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ES to neuron

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Series:IN_VITRO DIFFERENTIATION SERIES
Species:Mouse (Mus musculus)
Genomic View:Zenbu
Expression table:TET
Link to TET:FILE
Sample providers :Ana Pombo
Germ layer:ectoderm
Primary cells or cell line:primary cells
Time span:4 days
Number of time points:5


Overview

The differentiation from Embryonic Stem (ES) cells to specialized cells is one of the most important fields of research in modern cell biology. Stem cell therapies promise cures for a plethora of complex diseases such as neurodegeneration. ES cells have the potential to differentiate into any of the 200 different cell types that make up higher organisms. A key challenge towards understanding the mechanisms by which the different programs of gene expression are established during differentiation is the development of stem cell therapies and differentiation protocols into specific cellular lineages. To this end, we are interested in understanding the mechanisms that control important developmental regulator genes in ES cells, and the dynamic changes that occur during early cell differentiation to neuronal lineages.

Sample description

Cells were grown at 37°C in a 5% (v/v) CO2 incubator. Mouse ES cells (cell line ES-46C; ES cell line E14tg2a expressing GFP under Sox1 [1] were grown in GMEM medium (Invitrogen, # 21710025), supplemented with 10% (v/v) fetal calf serum (FCS; PAA, # A15-151), 2 U/ml LIF (Millipore, # ESG1107), 0.1 mM β-mercaptoethanol (Invitrogen, # 31350-010), 2 mM L-glutamine (Invitrogen, # 25030-024), 1 mM sodium pyruvate (Invitrogen, # 11360039), 1% penicillin-streptomycin (Invitrogen # 15140122), 1% MEM Non-Essential Amino Acids (Invitrogen, # 11140035) on gelatin-coated (0.1% (v/v)) Nunc T25 flasks. The medium was changed every day and cells were split every other day.

To investigate the early phases of neuronal commitment immediately after the cells exit from pluripotency, we followed a previously published protocol that provides a highly synchronous differentiation [2]. For the first time point, corresponding to day 0, 1.6x106 ES-46C cells were plated on gelatin-coated (0.1% (v/v)) Nunc 10 cm dishes in serum-free ESGRO Complete Clonal Grade Medium (Millipore, # SF001-500), containing 1U/ml LIF. For samples from day 1 to day 4, we started a monolayer protocol, where ES-46C cells are plated in serum-free medium ESGRO Complete Clonal Grade medium at high density (1.5x105 cells/cm2). After 24 hours, ES-46C cells were gently dissociated and plated onto 0.1% (v/v) gelatin-coated Nunc 10 cm dishes (1.6x106 cells per dish) in RHB-A media (StemCell Science Inc., # SCS-SF-NB-01). Media was changed every day.

All time point samples were processed for RNA at the same time relative to medium-change. After medium removal, TRIzol (Invitrogen, # 15596-018) was added directly to the dish and samples were treated following the manufacturer's instructions. Total RNA was treated with TURBO DNase I (Ambion, # AM1907) according to the manufacturer’s instructions. Treated RNA (1 μg) was reverse transcribed with 50 ng random primers and 10 U reverse transcriptase (Superscript II kit, Invitrogen, # 18064-014) in a 20 μl reaction. The synthesized cDNA was diluted 1:10, and 2.5 μl used for qRT-PCR for quality control.

References:
[1] Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Nat Biotechnol. 2003 Feb;21(2):183-6. Epub 2003 Jan 13.
[2] Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo. Abranches E, Silva M, Pradier L, Schulz H, Hummel O, Henrique D, Bekman E. PLoS One. 2009 Jul 21;4(7):e6286. doi: 10.1371/journal.pone.0006286.

Quality control


Figure 1: qRT-PCR expression of stage-specific markers

The time course was tested by qRT-PCR using stage-specific markers accordingly with [2]. QC shows that we achieved a synchronous and efficient progression of ES cells through the early differentiation steps into neuronal lineage. After 24 h, the levels of expression of the pluripotency marker Nanog is consistently decreased. Another relevant pluripotency marker, Oct4, is down-regulated at a slower rate, but shut down by day 4. A peak of expression of Fgf5, primitive ectoderm (PE) marker, shows an expected intervening state, before increasing levels of Blbp and Hes5 from day 2 to day 4 confirms the switch from ES cell identity to neural precursor cells by day 4. Results are normalized to ß-actin, and represent the mean and standard deviations from two biological replicates.

Figure 2: CAGE expression of marker genes in TPM.

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples



Sample Name Time point Donor/Replica
14357-155I1 ES-46C embryonic stem cells, neuronal differentiation day00 biol_rep1
14358-155I2 ES-46C embryonic stem cells, neuronal differentiation day01 biol_rep1
14359-155I3 ES-46C embryonic stem cells, neuronal differentiation day02 biol_rep1
14360-155I4 ES-46C embryonic stem cells, neuronal differentiation day03 biol_rep1
14361-155I5 ES-46C embryonic stem cells, neuronal differentiation day04 biol_rep1
14362-155I6 ES-46C embryonic stem cells, neuronal differentiation day00 biol_rep2
14363-155I7 ES-46C embryonic stem cells, neuronal differentiation day01 biol_rep2
14364-155I8 ES-46C embryonic stem cells, neuronal differentiation day02 biol_rep2
14365-155I9 ES-46C embryonic stem cells, neuronal differentiation day03 biol_rep2
14366-156A1 ES-46C embryonic stem cells, neuronal differentiation day04 biol_rep2