Journal: iScience

Article Title: TET3 protects the Dlk1-Dio3 imprinted locus from DNA hypomethylation during adult NSC reprogramming

doi: 10.1016/j.isci.2025.113994

Figure Lengend Snippet: NSCs from the adult SVZ are reprogrammed into iPSCs by the exogenous expression of Oct4 and Klf4 (A) Quantitative PCR (qPCR) of the neural genes Pax6 and Olig2, and the pluripotency genes Nanog and Zfp42 in ESCs and adult NSCs. qPCR analysis of the endogenous expression of the reprogramming transcription factors Oct4 , Sox2 , Klf4, and c-Myc in ESCs and adult NSCs is also shown. (B) Schematic representation of the protocol used to reprogram adult NSCs into iPSCs. NSCs were infected with retroviruses encoding Oct4 , Klf4, and the fluorescent protein mCherry. After five days in vitro (DIV) in NSCs medium, neurospheres formed by post-infected NSCs (PI-NSCs) were dissociated into single cells and plated on murine embryonic fibroblasts using ESC/LIF medium. Five days after dissociation, mCherry + and SSEA1 + clone-like aggregates containing pre-iPSCs started to appear. Medium was then changed to 2i/LIF medium to complete the reprogramming process. After ten more DIVs, cells became full iPSCs, and ten single clones of each culture were picked and subcultured for further analysis. (C) qPCR analysis of retroviral Klf4 and Oct4 expression in adult NSCs, PI-NSCs, pre-iPSCs, and iPSCs. (D) qPCR analysis of the neural genes Nes and Olig2 (upper panel) and the pluripotency-related genes Oct4 , Nanog, and Zfp42 (lower panel) in NSCs, pre-iPSCs, and iPSCs. ESCs were used as a control of the pluripotent state. (E) Immunocytochemistry (ICC) images of SSEA1, OCT4 (green) and SOX2 (red) in ESCs and adult NSCs. ICC for the pluripotency marker NANOG (red) in ESCs and for the neural marker OLIG2 in NSCs are also shown. (F) ICC images for SSEA1 and OCT4 (green) in pre-iPSCs and iPSCs. mCherry fluorescence in pre-iPSCs, ICC for the pluripotency marker NANOG (red) and the neural marker OLIG2 (blue) in iPSCs (middle panel) are also shown. Phase contrast images for fully reprogrammed iPSC clones are also included. Gapdh was used as a housekeeping gene for qPCR normalization. DAPI was used to counterstain nuclei in immunofluorescence images. Scale bars in E and F: 20 μm; phase contrast images in F: 40 μm (upper panel) and 5 μm (lower panel). Significance was evaluated using unpaired two-tailed t test, Mann-Whitney, ANOVA, and Kruskal-Wallis tests. p -values and number of samples are indicated. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001, n.s.: non significant. In box and whisker plots, the mean is marked with a (+) and whiskers represent the minimum and maximum values. In bar plots, mean and s.e.m. are shown. Each dot represents an independent culture. See also .

Article Snippet: To produce retroviruses expressing Oct4 and Klf4 , PlatinumE (Plat-E) retroviral packing cells (RV-101, Cell Biolabs) were transfected with a plasmid solution containing 1 mL of Opti-MEMTM (Gibco), 60 μL of 1mg mL -1 polyethylenimine (PEI, Polysciences) and 20 μg of the retroviral vectors pMXs- Oct4 (#13366, Addgene), pMXs- Klf4 (#13370, Addgene) and pMXs- mCherry (pMX-2A-CH, designed and kindly provided by Dr. Jose Manuel Torres).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, In Vitro, Clone Assay, Retroviral, Control, Immunocytochemistry, Marker, Fluorescence, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Whisker Assay