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: American Journal of Human Genetics

Article Title: Identification and single-base gene-editing functional validation of a cis-EPO variant as a genetic predictor for EPO-increasing therapies

doi: 10.1016/j.ajhg.2022.08.004

Figure Lengend Snippet: Establishment of heterozygous knockin at rs1617640 (A/C) in HEK-293 cells using CRISPR-Cas9 gene-editing technology alongside piggyBac transposon system (A) Schematic of the generation of a SNP knockin model of rs1617640 using CRISPR-Cas9 gene editing alongside the piggyBac transposon system. The location of rs1617640 (A/A) and the site at which the gRNA is designed to introduce a DSB within the wild-type sequence of HEK-293 cells is shown in the top panel. Two homology arms either side of the closest TTAA site are designed with one (5′ arm) containing the desired gene edit at rs1617640 (C/C). The two homology arms are cloned into the targeting construct either side of the piggyBac transposon carrying the selectable markers, puroΔtk. Upon introduction of a DSB at the site targeted by the gRNA, DNA repair via homologous recombination due to the presence of the homologous arms is initiated and the the piggyBac transposon becomes integrated into the genomic DNA at the TTAA site. After selection with puromycin, clones with mutation-corrected lines were identified and transiently transfected with piggyBac transposase plasmid, followed by FIAU treatment to eliminate piggyBac -containing clones. Mutation-corrected heterozygous clones for rs1617640 (A/C genotype) were isolated with no marks remaining within the genomic DNA. (B) Sanger sequencing results of the sequence at each stage of the CRISPR-Cas9 and piggyBac transposon technique. The top panel represents the wild-type sequence at rs1617640 and the wild-type sequences up- and downstream of the TTAA site prior to gene editing. The middle panel represents the mutated sequence at rs1617640 and the insertion of the piggyBac transposon sequence at the TTAA site following homologous recombination after successful introduction of the DSB. The bottom panel represents the seamless excision of the piggyBac transposon from the genomic DNA. The yellow highlighted region represents the gRNA sequence. (C) PCR gel electrophoresis confirming the insertion of the piggyBac transposon in clones 61 and 67. (D) PCR gel electrophoresis confirming successful removal of the piggyBac transposon from the genome following treatment with the transposase in clone 7-3. (E) qRT-PCR of EPO to validate rs1617640 as causal in controlling EPO mRNA expression levels. The graph shows the relative change in mRNA expression levels (+/− SEM) between genotypes (A/A and A/C). (F) qRT-PCR of genes involved in the Notch signaling pathway ( HEY2 , DTX3L , and PARP9) and two control genes ( PPIA and POLR2A ). EPO was repeated again as a positive control for altered expression for comparison. The graph shows the relative change in mRNA expression levels (+/− SEM) between genotypes. Columns and error bars in (E) and (F) represent mean and SEM values. Paired t test was performed. ns, non-significant; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: 48 h after transfection, cells were subject to 200 nM Fialuridine (FIAU) (Sigma-Aldrich, Missouri, USA) selection for 7 days, replacing media every 2–3 days, to select for cells no longer carrying the piggyBac transposon cassette.

Techniques: Knock-In, CRISPR, Introduce, Sequencing, Clone Assay, Construct, Homologous Recombination, Selection, Mutagenesis, Transfection, Plasmid Preparation, Isolation, Nucleic Acid Electrophoresis, Quantitative RT-PCR, Expressing, Positive Control

Journal: Gels

Article Title: An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

doi: 10.3390/gels8070404

Figure Lengend Snippet: Evaluation of FIAU-induced cytotoxicity in B5-CF scaffolds at day 1, day 3, day 5, day 7, and day 14. ( A ) Experimental timeline for the drug administration. ( B ) Cell proliferation. ( C ) Cytotoxicity (LDH release). ( D ) Albumin expression was assessed at different time points after FIAU treatment ( n = 3, *: p < 0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001), compared to the same cryogel type at day 1. ( E ) Evaluation of FIAU-induced human-specific toxicity, mitochondrial disfunction in B5-CF scaffolds. ATP content in cell-laden B5-CF was assessed at the end of 14 days after FIAU treatment. n = 3, ***: p < 0.005, ****: p < 0.001, compared to the group without FIAU treatment. ###: p < 0.005, between the samples at the extremities of the line. The red dashed line indicates the 50% relative cell proliferation.

Article Snippet: Fialuridine (FIAU) was ordered from Tokyo Chemical Industry (Tokyo, Japan).

Techniques: Expressing

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