pmxs oct3 4  (Addgene inc)

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

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: iPSCs generated from NSCs are able to differentiate into cells of the three germ layers in vitro and in vivo (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 three days in vitro (DIVs) in EB medium. Incipient EBs were incubated for four more DIVs in floating conditions. Then, EBs were seeded in gelatin pre-treated plates to allow differentiation for three more DIVs before analysis. (B) qPCR expression analysis of the pluripotency-related genes Oct4 , Nanog, and Zfp42 in NSCs, iPSCs, and iPSCs-derived EBs. Phase contrast image of a representative EB is shown. (C) qPCR analysis of Kdr and Afp (mesoderm), Foxa2 and Meox1 (endoderm), and Zic1 and Cer1 (ectoderm) in iPSCs and iPSCs-derived EBs. (D) ICC detection of the pluripotency marker NANOG (red) and the different germ layer markers: α-fetoprotein (green, endoderm), βIII-tubulin (green, ectoderm), and Brachyury (red, mesoderm); α-SMA (green, mesoderm) and GATA4 (red, endoderm) in iPSCs-derived EBs. (E) Image of the dorsolateral area of immunocompromised Nude mice two weeks after the injection of iPSCs, including a detailed image of the formed teratoma after its extraction (left panel). Histological analysis of teratomas using hematoxylin-eosin staining (right panel). Muscle fibers 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). Significance was evaluated using Mann-Whitney, ANOVA, or 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 maximum and minimum values. In bar plots, mean and s.e.m. are shown. Each dot represents an independent sample. 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: Generated, In Vitro, In Vivo, Suspension, Incubation, Expressing, Derivative Assay, Marker, Injection, Extraction, Staining, Immunofluorescence, MANN-WHITNEY, Whisker Assay

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: Expression of imprinted genes in adult NSCs is regulated during the reprogramming process (A) Principal component analysis (PCA) generated with the top 500 most variable genes obtained from RNA-seq of 3 NSCs and 2 iPSCs cultures, using normalized (variance stabilized transformation) gene counts. Principal components 1 and 2 are shown. (B) Heatmap displays the Z-score-scaled expression levels of all differentially expressed genes (DEGs) identified by comparing iPSCs and adult NSCs transcriptomes. (C) Volcano plot for all expressed genes based on RNA-seq data. The number of significantly downregulated (blue) and upregulated (red) genes in iPSCs relative to NSCs is indicated within the corresponding circles. (D) RNA-seq log 2 (fold change) in gene expression levels in iPSCs relative to NSCs for three pluripotency-associated genes, Oct4, Zfp42, and Nanog, and three neural lineage markers, Nes , Zic1, and Olig2 . (E) Heatmap displays the Z-score-scaled expression levels of the imprinted DEGs in iPSCs relative to NSCs. (F) Volcano plot of all expressed imprinted genes based on RNA-seq data. The number of significantly downregulated (blue) and upregulated (red) genes in iPSCs relative to NSCs is indicated within the corresponding circles. (G) Representation of the log 2 (fold change) of all differentially expressed imprinted genes in iPSCs relative to NSCs. Maternally and paternally expressed genes are shown separately. (H) qPCR validation of selected imprinted DEGs identified by RNA-seq data. Gapdh was used to normalize gene expression data. Significance was evaluated using the unpaired two-tailed t test and the Mann-Whitney test. 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 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, Generated, RNA Sequencing, Transformation Assay, Gene Expression, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY

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: TET3 mediates IG-DMR methylation protection by regulating Trim28 and Oct4 gene expression (A) qPCR quantification of Tet1 , Tet2, and Tet3 in NSCs, pre-iPSCs, and iPSCs. (B) qPCR quantification of the neural marker Nes 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 (upper panel). Percentage of Nestin and βIII-tubulin positive cells in iPSCs and NE cultures of both genotypes (lower left panel). Immunocytochemistry images of Nestin (red) and βIII-tubulin (green) in NE cultures of both genotypes (lower right panel). (D) Quantification by the pyrosequencing of the percentage of methylation at the Snurf-Snrpn ICR and at the IG-DMR in NSCs and iPSCs from both WT and Tet3KO cultures. Gray dashed line indicates the percentage of methylation in control brain samples. (E) qPCR analysis of Trim28 , Oct4 and Zfp57 in NSCs and iPSCs from WT and Tet3KO mice. (F) Schematic representation of Trim28 and Oct4 promoter regions and amplicons used in the TET3 ChIP-qPCR assay. (G) qPCR analysis after ChIP with TET3 antibody in wild-type adult NSCs. Trim28 and Oct4 promoters were analyzed. The Snrpn promoter was used as a positive control for TET3 binding, while a distal, unrelated genomic region served as a negative control. Enrichment values represent the proportion of immunoprecipitated DNA relative to the input (%ChIP signal/input). Gapdh was used as a housekeeping gene for qPCR analysis. DAPI was used to counterstain DNA. Scale bars in C: 20 μm. Significance was evaluated using the unpaired two-tailed t test and the Mann-Whitney test. 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 indicated as +, and whiskers represent the maximum and minimum 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: Methylation, Gene Expression, Marker, Immunocytochemistry, Control, ChIP-qPCR, Positive Control, Binding Assay, Negative Control, Immunoprecipitation, Two Tailed Test, MANN-WHITNEY, Whisker Assay

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: pMXs- Oct4 , Addgene , Cat#13366; RRID: Addgene_13366.

