Journal: Cell Regeneration

Article Title: Modeling drug-induced liver injury and screening for anti-hepatofibrotic compounds using human PSC-derived organoids

doi: 10.1186/s13619-022-00148-1

Figure Lengend Snippet: Modeling DILI with diverse phenotypes using HLOs. A Albumin secretion after administration of APAP at 0.5, 3, or 10 mM for 7 days in HLOs. n = 3, * P < 0.05. B ATP content analysis after administration of APAP at 0.5, 3, or 10 mM for 7 days in HLOs. n = 3, * P < 0.05. C Representative images of ROS intensity (CellROX in red, and DAPI in blue) after administration of APAP at 0.5, 3, or 10 mM for 7 days in HLOs. Scale bar, 50 μm. Right: Quantification of the number of CellROX-positive events per organoid. n = 15, * P < 0.05. D Albumin secretion after administration of FIAU at 1, 10, or 30 μM for 10 days. n = 3. E Representative images of lipid droplets (Bodipy in green, and DAPI in blue) and mitochondrial depolarization (TMRM in red) in the HLOs after administration of FIAU at 1, 10, or 30 μM for 10 days. Scale bars, 50 μm. Right: Quantification mean of Bodipy fluorescent intensity and quantification of the number of depolarization events per organoid. n = 15, * P < 0.05. F Representative images of lipid droplets (Bodipy in green, and DAPI in blue) and fibrosis (Collagen I in green, and DAPI in blue) in HLOs after administration of MTX at 1, 10, or 30 μM for 7 days. Scale bars, 50 μm. Right: Quantification mean of Bodipy and Collagen fluorescent intensity. n = 15, * P < 0.05. G Expression of fibrogenic marker genes in HLOs after treatment with MTX at 1, 10, or 30 μM for 7 days. n = 3, * P < 0.05. H Albumin secretion after MTX treatment for 7 days in HLOs. n = 3. I Representative images of ROS (CellROX in red, and DAPI in blue), after administration of TAK-875 at 3, 10 or 30 μM for 7 days in HLOs. Scale bars, 50 μm. Right: Quantifications of CellROX-positive events per organoid. n = 15. J Released MCP-1 and IL-6 in HLOs after 7 days of TAK-875 treatment. n = 3. * P < 0.05. K Albumin secretion after TAK-875 treatment for 7 days in HLOs. n = 3. Values represent means with SEM. P values were assessed by one-way ANOVA with Dunnett’s multiple comparisons test ( A , B , D , G , H , J , K ), Kruskal–Wallis tests ( C , E , F , I )

Article Snippet: About 100 HLOs per well were incubated with a culture medium containing 5% Matrigel and acetaminophen (APAP; MCE, T0065), methotrexate (MTX; Selleck, S1210), fasiglifam (TAK-875; MCE, 1000413-72-8) for 7 days, fialuridine (FIAU; Sigma, SML0632) for 10 days, or TGFβ1 (Peprotech, AF-100-21C) /LPS (Sigma, L4391) for 3 days, in 96 well ultra-low attachment multiwell plate (Corning, 3473), respectively.

Techniques: Expressing, Marker

Journal: Frontiers in Genome Editing

Article Title: Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events

doi: 10.3389/fgeed.2022.843885

Figure Lengend Snippet: PBase Removal of the Puro∆TK Cassette from Vector Replacement Cell Lines. (A) Graphic illustration of the Puro∆TK cassette excision. (B) Colonies surviving GCV or FIAU selection excision were confirmed as Puro∆TK cassette negative clones by PCR using P8/P4 and P7f/P6r primers to generate the PCR results as shown in gel pictures. M; 100 bp DNA ladder plus. (C) Sequence of excised clones screened from PBase-transfected Ic8 clones selected with 0.2 μM of GCV (Ic8GCVe2, top clone) or with FIAU (Ic8FIAUe11, bottom clone). Adenine (A) and Guanine (G) capitalized and highlighted in red representing W1282X mutation (G > A) and the correction (A > G), respectively. Dot represents deleted nucleotide.

Article Snippet: In order to remove the Puro∆TK cassette containing the drug selection markers from modified CF2-iPS3, a PiggyBac transposase (PBase) expression vector was transfected into recombinant cells, followed by negative selection with Fialuridine (FIAU) (Santa Cruz Biotechnology Inc., Dallas, TX) or Ganciclovir (GCV) (Sigma-Aldrich) ( ).

Techniques: Plasmid Preparation, Selection, Clone Assay, Sequencing, Transfection, Mutagenesis

Journal: PLoS Genetics

Article Title: Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

doi: 10.1371/journal.pgen.1004951

Figure Lengend Snippet: A. PCR analyses of CRE-transfected, FIAU selected ES cell clones. F, PCR amplification of the 4.9Mb borders using forward primers (RP24-F1 (blue arrow) and RP23-F2 (red arrow)); R, PCR amplification of the 4.9Mb borders using reverse primers (RP24-R1 (blue arrow) and RP23-R2 (red arrow)). Presence of PCR fragments in both the F and R PCR amplifications indicates the presence of the inverted 4.9M syntenic region. The scheme below the photograph shows the relative positions of the PCR primers on the wild type (wt) and inverted (inv) chromosomes, respectively. Arrowheads indicate the position of the remaining incompatible LoxP sites. Shaded blue box represents the 4.9Mb syntenic region, red arrows indicate the position and transcriptional orientation of Foxo1 . B. FISH analyses of metaphase and interphase chromosomes of one of the ES cell clones carrying the 4.9Mb inversion using the RP24–391O12 (green = centromeric border) and RP23–422I13 (red = telomeric border) BAC probes. The wild type chromosome 3 (wt) produced contiguous red and green hybridization signals, the chromosome 3 with the inversion (inv) shows split hybridization signals. Left—metaphase spread; right—interphase nucleus. Scheme below the micrographs shows the positions of the two BAC probes on the wild type (wt) and inverted (inv) chromosomes, respectively. C. Western blot of cell lysates from Foxo1-inv +/+ and wild type (WT) myoblasts probed with a Foxo1 antibody (Foxo1). Detection of actin serves as a loading control. D. Interphase nuclei of fibroblasts from a mouse homozygous for the 4.9 Mb syntenic region inversion (Foxo1-inv +/+ ) showing split signals after in situ hybridization with the RP23–422I13 (shown in green) and RP24–391O12 (shown in red) BAC probes.

Article Snippet: After 5 days of selection with 0.2μM of fialuridine (FIAU) (a kind gift of Bristol Myers) ES cells were collected for DNA isolation as a pool or as single clones.

Techniques: Transfection, Clone Assay, Amplification, Produced, Hybridization, Western Blot, Control, In Situ Hybridization