SAMN45108900
External Id
Istituto Italiano di Tecnologia
INSDC center name
2024-11-30T00:00:00Z
INSDC first public
2024-11-30T08:01:04.160Z
INSDC last update
SRS23401014
INSDC secondary accession
live
INSDC status
Model organism or animal
NCBI submission model
Model.organism.animal.1.0
NCBI submission package
Experimental Replicate 1
Replicate
SRS23401014
SRA accession
E14TG2a
cell_line
Mouse Embryonic Stem Cells
cell_type
Not collected
collection_date
Experimental Replicate 1
description
25 Days in vitro
dev_stage
Wild Type
genotype
N/A
geo_loc_name
For neural induction, a chemically defined minimal medium (CDMM) containing DMEM/F12 (Gibco, 11320033), 2mM Glutamine, 1mM Sodium Pyruvate, 0.1mM NEAA, 0.05mM β-mercaptoethanol, 1x N-2 Supplement, and 1x B-27 Supplement minus Vitamin A was used. The ES neuralization protocol comprised four steps. STEP 1: mESCs were dissociated and seeded on 0.25% gelatin-coated culture dishes (30000 cells/cm²) in 2i+Lif medium for one day (DIV -1). Next day, the medium was changed to Wnt/Bmp double inhibition (WiBi) medium: CDMM with 2.5μM 53AH (Wnt pathway inhibitor, Cellagen Technology, C5324-2s) and 0.25μM LDN193189 hydrochloride (Bmp inhibitor, Sigma-Aldrich, SML0559). The day of shift from 2i+Lif medium to WiBi medium is DIV 0. Cells were cultured in WiBi medium for 3 days (DIV0 - DIV3). On DIV3, differentiating ES cells were dissociated and seeded (30000 cells/cm²) in CDMM on dishes coated with Poly-ornithine (PLO, Sigma; 4 μg/cm² in sterile water, 3 hours coating at 37°C) and purified mouse Laminin (msLam, Sigma-Aldrich, CC095-M; 1 μg/cm² in PBS, O/N coating at 37°C). Next day, the medium was changed to WiBi medium, and cells were cultured for an additional 4 days with daily medium changes (DIV4 - DIV7). STEP 2: on DIV7, NPCs were dissociated and seeded (100000 cells/cm²) in CDMM on PLO/msLam coated dishes/wells for one day to recover from splitting. From the next day, isocortical culture was maintained in WiBi medium for four additional days (DIV8 - DIV11), whereas hippocampal culture (CHIR) was grown in CDMM supplemented with 3μM CHIR990212. During this time window, other combinations of morphogens were tested to induce an entorhinal/allocortical identity in NPCs: 1μM MEK inhibitor PD0325901, 2.5μM Wnt inhibitor 53AH, 0.25μM Bmp inhibitor LDN193189 hydrochloride, 0.1μM Smoothened agonist SAG (Santa Cruz Biotechnology, sc-212905), 100ng/mL recombinant Mouse FGF-8c Protein (Thermo Fisher Scientific, 424FC025CF). On the eleventh day of differentiation, DMEM/F12 was replaced with Neurobasal (Gibco, 21103049), and NEAA were removed from the CDMM to prevent glutamate-induced excitotoxicity. STEP 3: on DIV12, medium was changed to half CDMM experimental medium and half embryonic neuronal medium (eNb), containing Neurobasal, 2mM Glutamine, 1mM sodium Pyruvate, 0.05mM β-mercaptoethanol, 0.2mM Ascorbic Acid (Vit. C), and B-27 Supplement minus Vitamin A 50x. From the next day until DIV20 (7 days), half of the eNb medium was changed daily to allow conditioning of the medium by the differentiating neurons. STEP 4: on DIV20, medium was changed to half eNb and half postnatal neuronal medium (pNb), containing Neurobasal-A (Gibco, 10888022), 2mM Glutamine, 1mM sodium Pyruvate, 0.05mM β-mercaptoethanol, 0.2mM Ascorbic Acid (Vit. C), B-27 Supplement 50x (Gibco, 17504044), and 20 ng/ml recombinant human BDNF protein (Novus Biologicals, NBP2-52006). Starting the next day, pNb medium was changed every 2 to 3 days to condition the medium and allow the differentiated neurons to mature.
growth_protocol
Mus musculus
http://purl.obolibrary.org/obo/NCBITaxon_10090
organism
Cell culture
sample_type
male
sex
129/Ola
strain
N/A
tissue
MiBi Replicate 1
title
MEK + BMP inhibition
treatment