Journal: Stem Cell Research & Therapy

Article Title: Evaluation of photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients with defects in the EYS gene: a possible cost-effective cellular model for mechanism-oriented drug

doi: 10.1186/s13287-022-02827-x

Figure Lengend Snippet: Effect of drugs on gene expression in photoreceptor-directed fibroblasts derived from an EYS-RP patient, Pt#1. The fibroblasts of a healthy individual (N#3) and fibroblasts of a patient suffering from retinitis pigmentosa due to a homozygous mutation in the EYS gene (Pt#1) were transdifferentiated to photoreceptor-like cells by retroviral transduction of four transcription factors. Gene expression was compared 2 weeks post-transduction. Differentiation medium was supplemented with four drugs, 4-phenylbutyric acid (4-PBA), metformin (Metf), rapamycin (Rapa), N-acetyl-L-cysteine (NAC) or the vehicle (water and ethanol (EtOH)) or none (-). The gene expression with each pharmacological treatment was compared to the gene expression in photoreceptor-like cells derived from fibroblasts of healthy individual without any supplement (-). Columns represent mean ± SEM. All data points are overlaid ( n = 6 (N#3 without any supplement (-)), n = 3 (N#3 with supplement and Pt#1). a = p > 0.05, b = p > 0.01, c = p > 0.001; One-way ANOVA followed by Tukey’s honest test. For the sake of simplicity, comparison with the gene expression level in N#3 without any supplement (-) is shown here. Comparison among other groups is shown in Additional file : Fig. S2

Article Snippet: After 5–6 h of retroviral transduction, the media in transduced fibroblast cells were replaced with differentiation media containing either one of the four drugs (rapamycin (Selleck chemicals, Cat#S1039), 4-PBA (WAKO, Cat#168-06471), NAC (WAKO, Cat#015-05132), metformin (Abcam, Cat#ab120847)), vehicle or no addition for Pt#1 and containing vehicle or no addition for N#3.

Techniques: Expressing, Derivative Assay, Mutagenesis, Transduction

Journal: JCI Insight

Article Title: Multidimensional analysis and therapeutic development using patient iPSC–derived disease models of Wolfram syndrome

doi: 10.1172/jci.insight.156549

Figure Lengend Snippet: ( A ) A schematic of Wolfram syndrome etiology and the targets to modulate by a combination treatment of 4-PBA and TUDCA (P+T). ( B ) Expression of HiBiT-tagged WFS1 protein after treatment with 500 μM 4-PBA and 50 μM TUDCA (P+T) for 24 hours. NanoLuc levels, expressed from an identical plasmid backbone, were examined ( n = 48, P value by unpaired t test). ( C ) (Left) Representative blotting images of WFS1 and α-Tubulin in iPSCs treated with or without P+T for 48 hours. (Right) Quantification of WFS1 protein levels normalized with α-Tubulin. ( n = 3, * P < 0.05 by unpaired t test compared with Ctrl.) ( D ) Relative mRNA levels of WFS1 in iPSCs treated with or without P+T for 48 hours ( n = 5, ** P < 0.01 by unpaired t test compared with Ctrl). ( E ) Representative immunofluorescence images of neural progenitor cell (NPC) markers in NPCs differentiated from patient-derived iPSCs. Scale bar: 100 μm. ( F ) Quantitative PCR analysis of ER stress–related genes in NPCs treated with or without P+T for 48 hours. ( n = 6, * P < 0.05, ** P < 0.01, and **** P < 0.0001 by unpaired t test compared with Ctrl.) ( G ) Mitochondrial respiration of NPCs treated with or without P+T for 48 hours represented as percentage of baseline oxygen consumption rate (OCR) measurements. Respiration was interrogated by measuring changes in relative OCR multiple times, every 8.5 minutes, after injection with oligomycin (OM), FCCP, and antimycin A (AA)/rotenone (R) ( n = 3, W024: *** P < 0.001, W392: * P < 0.05, and W121: * P < 0.05 by unpaired t test compared with Ctrl AUC). ( H ) Caspase-3/7 activity normalized by cell viability in NPCs treated with or without either of 4-PBA, TUDCA, or P+T for 48 hours. ( n = 7; *** P < 0.001 and **** P < 0.0001 by 1-way ANOVA compared with Ctrl; # P < 0.05, ## P < 0.01, and #### P < 0.0001 by 1-way ANOVA.)

Article Snippet: We obtained 4-PBA and TUDCA from Amylyx Pharmaceuticals Inc. We dissolved 4-PBA and TUDCA in PBS and used them at final concentrations of 500 μM and 50 μM, respectively.

Techniques: Expressing, Plasmid Preparation, Immunofluorescence, Derivative Assay, Real-time Polymerase Chain Reaction, Injection, Activity Assay

Journal: JCI Insight

Article Title: Multidimensional analysis and therapeutic development using patient iPSC–derived disease models of Wolfram syndrome

doi: 10.1172/jci.insight.156549

Figure Lengend Snippet: ( A ) Intraperitoneal glucose tolerance test (IP-GTT) with WT or Wfs1 -KO mice at baseline and 1 month after feeding with either control chow or food containing 4-PBA: 0.338% and TUDCA: 0.225% (P+T chow). ( B ) AUCs of the IP-GTT (KO, Ctrl: n = 12; KO, P+T: n = 12; WT: n = 7; ** P < 0.01 and *** P < 0.001 by 1-way ANOVA; ## P < 0.01 and #### P < 0.0001 by 1-way ANOVA compared with WT: Baseline; †††† P < 0.0001 by 1-way ANOVA compared with WT: 1 month).

Article Snippet: We obtained 4-PBA and TUDCA from Amylyx Pharmaceuticals Inc. We dissolved 4-PBA and TUDCA in PBS and used them at final concentrations of 500 μM and 50 μM, respectively.

Techniques: