mESCs were sub-cultured in gelatinised six-well tissue culture plates for 48 h to deplete feeder cells. Cells were collected and plated into gelatinised six-well plates at 1×105 cells per cm2 for 24 h. 2-D induction culture was based on the protocols previously described (Turner et al., 2014a (link),b (link)). Briefly, cells were then harvested and re-plated into 60-mm tissue culture dishes at a density of 4.7×103 cells per cm2 with overnight incubation in mESC culture medium. The following morning, medium was changed to NDiff® 227 (Clontech, Saint-Germain-en-Laye, France; Y40002) for 48 h and then to NDiff® 227 supplemented with Activin-A (R&D Systems, Abingdon, UK; 338-AC) and CHIR 99021 (Tocris, Abingdon, UK; 4423) to a final concentration of 100 ng ml−1 and 3 µmol l−1, respectively, for a further 48 h incubation. Medium was changed on a daily basis. Experiments were carried out in three independent biological replicates.
Ndiff227
Manufactured by Takara Bio
Sourced in France, United Kingdom
Ndiff227 is a cell line derived from human embryonic stem cells. It is designed for use in research applications requiring the maintenance and differentiation of pluripotent stem cells.
Automatically generated - may contain errors
Lab products found in correlation
24 well plate, by Thermo Fisher Scientific (3 mentions)
Gelatin, by Merck Group (3 mentions)
Dbcamp, by Merck Group (3 mentions)
Lm 22a4, by R&D Systems (3 mentions)
Noggin, by R&D Systems (3 mentions)
Chir99021, by Bio-Techne (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pd0325901, by Selleck Chemicals (2 mentions)
Activin a, by R&D Systems (1 mentions)
Slide 4 well, by Ibidi (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Valproic acid sodium salt vpa, by Merck Group (1 mentions)
Ko dmem, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Selleck Chemicals (1 mentions)
Chir90021, by Selleck Chemicals (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
13 protocols using ndiff227
1
Directed Differentiation of mESCs
2
Direct Reprogramming of Fibroblasts to Neurons
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Neurons were generated from PKAN and control fibroblasts by direct reprogramming as previously described by Drouin-Ouellet et al. [37 (link)–39 (link)]. Controls and patients-derived fibroblasts were seeded in µ-Slide 4 Well (Ibidi Inc., Martinsried, Germany). The day after, dermal fibroblasts were infected with one-single lentiviral vector containing neural transcription factors (Acsl1 and Brn2) and two shRNA against the REST complex, generated as previously described [40 (link)]. Cells were infected with a multiplicity of infection (MOI) of 30. The plasmids were a gift from Dr. Malin Parmar (Developmental and Regenerative Neurobiology, Lund University, Sweden). After 24 h, medium was replaced with fresh fibroblast medium. Fibroblasts medium was replaced with neural differentiation medium after 48 h (NDiff227; Takara-Clontech) as described. Twenty-seven days post-infection neuronal cells were identified by the expression of Tau. DAPI + and Tau + cells were considered induced neurons. Images were taken at DeltaVision system with an Olympus IX-71 fluorescence microscope with 60 × oil objective and analysed by Fiji-ImageJ software.
3
Direct Neuronal Reprogramming of Fibroblasts
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For direct neural reprogramming, fibroblasts were plated at a density of 27,800 cells per cm2 in 24‐well plates (Nunc) coated with 0.1% gelatin (Sigma). Three days after viral transduction, fibroblast medium was replaced by neural differentiation medium (NDiff227; Takara‐Clontech) supplemented with growth factors at the following concentrations: LM‐22A4 (2 μM, R&D Systems), GDNF (2 ng/ml, R&D Systems), NT3 (10 ng/μl, R&D Systems) and db‐cAMP (0.5 mM, Sigma) and the small molecules CHIR99021 (2 μM, Axon), SB‐431542 (10 μM, Axon), noggin (0.5 μg/ml, R&D Systems), LDN‐193189 (0.5 μM, Axon), as well as valproic acid sodium salt (VPA; 1 mM, Merck Millipore). Half of the neuronal conversion medium was replaced every 2–3 days. Cells were replated onto a combination of polyornithine (15 μg/ml), fibronectin (0.5 ng/μl), and laminin (5 μg/ml) coated 24‐well plates at day 12 post‐transduction. Eighteen days post‐transduction, the small molecules were stopped and the neuronal medium was supplemented with only the growth factors (LM‐22A4, GDNF, NT3, and db‐cAMP) until the end of the experiment.
