Journal: Stem Cell Research & Therapy

Article Title: Footprint-free induced pluripotent stem cells can be successfully differentiated into mesenchymal stromal cells in the feline model

doi: 10.1186/s13287-025-04325-2

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: Retroviral plasmids based on the Moloney murine leukemia virus (MMLV) were purchased from Addgene and contained the coding sequences for the human transcription factors: OCT4 (Addgene #17217), SOX2 (Addgene #17218), NANOG (Addgene #18115), c-MYC (Addgene #17220), and KLF4 (Addgene #17219).

Techniques: Virus

Journal: Stem Cell Research & Therapy

Article Title: Footprint-free induced pluripotent stem cells can be successfully differentiated into mesenchymal stromal cells in the feline model

doi: 10.1186/s13287-025-04325-2

Figure Lengend Snippet: Feline iPSCs express pluripotent characteristics, pluripotency markers, and silencing of exogenous transgenes. ( A ). Characteristic morphology of established iPSC colonies after being passaged onto feeder cells. Arrows indicate cells with a high nuclear-to-cytoplasmic ratios. ( B & C ). Conventional RT-PCR analysis of endogenously expressed feline pluripotency markers SOX2 , NANOG and OCT4 , and the loading control GAPDH , in feline iPSCs at passage (P) 0, 3, 6, 9 and 12 ( B ) and P15 and 25 ( C ). Full-length gels are presented in Additional File 9: Fig. ). ( D ). Conventional RT-PCR analysis of human exogenous transcription factors c-Myc , KOS , KLF4 , and Sendai virus (SV), in feline iPSCs at P 0, 3, 6, 9 and 12. Feline GAPDH was included as loading control. ( E ). Expression of SeV using qPCR analysis at different passages

Article Snippet: Retroviral plasmids based on the Moloney murine leukemia virus (MMLV) were purchased from Addgene and contained the coding sequences for the human transcription factors: OCT4 (Addgene #17217), SOX2 (Addgene #17218), NANOG (Addgene #18115), c-MYC (Addgene #17220), and KLF4 (Addgene #17219).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Virus, 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) 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