Journal: Cell reports
Article Title: Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation
doi: 10.1016/j.celrep.2024.113774
Figure Lengend Snippet: (A) A schema of the retroviral labeling procedure used to manipulate the levels of L1 in RGLs of forebrain organoids. (B) Representative confocal images of VZ-like regions of retrovirally labeled RGL-derived cells that are migrating into the developing cortical plate of forebrain organoids. Sections were stained with anti-GFP (green) and DAPI. Scale bar, 50 μm. (C) Fold change of retrovirally labeled RGLs 3 days post infection (dpi) observed in CP-like over VZ-like regions. Mann-Whitney U test, *p < 0.05. Boxplots with whiskers indicate minimum to maximum values, with box limits for 25 th to 75 th percentiles, and a centerline for the median. (D) Relative normalized position of GFP-positive migratory cells within the evolving cortical plate of forebrain organoids. Mann-Whitney U test, *p < 0.05. Boxplots with whiskers indicate minimum to maximum values, with box limits for 25 th to 75 th percentiles, and a centerline for the median. (E) Distribution of GFP-positive cells in the CP-like structures. Kolmogorov-Smirnov test, NTC (non-targeting control) vs. shL1HS-2: p = 0.0177, NTC vs. shL1HS-1: p < 0.0001. (C–E) N NTC = 56 cells (from n = 4 independent organoids; 2 organoids per iPSC, 2 organoids per ESC line), N shL1HS-2 = 78 cells (from n = 4 independent organoids; 2 organoids per iPSC, 2 organoids per ESC line), and Ns hL1HS-1 = 113 cells (from n = 4 independent organoids; 2 organoids per iPSC, 2 organoids per ESC line). (F) Representative images of electroporated cells in the CP-like layer 2 weeks after the electroporation of shRNA plasmids. Scale bar, 25 μm. (G) Quantification of dendritic length of electroporated cells. Data are presented as mean ± SEM. Mann-Whitney test, ***p = 0.0019. (H) Sholl analysis for dendritic complexity of neurons in human cortical organoids. Two-way ANOVA followed by Holm-Sidak’s multiple comparisons test, ***p < 0.001, **p < 0.01, and *p < 0.05. N NTC = 29 cells (from n = 2 independent organoids), N shL1HS-2 = 69 cells (from n = 2 independent organoids), and N shL1M = 17 cells (from n = 2 independent organoids). Data are presented as mean ± SEM.
Article Snippet: Briefly, human iPSC or ESC colonies were detached with collagenase Type IV (Gibco) and transferred to an Ultra-Low attachment 10-cm plate (Corning Costar) containing hPSC medium that consisted of DMEM:F12 (Invitrogen), 20% Knockout Serum Replacer (Gibco), 1x Non-essential Amino Acids (Invitrogen), 1x β-mercaptoethanol (Gibco), 1x GlutaMAX (Invitrogen), 10 ng/mL FGF-2 (Peprotech) and ROCK inhibitor Y27632 (10 μM).
Techniques: Retroviral, Labeling, Derivative Assay, Staining, Infection, MANN-WHITNEY, Control, Electroporation, shRNA