4
Direct Neuronal Reprogramming from Fibroblasts
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For direct neuronal reprogramming, fibroblasts were plated at a density of 26,300 cells per cm2 in 24-well plates (Nunc) coated with 0.1% gelatin (Sigma). On the next day, cells were transduced with shRNAs against REST or nPTB and the medium was changed the following day. Three days after this viral transduction, cells were transduced with the pB.pA vector. Fibroblast medium was replaced by neural differentiation medium (NDiff227; Takara-Clontech) supplemented with growth factors at the following concentrations: LM-22A4 (2 µM, R&D Systems), GDNF (2 ng/mL, R&D Systems), NT3 (10 ng/mL, R&D Systems) and the small molecules CHIR99021 (2 µM, Axon), SB-431542 (10 µM, Axon), noggin (0.5 µg/mL, R&D Systems), LDN-193189 (0.5 µM, Axon), as well as valproic acid sodium salt (VPA; 1 mM, Merck Millipore) and db-cAMP (0.5 mM, Sigma), 3 days after the second transduction. Half of the neuronal conversion medium was replaced every 2–3 days. 18 days post-transduction, the small molecules were stopped, and the neuronal medium was supplemented with only the factors LM-22A4, GDNF, NT3 and db-cAMP until the end of the experiment.
5
Direct Neural Reprogramming of Fibroblasts
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For direct neural reprogramming, fibroblasts were plated at a density of 26 300 cells per cm 2 in 24-well plates (Nunc) coated with 0.1% gelatin (Sigma). On the next day, cells were transduced with shRNAs against REST and the medium was changed the following day. Three days after this viral transduction, cells were transduced with the pB.pA, Lmx1a and FoxA2 constructs. Fibroblast medium was replaced by neural differentiation medium (NDiff227; Takara-Clontech) supplemented with growth factors at the following concentrations: LM-22A4 (2 µM, R&D Systems), GDNF (2 ng/mL, R&D Systems), NT3 (10 ng/µL, R&D Systems) and the small molecules CHIR99021 (2 µM, Axon), SB-431542 (10 µM, Axon), noggin (0.5 µg/ml, R&D Systems), LDN-193189 (0.5 µM, Axon), as well as valproic acid sodium salt (VPA; 1mM, Merck Millipore) and db-cAMP (0.5 mM, Sigma), three days after the second transduction. Half of the neuronal conversion medium was replaced every 2-3 days. 18 days post-transduction, the small molecules were stopped, and the neuronal medium was supplemented with only the factors LM-22A4, GDNF, NT3 and db-cAMP until the end of the experiment.
6
Maintaining Pluripotent Mouse ESCs
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Mouse embryonic stem cells E14Tg2a (E14) were purchased from ATCC and cultured without feeders in the presence of LIF. SL-ESCs were maintained in DMEM with 15% ES grade fetal calf/bovine serum. 2iL-ESCs were maintained in Ndiff227 (Takara Bio, Inc.) medium supplemented with 1-μM MEK inhibitor PD0325901 and 3-μM GSK inhibitor CHIR99021. Zic3 KO and Trp53 KO ESCs were tested by western blot. E14 cells were purchased from ATCC and were not authenticated.
7
Culturing and Differentiating Rex1-GFP mESCs
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A Rex1GFP mESC line (Wray et al., 2011 (link)), was a gift from Austin Smith and cultured on feeder cells in SL: KO-DMEM (Thermo Fisher Scientific) supplemented with 15% fetal bovine serum (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), 1% nonessential amino acid (NEAA) (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (Sigma), and 1,000 U mL−1 LIF (Millipore). Where indicated, mESCs were cultured on gelatin-coated plates in 2iL medium: NDiff227 (Takara) supplemented with 1% KO serum replacement (KSR) (Thermo Fisher Scientific), 5% BSA (Thermo Fisher Scientific), 1% NEAA, 0.1 mM 2-mercaptoethanol, 1,000 U mL−1 LIF, 1.0 μM PD0325901 (Selleck), and 3.0 μM CHIR90021 (Selleck). Differentiation was induced in the NDif227 medium supplemented as mentioned earlier but without the 2 inhibitors and LIF.
