Journal: Gene therapy

Article Title: Engineered stem cell-derived microglia as therapeutic vehicle for experimental autoimmune encephalomyelitis.

doi: 10.1038/gt.2012.100

Figure Lengend Snippet: Figure 6. Growth-promoting effect of NT3-transduced microglia. (a) Whole DRG derived from embryonic mice were cultured in chemically defined medium and supplemented with supernatant of NT3-GFP-ESdM, GFP-ESdM, recombinant NT3 or medium as control. Representative pictures of DRG outgrowth are shown. Supernatant derived from NT3-GFP-ESdM cultures promoted outgrowth. Bar ¼ 500 mm. (b) The bioactivity of NT3-GFP-ESdM and GFP-ESdM-conditioned medium was assessed in vitro by determination of neurite outgrowth in relation to the control medium. Medium was supplemented with medium as a control, supernatant of GFP-ESdM, supernatant of NT3-GFP-ESdM or recombinant NT3 protein (10 ng ml 1). Supernatant of NT3-GFP-ESdM promoted DRG neuron outgrowth in comparison to supernatant of GFP-ESdM or control medium. Data are presented as mean±s.e.m. of three independent experiments. Statistical analysis via analysis of variance (ANOVA) *Po0.05; ***Po0.001. (c) Increased expression of GAP43 in the white matter of the thoracic spinal cord of EAE mice treated with NT3-GFP-ESdM. EAE was induced by immunization with MOG peptide. NT3-GFP-ESdM, GFP-ESdM or PBS were intravenously applied 20 days after immunization. Spinal cord sections were immunostained with antibodies directed against GAP43 at 4 days after transplantation of NT3-GFP-ESdM and counterstained with DAPI to label nuclei. Staining of GAP43 was detected in the spinal cord and associated with GFP þ cells. Bar ¼ 20 mm. (d) Quantification of GAP43 þ cells detected in the spinal cord of EAE mice at 1 day, 4, 10 or 21 days after treatment with NT3-GFP-ESdM, GFP-ESdM or PBS. Treatment with NT3-GFP-ESdM increased the density of GAP43 þ cells. Data are presented as mean ±s.e.m. of three independent experiments. Statistical analysis via ANOVA followed by Bonferroni, *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: Filtered cell culture supernatant of NT3-GFP-ESdM or GFP-ESdM (diluted 1:10 with neural medium), recombinant mouse NT3 (10 ng ml 1, R&D Systems) or medium alone as control were added to the DRG or hippocampal neurons for 4 h. In five randomly chosen areas, neurons that stained with an intensity above a set threshold were counted using ImageJ software.

Techniques: Derivative Assay, Cell Culture, Recombinant, Control, In Vitro, Comparison, Expressing, Transplantation Assay, Staining

Journal: bioRxiv

Article Title: A human-specific, concerted repression of microcephaly genes contributes to radiation-induced growth defects in forebrain organoids

doi: 10.1101/2024.06.27.600564

Figure Lengend Snippet: Early human cortical development was modeled using forebrain organoids derived from embryonic stem cells (hESCs), following the methodology by Sloan et al., 2019. (A) Every organoid batch was generated using 600 microwells of an AggreWell™ 800 plate. Double SMADi inhibition with 5 µM Dorsomorphin and 10 µM SB-431542 was applied from Day 0 to Day 5, followed by exposure to 20 ng/ml EGF and bFGF from Day 6 to Day 24. Subsequently, 20 ng/ml BDNF and NT-3 were applied from Day 25 to Day 43. From Day 43, cell culture medium was free from growth factors. Organoid growth was followed up from Day 1 to Day 54, and the diameter of at least 10 organoids per time point was quantified using the ImageJ software. Scale bar = 500 µm. (B) Forebrain organoid characterization via immunohistochemistry revealed the expression of classical markers present throughout normal human cortical development: SOX2, Nestin, and PAX6 staining for neural progenitor cells, DCX and TUJ1 staining for post-mitotic neurons, OLIG2 for oligodendrocytes and oligodendrocyte precursors. Scale bar = 50 µm. (C) Deconvolution analysis based on single cell RNA-seq data (from ) showed the abundance of different cell types during organoid development. (D) Forebrain organoids were exposed to ionizing radiation (Sham-irradiated x 0.5 Gy x 2 Gy) at developmental Days 14 and 56. Samples were processed for immunostaining (2 h, 6 h, 24 h, 14 d), RNA-sequencing (6 h, 24 h, 14 d), and size follow up (14 d). (E) Brain organoid size was monitored every 2 days. 24 organoids were analyzed per condition. Scale bar = 1000 µm. Statistical analysis was performed using Two-way ANOVA test followed by Tukey’s test for multiple comparisons. **p< 0.001; ***p< 0.0004; ****p< 0.0001. See also -S3.

Article Snippet: On Day 6 of culture, Neural Induction Medium was replaced by Neural Differentiation Medium (Neurobasal-A Medium (Gibco, #10888022), 2% B-27 supplement without vitamin A (Gibco, #12587010), 0.5% GlutaMAX, 1% penicillin/streptomycin, and 0.1% MycoZap) supplemented with 20 ng/ml mouse recombinant EGF (PeproTech, # 315-09-1MG) and 20 ng/ml human recombinant FGF-basic (PeproTech, #100- 18B) until Day 24, which was then replaced by 20 ng/ml human/mouse recombinant NT-3 (Stem Cell Technologies, #78074) and 20 ng/ml human recombinant BDNF (Stem Cell Technologies, #78005) until Day 43 of culture.

Techniques: Derivative Assay, Generated, Inhibition, Cell Culture, Software, Immunohistochemistry, Expressing, Staining, RNA Sequencing Assay, Irradiation, Immunostaining