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

Journal: Nature medicine

Article Title: Multimodel preclinical platform predicts clinical response of melanoma to immunotherapy

doi: 10.1038/s41591-020-0818-3

Figure Lengend Snippet: a, Expression of indicated MHC class I- and class II-related genes in the four models. Heat map depicts fragments per kilobase of transcript per million (FPKM) obtained from RNA-seq of untreated (N = 4), isotype control (N = 3 for M1–3, N = 4 for M4) or anti-CTLA-4 treated (N = 3 for M1 and M2, N = 6 for M3 and N = 5 for M4) cell line-derived allografts. b, IFN-γ concentration in the medium from 24-h co-cultures of DCT-reactive T cells and the four model cells expressing GFP–DCT peptide35,36, GFP empty vector or nontransduced. CD8+-sorted T cells from C57BL/6 splenocytes were transduced with recombinant T cell receptor (TCR) Vβ3 for the recognition of 9aa DCT peptide (DCT-Vβ3) or Thy1.1 as control. Graph depicts IFN-γ pg ml−1 measured by ELISA. c, Representative flow cytometry plots showing 41BB expression in DCT-reactive T cells after 24-h co-culture with indicated melanoma cell lines (N = 3 co-cultures). Experiments were repeated twice with similar results. NT, nontransduced. d, Percentage of 41BB-positive cells obtained from c. Data were representative of two independent experiments and are depicted as mean (N = 3 co-cultures) and error bars represent s.e.m. (b,d).

Article Snippet: For the validation of MHC-I-related gene expression in the cell lines from the four models, cells were cultured for 24 h in medium containing 20 ng ml −1 of mouse recombinant IFN-γ (MBL International, JM-4117–100).

Techniques: Expressing, RNA Sequencing Assay, Derivative Assay, Concentration Assay, Plasmid Preparation, Transduction, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Co-Culture Assay