episomal vectors encoding reprogramming factors pclxe-h oct4 / shp53  (Addgene inc)

Journal: medRxiv

Article Title: Investigating the pathogenicity of the recessive HNF1A p.A251T variant in monogenic diabetes using iPSC-derived beta-like cells

doi: 10.1101/2024.12.10.24318788

Figure Lengend Snippet: Reprogramming and characterisation of iPSC carrying p.A251T HNF1A variant . A: Overview of the iPSC reprogramming protocol. Somatic fibroblasts were transfected with episomal reprogramming vectors at day 4. After transfection, cells were cultured in N2B27 media with bFGF for 14 days. Media was then switched to stem cell conditions, and colonies were picked around day 21 for further analysis. B: Confocal microscopy images showing OCT4 expression in reprogrammed iPSC colonies. Cells were stained with DAPI (blue, 405 nm) to label nuclei and OCT4 (green, 488 nm) to assess pluripotency. Imaging was performed using the Leica Stellaris 8 Confocal Microscope. Co-localisation of DAPI and OCT4 indicates pluripotency. C: Quantification of OCT4-positive cells. OCT4-positive cells were identified by co-localisation with DAPI and quantified using an automated macro. The graph shows the percentage of OCT4-positive cells relative to total cells in the colonies. D: Morphological changes during the differentiation process observed under brightfield microscopy. Day 1: Fibroblast-like cells (top left). Day 12: Intermediate cell morphology (top right). Day 15: iPSC-like colony formation. Day 18: Growth phase, ready for picking. Arrows indicate changes in cell morphology over time. Scale bar = 100 µm. E: Karyotyping analysis of selected iPSC clones. G-banding of chromosomes revealed a normal karyotype with 46 chromosomes, including two X chromosomes, consistent with the donor’s female sex.

Article Snippet: A minimum of 3×10 6 fibroblast cells were electroporated (1650V, 10ms, 3 pulses) with 3ug of three 3 reprogramming plasmids containing the episomal factors Oct4, Sox2, Klf4, Lin28, and L-Myc (OSKM): pCXLE-hSK (Addgene), pCXLE-hUL (Addgene) and pCXLE-hOCT3/4-shp53-F (Addgene) ( ).

Techniques: Variant Assay, Transfection, Cell Culture, Confocal Microscopy, Expressing, Staining, Imaging, Microscopy, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Monodelphis domestica Induced Pluripotent Stem Cells Reveal Metatherian Pluripotency Architecture

doi: 10.3390/ijms232012623

Figure Lengend Snippet: POU5F1 and splice-variant-specific expression of POU5F3 synergistically regulate core pluripotency genes in monoiPSCs. ( a ) Gene/exon structures of the M. domestica genome transcribed POU5F3 splice variants; the XM_016427856.1 splice variant encodes full length transcript that is comprised of five exons; the XM_003339642.2 has exon 4 truncated and XM_007475408.1 lacks exon 4 completely; XM_16427857.1 has exon 1 truncated. ( b ) Line plot showing normalized expression of POU5F1 and POU5F3 splice variants in M. domestica embryonic cells, in brain, heart, liver, and gonadal developmental stages, and in neoSF and established monoiPSCs. ( c ) Line plot of core pluripotency transcription factors in M. domestica embryonic cells, in brain, heart, liver, and gonadal developmental stages, and in neoSF and established monoiPSCs. ( d ) ICC-based expression and localization of POU5F1, NANOG and SOX2 in a partially reprogrammed monoiPSC clone. ( e ) Successful differentiation of the partially reprogrammed monoiPSC clone into SOX17-expressing and CXCR4-expressing multipotent definitive endodermal cells.

Article Snippet: One million M. domestica neoSF cells in log growth phase were harvested and then nucleofected with 1 μg each of the purified OriP/EBNA episomal plasmids encoding human reprogramming factors OCT4 , SOX2 , NANOG , KLF4 , LIN28 and cMYC (all from Addgene, Watertown, MA, USA) [ , ], using Amaxa nucleofector technology on a 4D-Nucleofactor X Unit and the protocol (P2 primary cell nucleofector solution and DT130 program) we have optimized for M. domestica neoSF (Lonza, http://www.lonza.com/ ).

Techniques: Variant Assay, Expressing