n2b27 medium with 2i  (Tocris)

Journal: bioRxiv

Article Title: KIF2A maintains cytokinesis in mouse embryonic stem cells by stabilising intercellular bridge microtubules

doi: 10.1101/2024.07.11.603034

Figure Lengend Snippet: A, B ) Pluripotency exit and colony formation assay. Wild type (WT) and Kif2a KO (KO) mESCs growing in naïve conditions, were trypsinised and replated in N2B27 exit medium (either lacking 2i/LIF or with IWP2 and LIF) for 24 hours followed by replating in naïve medium for 5 days. mESC colonies were then stained using alkaline phosphatase. In ( A ) representative alkaline phosphatase stainings are shown. The number of alkaline phosphatase-positive colonies was counted and plotted as a fraction of the number of colonies formed by naïve cells (set to 100%); this is shown in ( B ). Scale bar = 2 mm. P-values shown are derived from T-tests (n = 5 for controls and LIF +IWP2, n = 6 2i/LIF, 2 independent experiments). C ) Differential gene expression analysis. We used DESeq2 to analyse differentially expressed genes (p<0.05) in WT versus KO mESCs. D ) Metascape analysis of differentially expressed genes in WT versus KO mESCs. The 87 mRNAs that were found to be significantly downregulated in Kif2a KO (KO) mESCs were analysed by Metascape. Selected pathways are shown.

Article Snippet: This was either N2B27 medium without 2i/L, or N2B27 medium with LIF + 1 μ M IWP2 (Stem Cell Technologies, 72124), as indicated.

Techniques: Colony Assay, Staining, Derivative Assay, Expressing

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Differentiated neuronal EVs taken up by mESCs. (A) Schematic of mESCs treated by PKH6 dye–labeled EVs. (B) Confocal images of differentiated mESCs incubated without EV (No-EV) or with PKH6 dye–labeled N6 or N9 EV. Nuclei were stained with DAPI. Scale bar, 10 µm. (C) Immunoblot of GFP and CD9 in EVs (GFP-EV) purified from GFP-expressing cells. 2 × 10 5 mESCs in a 35-mm dish were incubated in 2 ml of N2B27 medium for 24 h with EVs purified from control or GFP-expressing cells. (D) Immunoblot of GFP from whole-cell lysate of mESCs treated with PBS or GFP-EV for 24 h. (E) Immunoblot of GFP from whole-cell lysate of mESCs treated with indicated doses of GFP-EV for 24 h. (F) Immunoblot of GFP from whole-cell lysate of mESCs treated with indicated time of incubation with GFP-EV. (G) Immunoblot of GFP from whole-cell lysate of mESCs treated with 10 ng GFP protein for indicated time or treated with GFP-EV containing 10 ng GFP for 24 h. The GFP protein amount within the GFP-EV was detected by quantitative immunoblot.

Article Snippet: For the neuronal differentiation, medium was harvested and replaced every 3 d. mESCs (R1, gift from Robert Tjian laboratory, University of California, Berkeley, Berkeley, CA) were maintained on N2B27 medium plus 2i (3 μM CHIR99021 and 1 μM PD0325901; Selleckchem) and leukemia inhibitory factor (Millipore Sigma).

Techniques: Labeling, Incubation, Staining, Western Blot, Purification, Expressing, Control

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Neuronal EVs promote neural induction and cell proliferation of mESCs. (A) Gene expression analysis of Nestin and Six3 in mESCs treated without (control) or with indicated EVs. EV number was quantified by Nanosight 2000. Data plotted were from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P < 0.05). (B) Gene expression analysis of Nestin and Six3 in mESCs treated for 4 d without (control) or with different doses of N6-EV. Data plotted were from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). (C) Gene expression analysis of Sox1 , Pax6 , and Tuj1 in mESCs treated for 4 d with 2× 10 9 PC12-EV, N6-EV, or N6-EV together with NGF neutralizing antibody (500 ng/ml). The values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (D) Immunostaining of Nestin (green, Alexa Fluor 488) and Pax6 (red, Alexa Fluor 568) in mESCs as described in B. Scale bar, 25 µm. (E) Quantitative analysis of the percentage of cells containing indicated markers compared with DAPI-stained cells. The values represent the mean ± SD, from three independent experiments (*, P < 0.05). Error bars represent SD from independent samples. (F) Gene expression analysis of Sox1 , Pax6 , and Nestin in mESCs treated for 4 d with 2 × 10 9 EVs from undifferentiated pluripotent ESCs (ES-D0), EBs of ES differentiated for 8 d in KSR medium (ES-D8), and EBs trypsinized in N2 medium for an additional 4 d (ES-D12). The values represent the mean ± SD, from two independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (G) The cellular morphology of mESCs treated with PC12-Exo or different doses of N6-EV in N2B27 medium for 4 d. Scale bars, 200 µm. (H) Quantitative analysis of mESC number treated with or without N6-EV. Data plotted were from two independent experiments. The values represent the mean ± SD (*, P < 0.05). Error bars represent SD from independent samples. (I) Proliferation analysis of mESCs with BrdU staining after EV treatment. The values represent the mean ± SD, from two independent experiments (*, P < 0.05). Error bars represent SD from independent samples.

Article Snippet: For the neuronal differentiation, medium was harvested and replaced every 3 d. mESCs (R1, gift from Robert Tjian laboratory, University of California, Berkeley, Berkeley, CA) were maintained on N2B27 medium plus 2i (3 μM CHIR99021 and 1 μM PD0325901; Selleckchem) and leukemia inhibitory factor (Millipore Sigma).

Techniques: Gene Expression, Control, Immunostaining, Staining, BrdU Staining