Journal: Stem Cells International

Article Title: Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method

doi: 10.1155/2016/6853081

Figure Lengend Snippet: RNA-mediated generation of iPS cells using an ECM-based xeno-free/feeder-free hPSC medium. (a) A schematic representation of the experimental design. Human adult dermal fibroblasts were seeded on 6-well plates (5 × 10 4 cells per well) and were subjected to multiple transfections as shown in the schedule. (b) The ESC-like colonies began to appear on day 11 after cell seeding (see the tip of the arrow at the center of the white circle, top right panel ). The number within the square at the right bottom corner of each panel indicates the number of days passed after cell seeding. Scale bar: 100 µ m. (c) The graph shows the number of ESC-like colonies generated per 10,000 fibroblasts initially seeded. (d) The percentage of ESC-like colonies among the total colonies is presented in graph format.

Article Snippet: Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Generated

Journal: Stem Cells International

Article Title: Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method

doi: 10.1155/2016/6853081

Figure Lengend Snippet: qRT-PCR analyses of the iPS cells. (a) The mRNA-iPSCs were analyzed for the expression of multiple representative lineage-specific markers. H9-hESCs and urine-derived iPS cells generated using the episomal plasmid method (UNFiPSC1) were used as positive controls, and EBs were used as negative controls. ∗∗∗ p < 0.01. (b–d) The expression levels of representative markers of derivatives of ectoderm (NCAM, Nestin, and Pax6) (b), mesoderm (FoxF1, Hand1, and Gata2) (c), and endoderm (AFP and GATA6) (d) were examined via qRT-PCR. In these experiments, H9-hESCs and UNFiPSC1 were used as controls for undifferentiated cells, whereas EBs and fibroblasts were used as controls for differentiated cells. ∗∗∗ p < 0.01.

Article Snippet: Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Generated, Plasmid Preparation

Journal: Stem Cells International

Article Title: Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method

doi: 10.1155/2016/6853081

Figure Lengend Snippet: Genome-wide gene expression profiling of the mRNA-iPSCs. (a-b) Scatterplot analysis showed that the global gene expression pattern of mRNA-iPSCs is similar to that of H9-hESCs (a). However, the gene expression patterns are clearly different between mRNA-iPSCs and their parental cells, fibroblasts (b). (c) Heatmap analysis indicated that the expression patterns of 100 hESC-enriched genes and 100 human fibroblast-enriched genes (Supplementary Table 4) in mRNA-iPSCs were similar to those in H9-hESCs but not fibroblasts. (d-e) PluriTest analysis showed that mRNA-iPSCs and other previously established iPS cells (UNFiPSC1 and ANiPSC1) were clustered with H9-hESCs in the pluripotent group, whereas fibroblasts and other primary cells were clustered in the nonpluripotent group. (f) Gene profiling-based hierarchical clustering analysis demonstrated the close association of the mRNA-iPSCs with H9-hESCs but not with the primary cells (fibroblasts).

Article Snippet: Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).

Techniques: Genome Wide, Gene Expression, Expressing

Journal: Stem Cells International

Article Title: Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method

doi: 10.1155/2016/6853081

Figure Lengend Snippet: Analyses of the methylation in the Oct4 and Nanog promoters and of chromosomal abnormality in the mRNA-iPSCs. (a) The bisulfite sequencing data indicated that the promoters of the Oct4 and Nanog genes in the mRNA-iPSCs were largely demethylated, similar to the methylation status of these promoters in hESCs. In contrast, their original cells, fibroblasts, were hypermethylated at these promoters. (b) G-banding analysis showed that no apparent chromosomal abnormality was generated during reprogramming and extended culture (for 35 passages) of the mRNA-iPSCs in our ECM-based xeno-free/feeder-free hPSC culture system.

Article Snippet: Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).

Techniques: Methylation, Methylation Sequencing, Generated

Journal: PLoS ONE

Article Title: Proteomic Profiling of Human Keratinocytes Undergoing UVB-Induced Alternative Differentiation Reveals TRIpartite Motif Protein 29 as a Survival Factor

doi: 10.1371/journal.pone.0010462

Figure Lengend Snippet: A: Immunofluorescent staining of TRIM29 (green) of histological sections of reconstructed epidermis and from human skin tissue were visualized by semi-quantitative confocal microscopy. Nuclei were stained with TO-PRO-3 (blue). The results are representative of three independent experiments. B: TRIM29 mRNA abundance is more important in keratinocytes than in other cell types. Total RNA was extracted from subconfluent N-hTERT and primary keratinocytes, from dermal fibroblasts AG04431 and from hepatocarcinoma cells (HepG2). Real-time RT-PCR was performed for human TRIM29. TRIM29 mRNA abundance in fibroblasts was considered as the reference. The results (means ± SD from triplicates) (N = 3) are presented on a logarithmic scale (** p<0.01 and *** p<0.001 vs fibroblasts).

Article Snippet: Human dermal fibroblasts AG04431 (Coriell Institute for medical Research, Camden NJ, USA) were grown classically in BME (Invitrogen, Carlsbad, CA, USA) containing 10% (v:v) fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), under 5% CO 2 .

Techniques: Staining, Confocal Microscopy, Quantitative RT-PCR