GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2728782

Query DataSets for GSM2728782

Status

Public on Jul 26, 2018

Title

A_0d-2

Sample type

SRA

Source name

Neural progenitor cells

Organism

Homo sapiens

Characteristics

cell type: hiPSC-derived neural progenitor cells
cell subtype: monolayer, day 0
cell line: hiPSC-NPC A

Treatment protocol

hiPSC-NPC were expanded and dislodged with trypsin into a cell suspension, as described above. This cell suspension was passed through a 70 µm nylon strainer (Millipore) prior to bioreactor inoculation, in order to eliminate cell clumps. The obtained single cell suspension was diluted for a cell density of 4x105 cell/mL in aggregation medium (AM), with the same composition as EM, except for reduced EGF/FGF concentration (5 ng/mL) and the addition of 5 μM Y-27632. Cells were then inoculated into software-controlled stirred-tank DASGIP® Bioblock bioreactor system (Eppendorf). Culture conditions were set to maintain cells under 3 % dissolved oxygen (15 % of air with 21 % of oxygen), pH 7.4, 37 ºC and a stirring rate of 70 rpm. In order to control the aggregate size and avoid aggregate fusion, the stirring rate was gradually increased up to 90 rpm with 10 rpm steps, based on visual inspection of the culture. After 48 hours of culture, perfusion operation mode was activated, with a dilution rate of 0.33 day-1 (i.e., 33 % working volume exchange per day) under gravimetric control. To prevent the loss of aggregates through the outlet perfusion line, a metallic filter of 20 μm pore size was adopted as cell retention device. After a 7 day aggregation period with AM, differentiation was induced by replacing the perfusion medium with differentiation medium (DM), maintain the culture for further 23 days (total of 30 days). DM was prepared by supplementing DMEM/F12 with Glutamax with 2 % B27 supplement, 1.6 μg/mL glucose, 10 μg/mL insulin, 10 μg/mL putrescin, 63 ng/mL progesterone, 50 μg/mL apotransferrin, 50 ng/mL sodium selenium (all from Sigma-Aldrich) and 200 mM ascorbic acid (Wako).

Growth protocol

Expansion of hiPSC-NPC was performed on poly-L-ornithine-laminin (PLOL)-coated surfaces. PLOL coating was prepared by performing a 3 hour incubation at 37°C with 0.16 mg/mL solution of poly-L-ornithine in PBS (with Ca2+ and Mg2+), followed by a washing step and a 3 hour incubation at 37°C with 1 μg/mL solution of laminin in PBS (with Ca2+ and Mg2+). hiPSC-NPC were cultured in expansion medium (EM), composed of DMEM/F12 media with Glutamax (Life Technologies) supplemented with 1% N2 supplement (Life Technologies), 0.1% B27 supplement (Life Technologies), 1.6 μg/mL glucose (Sigma-Aldrich), 20 μg/mL insulin (Sigma-Aldrich), 20 ng/mL rhu-bFGF (Peprotech), 20 ng/mL rhu-EGF (Sigma-Aldrich). Cells were split typically every 4-5 days at 90-100% confluence. Cells were dislodged through incubation with 0.05% Trypsin-EDTA for 1-2 minutes. Cells were ressuspended in DMEM supplemented with 10% FBS (Life Technologies), sedimented by centrifugation and the resultant pellet ressuspended in EM. Cell concentration and viability were determined by the trypan blue exclusion method in a Fuchs-Rusenthal hemocytometer. The cell suspension was used to inoculate PLOL-coated T-flasks, at a cell density of 3 x 104 cell/cm2. A 50 % media exchange was performed at day 2 of culture. Cells were maintained under humidified atmosphere, in a multi-gas cell incubator (Sanyo), with 5 % CO2 and 3 % O2, at 37 °C.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions and quantified using a NanoDrop 2000c (Thermo Scientific)
Libraries were prepared using the TruSeq RNA Sample Preparation v2 kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

hiPSC-NSC_3D diff_processed.txt

Data processing

The raw sequences were mapped to the Ensembl Human Genome Assembly GRCh38.p10 using StrangNGS software (http://www.strand-ngs.com/).
After mapping and counting, the data were normalized using quantile using StrandNGS.
Genome_build: Ensembl Human Genome Assembly GRCh38.p10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

Submission date

Aug 01, 2017

Last update date

May 15, 2019

Contact name

Daniel Simão

E-mail(s)

dsimao@itqb.unl.pt

Organization name

iBET, Instituto de Biologia Experimental e Biológica

Street address

Av. República, Qta. do Marquês, Estação Agronómica Nacional, Edificio IBET/ITQB