newborn human dermal fibroblast nhdf  (ATCC)

Journal: Genes

Article Title: A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors

doi: 10.3390/genes15050575

Figure Lengend Snippet: SMAR vectors are capable of reprogramming human dermal fibroblasts. ( A ) Scheme depicting episomal reprogramming by EBNA or SMAR vectors in neonatal human dermal fibroblasts (NHDFs) by transfection with SMAR or EBNA reprogramming vectors at 1×, 2×, or 5× doses of vector by mass. Cells were cultured for 30–40 days post-transfection and monitored for the formation of colonies. ( B ) GFP positivity of transfected NHDFs was quantified 24 h and 48 h post-transfection using an Incucyte SX5. Scale = 500 µm. Data represent mean ± standard deviation; significance was measured by a two-way ANOVA; ns = non-significant; **** = p ≤ 0.0001. ( C ) Brightfield images of NHDFs after transfection over time. Scale = 500 µm. Data shows three time points from one representative experiment.

Article Snippet: Neonatal human dermal fibroblasts (NHDF, newborn male) were obtained from Thermo Fisher Scientific (lot No. 2456041, Carlsbad, CA, USA) and cultured on gelatine-coated dishes (0.1% gelatine in distilled water) under DMEM (4500 mg/L glucose, 2 mM L-glutamine, sodium bicarbonate, without sodium pyruvate) supplemented with 10% FBS (Gibco), 1% Pen/Strep (Gibco) and 1% non-essential amino acids (NEAA, Gibco, Life Technologies Europe, Bleiswijk, The Netherlands).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Standard Deviation

Journal: Genes

Article Title: A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors

doi: 10.3390/genes15050575

Figure Lengend Snippet: Optimisation of SMAR reprogramming yields high-quality SMAR-iPSCs with similar gene expression to human embryonic stem cells. Three EBNA clones and four SMAR5x clones were analysed ( A ) by immunofluorescence for the expression of stem cell markers SSEA-4 (green), Nanog (orange), Tra-1-81 (green), and Oct3/4 (red), ( B ) by qRT-PCR for their expression of stemness genes compared with three human embryonic stem cell (HuES) lines. Data shown are a z-transformation of the ddCT of gene expression to the negative control fibroblast line. ( C ) Principal component analysis (PCA) of delta Ct expression of stemness genes. Arrows represent the contribution of each gene to the principal component. ( D ) iPSC clones were differentiated into cells of three distinct lineages and analysed by immunofluorescence for the expression of lineage-specific factors. Sox17 (yellow) was used as an endoderm marker, NCAM (green) as a mesoderm marker, and β-tubulin III (magenta) as an ectoderm marker. ( E ) qRT-PCR for lineage-specific markers after directed differentiation of iPSC lines compared with HuES lines. Data shown are a z-transformation of the ddCT of gene expression to the negative control fibroblast line. ( F ) Principal component analysis (PCA) of delta Ct expression of lineage-specific genes. Arrows represent the contribution of each gene to the principal component. Scale = 200 µm. Heatmap data represent mean values, and significance was measured for each group compared to negative control fibroblasts by a one-sample t -test assuming 0 as the theoretical mean or between groups (indicated by brackets) by a one-way ANOVA; ns = non-significant; * = p ≤ 0.05.

Article Snippet: Neonatal human dermal fibroblasts (NHDF, newborn male) were obtained from Thermo Fisher Scientific (lot No. 2456041, Carlsbad, CA, USA) and cultured on gelatine-coated dishes (0.1% gelatine in distilled water) under DMEM (4500 mg/L glucose, 2 mM L-glutamine, sodium bicarbonate, without sodium pyruvate) supplemented with 10% FBS (Gibco), 1% Pen/Strep (Gibco) and 1% non-essential amino acids (NEAA, Gibco, Life Technologies Europe, Bleiswijk, The Netherlands).

Techniques: Gene Expression, Clone Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Transformation Assay, Negative Control, Marker