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

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: iPSCs generated from NSCs are able to differentiate into cells of the three germ layers in vitro and in vivo (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 three days in vitro (DIVs) in EB medium. Incipient EBs were incubated for four more DIVs in floating conditions. Then, EBs were seeded in gelatin pre-treated plates to allow differentiation for three more DIVs before analysis. (B) qPCR expression analysis of the pluripotency-related genes Oct4 , Nanog, and Zfp42 in NSCs, iPSCs, and iPSCs-derived EBs. Phase contrast image of a representative EB is shown. (C) qPCR analysis of Kdr and Afp (mesoderm), Foxa2 and Meox1 (endoderm), and Zic1 and Cer1 (ectoderm) in iPSCs and iPSCs-derived EBs. (D) ICC detection of the pluripotency marker NANOG (red) and the different germ layer markers: α-fetoprotein (green, endoderm), βIII-tubulin (green, ectoderm), and Brachyury (red, mesoderm); α-SMA (green, mesoderm) and GATA4 (red, endoderm) in iPSCs-derived EBs. (E) Image of the dorsolateral area of immunocompromised Nude mice two weeks after the injection of iPSCs, including a detailed image of the formed teratoma after its extraction (left panel). Histological analysis of teratomas using hematoxylin-eosin staining (right panel). Muscle fibers 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). Significance was evaluated using Mann-Whitney, ANOVA, or 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 maximum and minimum values. In bar plots, mean and s.e.m. are shown. Each dot represents an independent sample. See also .

Article Snippet: pMXs- Oct4 , Addgene , Cat#13366; RRID: Addgene_13366.

Techniques: Generated, In Vitro, In Vivo, Suspension, Incubation, Expressing, Derivative Assay, Marker, Injection, Extraction, Staining, Immunofluorescence, MANN-WHITNEY, Whisker Assay

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: Expression of imprinted genes in adult NSCs is regulated during the reprogramming process (A) Principal component analysis (PCA) generated with the top 500 most variable genes obtained from RNA-seq of 3 NSCs and 2 iPSCs cultures, using normalized (variance stabilized transformation) gene counts. Principal components 1 and 2 are shown. (B) Heatmap displays the Z-score-scaled expression levels of all differentially expressed genes (DEGs) identified by comparing iPSCs and adult NSCs transcriptomes. (C) Volcano plot for all expressed genes based on RNA-seq data. The number of significantly downregulated (blue) and upregulated (red) genes in iPSCs relative to NSCs is indicated within the corresponding circles. (D) RNA-seq log 2 (fold change) in gene expression levels in iPSCs relative to NSCs for three pluripotency-associated genes, Oct4, Zfp42, and Nanog, and three neural lineage markers, Nes , Zic1, and Olig2 . (E) Heatmap displays the Z-score-scaled expression levels of the imprinted DEGs in iPSCs relative to NSCs. (F) Volcano plot of all expressed imprinted genes based on RNA-seq data. The number of significantly downregulated (blue) and upregulated (red) genes in iPSCs relative to NSCs is indicated within the corresponding circles. (G) Representation of the log 2 (fold change) of all differentially expressed imprinted genes in iPSCs relative to NSCs. Maternally and paternally expressed genes are shown separately. (H) qPCR validation of selected imprinted DEGs identified by RNA-seq data. Gapdh was used to normalize gene expression data. Significance was evaluated using the unpaired two-tailed t test and the Mann-Whitney test. 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 bar plots, mean and s.e.m. are shown. Each dot represents an independent culture. See also .

Article Snippet: pMXs- Oct4 , Addgene , Cat#13366; RRID: Addgene_13366.

Techniques: Expressing, Generated, RNA Sequencing, Transformation Assay, Gene Expression, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY

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: TET3 mediates IG-DMR methylation protection by regulating Trim28 and Oct4 gene expression (A) qPCR quantification of Tet1 , Tet2, and Tet3 in NSCs, pre-iPSCs, and iPSCs. (B) qPCR quantification of the neural marker Nes 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 (upper panel). Percentage of Nestin and βIII-tubulin positive cells in iPSCs and NE cultures of both genotypes (lower left panel). Immunocytochemistry images of Nestin (red) and βIII-tubulin (green) in NE cultures of both genotypes (lower right panel). (D) Quantification by the pyrosequencing of the percentage of methylation at the Snurf-Snrpn ICR and at the IG-DMR in NSCs and iPSCs from both WT and Tet3KO cultures. Gray dashed line indicates the percentage of methylation in control brain samples. (E) qPCR analysis of Trim28 , Oct4 and Zfp57 in NSCs and iPSCs from WT and Tet3KO mice. (F) Schematic representation of Trim28 and Oct4 promoter regions and amplicons used in the TET3 ChIP-qPCR assay. (G) qPCR analysis after ChIP with TET3 antibody in wild-type adult NSCs. Trim28 and Oct4 promoters were analyzed. The Snrpn promoter was used as a positive control for TET3 binding, while a distal, unrelated genomic region served as a negative control. Enrichment values represent the proportion of immunoprecipitated DNA relative to the input (%ChIP signal/input). Gapdh was used as a housekeeping gene for qPCR analysis. DAPI was used to counterstain DNA. Scale bars in C: 20 μm. Significance was evaluated using the unpaired two-tailed t test and the Mann-Whitney test. 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 indicated as +, and whiskers represent the maximum and minimum values. In bar plots, mean and s.e.m. are shown. Each dot represents an independent culture. See also .

Article Snippet: pMXs- Oct4 , Addgene , Cat#13366; RRID: Addgene_13366.

Techniques: Methylation, Gene Expression, Marker, Immunocytochemistry, Control, ChIP-qPCR, Positive Control, Binding Assay, Negative Control, Immunoprecipitation, Two Tailed Test, MANN-WHITNEY, Whisker Assay