8
Generating mouse gastruloids with pre-treatment
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Gastruloids were prepared by aggregating 300 mouse embryonic stem cells in 40 μl droplets of N2B27 (NDiffⓇ 227, Takara Bio Inc. Y40002) per well of U-bottomed 96-well plates (Greiner 650185). The full protocol for generating mouse gastruloids can be found on Protocol Exchange (DOI:https://doi.org/10.1038/protex.2018.094 ) and a full characterisation has been described previously [13 (link)]. The following modifications were made to the protocol. Cell cultures were plated into new flasks at a density of 8 × 103 cells/cm2 in ES + LIF medium two days prior to preparing gastruloids. The culture medium was changed fully to 2i + LIF medium the following day as a 24 h pre-treatment.
The compounds were administered to the gastruloids on plating and daily after aggregation was complete (at 0 h, 48 h, 72 h and 96 h post-plating). Between 48 and 72 h, gastruloids were exposed to CHIR-99,021, as described previously [13 (link)]. The compounds were prepared fresh from frozen stocks in N2B27 culture medium at each time point.
The compounds were administered to the gastruloids on plating and daily after aggregation was complete (at 0 h, 48 h, 72 h and 96 h post-plating). Between 48 and 72 h, gastruloids were exposed to CHIR-99,021, as described previously [13 (link)]. The compounds were prepared fresh from frozen stocks in N2B27 culture medium at each time point.
9
Murine Embryonic Stem Cell Culture
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J1, CJ7, and E14TG2a (E14) male mESCs were cultured on tissue culture-treated plates coated in 0.1% gelatin with +LIF medium, which is composed of Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 18% fetal bovine serum (BioWest), EmbryoMax nucleosides (Millipore), MEM nonessential amino acids (Gibco), 100 μM β-mercaptoethanol, 50 U/mL penicillin/streptomycin/L-glutamine (PSG, Gibco), as well as 1000 U/mL recombinant mouse LIF (Millipore) in a 37°C incubator with 5% CO2. Cells were passaged every other day and media was changed daily. For cell culture experiments, cells were routinely seeded at 1.9×105 cells/cm2 for high density (HD) and 2.3×104 cells/cm2 for low density (LD). For differentiation, cells were passaged on d0 to transfer into -LIF medium (same as +LIF, except without LIF) and seeded on d1 at the appropriate densities (high or low). J1 and E14 mESCs and HEK293T cells were from ATCC, whereas CJ7 mESCs were obtained from Dr. Stuart Orkin. PCR did not detect mycoplasma contamination. For serum-free culture, mESCs were maintained in NDiff 227 (Takara Bio) supplemented with 1 μM PD0325901 (Selleck Chemicals), 3 μM CHIR99021 (Selleck Chemicals), and 1000 U/mL recombinant mouse LIF (2i media) and differentiated by withdrawal of LIF and both inhibitors.
10
Mouse Embryonic Stem Cell Culture
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The following mESC lines were used: Blimp1:GFP (Ohinata et al., 2005 (link)) (kindly provided by Azim Surani, University of Cambridge, UK), Blimp1:mVenus Stella:eCFP (BVSC) (Ohinata et al., 2008 (link)) (kindly provided by Mitinori Saitou, ASHBi Institute for the Advanced Study of Human Biology, Kyoto, Japan), Sox17−/− (as described below), Foxa2−/− (Cernilogar et al., 2019 (link)) (kindly provided by Heiko Likert, Helmholtz Munich, Germany), Bmpr1a−/− (Di-Gregorio et al., 2007 (link)) (kindly provided by Tristan Rodriguez, Imperial College London, UK), and Spry4:Venus and Spry4:Venus Fgf4−/− (Morgani et al., 2018 (link)) (kindly provided by Christian Schroeter, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany). All mESC lines were cultured in 2iLIF in N2B27 medium [NDiff227, Takara Bio, Y40002; supplemented with 3 µM CHIR-99021 (CHI), 1 µM PD0325901 (PD03) and 11 ng/ml mLIF (Merck Millipore ESG1106 and made in-house by the Department of Biochemistry, Cambridge University, UK)] on gelatinised (0.1% gelatin) tissue culture flasks or six-well plates kept in humidified incubators at 37°C and 5% CO2. Cells were passaged into new flasks or plates every two days with the medium exchanged daily.
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