pmxs oct3  (Addgene inc)

Journal: bioRxiv

Article Title: TET3 protects the Dlk1-Dio3 Imprinted Locus from DNA hypomethylation during adult NSC Reprogramming

doi: 10.1101/2025.02.13.638077

Figure Lengend Snippet: (A) Quantitative PCR (qPCR) of the neural genes Pax6 and Olig2, and the pluripotency genes Nanog and Zfp42 in ESCs (blue) and adult NSCs (beige) (left and right panels). 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 (middle panel). (B) Schematic representation of the protocol used to reprogram adult NSCs into iPSCs. NSCs are infected with retroviruses encoding Oct4 , Klf4 and the fluorescent protein mCherry. After 5 days in vitro (DIV) in NSCs medium, neurospheres formed by post-infected NSCs (PI-NSCs) are dissociated into single cells and plated on murine embryonic fibroblasts using ESC/LIF medium. 5 days after dissociation, mCherry + and SSEA1 + clone-like aggregates containing pre-iPSCs start to appear. Medium is then changed to 2i/LIF medium to complete the reprogramming process. After 10 more DIVs, cells have become full iPSCs and 10 single clones of each culture are picked and subcultured independently for further analysis. (C) qPCR expression analysis of retroviral Klf4 and Oct4 expression in adult NSCs, PI-NSCs, pre-iPSCs and iPSCs . (D) qPCR expression analysis of the neural genes Nestin 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 (left panel) and adult NSCs (right panel). ICC for the pluripotency marker NANOG (red) for ESCs (left panel) and for the neural marker OLIG2 (blue) in NSCs (right panel) are also shown. (F) ICC images for SSEA1 and OCT4 (green) in pre-iPSCs (left panel) and iPSCs (middle panel). mCherry fluorescence for pre-iPSCs (left panel) and ICC for the pluripotency marker NANOG (red) and the neural marker OLIG2 (blue) for iPSCs (middle panel) are shown. Phase contrast images for fully reprogrammed iPSCs clones (right panel) are also shown. Gapdh was used as a housekeeping gene for qPCR normalization. DAPI was used to counterstain nuclei in immunofluorescence images. Scale bars for ICCs in e and f: 20 μm; and for phase contrast images in e 40 μm in upper panel and 5 μm in lower panel. P-values and number of samples are indicated. In box and whiskers plots, the mean is indicated as + and whiskers represent the maximum and minimum values. In bar plots, error bars show s.e.m.

Article Snippet: To produce retroviruses expressing Oct4 and Klf4 , Platinum-E (Plat-E) retroviral packing cells (Cell Biolabs) were transfected with a plasmid solution containing 1 mL of Opti-MEM TM (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: Real-time Polymerase Chain Reaction, Expressing, Infection, In Vitro, Clone Assay, Retroviral, Control, Immunocytochemistry, Marker, Fluorescence, Immunofluorescence

Journal: bioRxiv

Article Title: TET3 protects the Dlk1-Dio3 Imprinted Locus from DNA hypomethylation during adult NSC Reprogramming

doi: 10.1101/2025.02.13.638077

Figure Lengend Snippet: (A) Schematic representation of the embryoid bodies (EB) assay using the “ hanging drops ” method. iPSCs were dissociated and the cell suspension (30 cells/μL) was distributed in drops in a plate that was incubated upside-down for 3 days in vitro (DIVs) in EB medium. Then, the plate was inverted and culture media was added. Incipient EBs were incubated 4 more DIVs in floating conditions. Then, EBs were seeded in gelatine pre-treated plates to allow differentiation for 3 more DIVs before analysis. (B) qPCR expression analysis of pluripotency-related genes Oct4 , Nanog and Zfp42 genes in NSCs (beige), iPSCs (green) and iPSCs-derived EBs (brown) (left panel). Phase contrast image of an EB is included (right panel). (C) qPCR analysis of Kdr1 and Afp (mesoderm), Foxa2 and Meox1 (endoderm), and Zic1 and Cer1 (ectoderm) in iPSCs (green) and iPSCs-derived EBs (brown). (D) ICC detection of the pluripotency marker NANOG (red) and the different germ layer markers α-fetoprotein (green, endoderm) (upper panel), βIII-tubulin (green, ectoderm) and Brachyury (red, mesoderm) (middle panel), and α-SMA (green, mesoderm) and GATA4 (red, endoderm) (lower panel) in iPSCs-derived EBs. (E) Image of the dorsolateral area of immunocompromised Nude mice 2 weeks after the injection of iPSCs, including a detailed image of the formed teratoma after its extraction (left panel). Histological analysis of teratomas using haematoxylin-eosin staining (right panel). Muscle fibres derived from mesoderm, columnar epithelium derived from endoderm and epithelial cells derived from ectoderm are indicated with arrowheads. Gapdh was used as a housekeeping gene for qPCR normalization. DAPI was used to counterstain nuclei in immunofluorescence images. Scale bars in b: 10 μm; in d: 50 μm; and in e: 1 cm (left panel) and 20 μm (right panel). P-values and number of samples are indicated. In box and whiskers plots, the mean is indicated as + and whiskers represent the maximum and minimum values. In bar plots, error bars show s.e.m.

