puromycin (1μg/ml, Gibco) selection of the transduced cells took place the first 5 days. At day 9 the GIM was replaced by glial differentiation medium (GDM) consisting of DMEM-F12, 1:200 N2 supplement, 1:100 B27 supplement lacking vitamin A, 1% penicillin/streptomycin/glutamine enriched by 0,5 μM SAG, 60 ng/mL T3, 10 ng/mL NT3 (Peprotech), 10 ng/mL IGF-I (Peprotech), 200 μM AA and 10 ng/mL PDGF. PDGF was replaced by 100 μM dbcAMP (Sigma) at day 12. Medium change was performed every other day. Conversion efficiency is defined as the number of O4+ cells divided by the total number of DAPI+ cells at day 16. Yield is defined as the number of O4+ cells at day 16 divided by the number of plated fibroblasts at day 0. Following total RNA extraction using the RNasy Mini Kit (Quiagen) and DNase (Quiagen) treatment the quality of the produced RNA was tested with Bioanalyzer analysis (Agilent). Samples with RIN value over 7 were used for RNA sequencing. The date of RNA Isolation is 06.09.2018. NEBNext® Poly(A) mRNA Magnetic Isolation Module was followed by a directional library preparation (NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®). The date of library preparation is 17.09.2018.
ENA-FIRST-PUBLIC
2020-01-31T04:02:42Z
ENA-LAST-UPDATE
2019-04-02T16:09:32Z
External Id
SAMEA5546666
INSDC center name
Max Planck Institute for Molecular Biomedicine
INSDC first public
2020-01-31T04:02:42Z
INSDC last update
2019-04-02T16:09:32Z
INSDC status
public
Submitter Id
E-MTAB-7838:dc-hiOL A
broker name
ArrayExpress
cell line
fibroblast derived cell line
cell type
directly converted O4+ human oligodendrocytes
common name
human
individual
dc-hiOL A
progenitor cell type
fibroblast of dermis
sample name
E-MTAB-7838:dc-hiOL A
scientific_name
Homo sapiens
sex
male