Article Snippet: To produce retroviruses expressing Oct4 and Klf4 , Platinum-E (Plat-E) retroviral packing cells (Cell Biolabs) were transfected with a plasmid solution containing 1 mL of Opti-MEM TM (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: Suspension, Incubation, In Vitro, Expressing, Derivative Assay, Marker, Injection, Extraction, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: TET3 protects the Dlk1-Dio3 Imprinted Locus from DNA hypomethylation during adult NSC Reprogramming

doi: 10.1101/2025.02.13.638077

Figure Lengend Snippet: (A) Principal component analysis (PCA) generated with top 500 most variable genes obtained from RNA-seq of 3 NSCs and 2 iPSCs cultures, using normalized (variance stabilized transformation) gene counts. It shows principal components 1 and 2. (B) Heatmap showing the scaled expression (Z-score) of all differentially expressed genes (DEGs) obtained comparing gene expression levels of iPSC to adult NSCs cultures. (C) Volcano plot for all expressed genes based on RNA-seq data. The number of significantly downregulated (blue) and upregulated (red) genes in iPSCs compared to NSCs is indicated inside the circles. (D) RNA-seq log 2 (fold change) of gene expression in iPSCs compared to NSCs for the three pluripotency-related genes Oct4, Zfp42 and Nanog and for the three neural genes Nestin (Nes) , Zic1 and Olig2 . (E) Heatmap showing the scaled expression (Z-score) of the imprinted DEGs in iPSCs compared to NSCs. (F) Volcano plot for all expressed imprinted genes based on RNA-seq data. The number of significantly downregulated (blue) and upregulated (red) genes in iPSCs compared to NSCs is indicated inside the circles. (G) Representation of the log 2 (fold change) of all imprinted DEGs in iPSCs compared to NSCs. Maternally (left panel) and paternally (right panel) expressed genes are shown separately.

Article Snippet: To produce retroviruses expressing Oct4 and Klf4 , Platinum-E (Plat-E) retroviral packing cells (Cell Biolabs) were transfected with a plasmid solution containing 1 mL of Opti-MEM TM (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: Generated, RNA Sequencing Assay, Transformation Assay, Expressing

Journal: bioRxiv

Article Title: TET3 protects the Dlk1-Dio3 Imprinted Locus from DNA hypomethylation during adult NSC Reprogramming

doi: 10.1101/2025.02.13.638077

Figure Lengend Snippet: (A) qPCR quantification of Tet1 , Tet2 and Tet3 in NSCs, pre-iPSCs and iPSCs. (B) qPCR quantification of the neural marker Nestin and the pluripotency marker Nanog in wild-type (WT) and Tet3 -deficient (Tet3KO) NSCs and iPSCs. (C) Schematic of the protocol used to differentiate iPSCs into neuroectoderm (NE). iPSCs are disaggregated and re-plated in gelatin-treated plates at a 1.5 ×10 4 cells/cm 2 density in N2B27 supplemented medium. Seven days after cells are analyzed. (D) Percentage of Nestin and βIII-tubulin positive cells in iPSCs and NE cultures of both genotypes (left panel). Immunocytochemistry images of Nestin (red) and βIII-tubulin (green) in NE cultures of both genotypes (right panel). (E) Quantification by pyrosequencing of the percentage of methylation Snurf-Snrpn DMR and IG-DMR in NSCs and iPSCs from both WT and Tet3KO cultures. Gray dashed line indicates the percentage of methylation in control brain samples. (F) Heatmap showing ChIP-seq peaks of transcription factors and other proteins in pluripotent stem cells (ChIP-Atlas). Colours correspond to the number of occurrences (number of peaks) each protein binds to different ICRs. (G) qPCR analysis of Trim28 , Oct4 and Zfp57 in NSCs and iPSCs from WT and Tet3KO mice. Gapdh was used as a housekeeping gene for qPCR analysis. DAPI was used to counterstain DNA. P-values and number of samples are indicated. In box and whiskers plots, the mean is indicated as + and whiskers represent the maximum and minimum values. In bar plots, error bars show s.e.m.

Article Snippet: To produce retroviruses expressing Oct4 and Klf4 , Platinum-E (Plat-E) retroviral packing cells (Cell Biolabs) were transfected with a plasmid solution containing 1 mL of Opti-MEM TM (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: Marker, Immunocytochemistry, Methylation, Control, ChIP-sequencing