Journal: Stem Cell Research & Therapy

Article Title: A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

doi: 10.1186/s13287-023-03311-w

Figure Lengend Snippet: hTERT-transduced cell lines are phenotypically stable without senescence traits. A Schematic representation of the different strategies employed to obtain hepatocyte-like cells (HLC) from human dermal fibroblasts (HDF), i.e., exogenous constitutive expression of HNF4A, HNF1A and FOXA3 using independent lentiviral vectors (HLC-3F), a doxycycline-inducible polycistronic lentiviral vector expressing HNF4A, HNF1A and FOXA3 (TetO-HHFG; diHLC), TetO-HHFG expression in large-T antigen immortalized HDF (diHLC-LT) and TetO-HHFG expression in hTERT-transduced HDF (diHLC-T). B Representative phase contrast images of iHDF and iHDF-T at PD v.low (just derived; approximately PD24), PD low (PD 30–40) and PD high (PD 100–110). Images depict confluent cultures when possible. Black bar equals 100 μm. C Estimated doubling time for PD low and PD high iHDF-T ( n = 6; three replicates each). D Volcano plot of all genes expressed in PD high vs. PD low iHDF-T (three replicates each) by RNAseq. Plotted genes were expressed in all samples of at least one group (absolute reads > 10). Red dots correspond to genes differentially expressed defined as FDR < 0.05 and fold change > 2 (0.6%). E mRNA level of CDKN1A and CDKN2A in iHDF-T at PD v.low , PD low and PD high . Gray bars represent primary human dermal fibroblasts at PD16 and PD54 (early senescent). Values correspond to the average of 2 experiments with four replicates each. *** p < 0.005. F None of the senescence-related gene sets was significantly different between PD low and PD high iHDF-T according to GSEA (see details in the text). Filled bars: NES > 0; Dashed bars: NES < 0. Red line marks FDR = 0.05

Article Snippet: For pathway analysis, normalized data from RNAseq was filtered for duplicate symbols and analyzed using GSEA software (Broad Institute) according to published methods [ ].

Techniques: Expressing, Plasmid Preparation, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

doi: 10.1186/s13287-023-03311-w

Figure Lengend Snippet: hTERT-transduced cell lines do not acquire neoplastic features at PD high . A iHDF-T karyotype is not altered after more than 70 population doublings: 46XX,del (15) (q11.2q15). Twenty-five cells per cell population were analyzed. B iHDF-T do not grow under soft matrix compared to carcinogenic HepG2 cells. A total of 100 cell aggregations per cell type were analyzed. C Percentage of Ki67-positive cells in cultures of PD low and PD high iHDF-T. Cells were cultured 6 days at 0.5% serum, 6 days at 10% serum and 2 days at 10% serum. A total of 10 images from cultured cells immunostained with anti-Ki67 antibody were analyzed using ImageJ software. Number of cells counted was 360, 470 and 300 for 6d-0.5%; 6d-10% and 2d-10%, respectively. * p < 0.05 D Most significant oncogenic molecular signatures in PD high vs. PD low iHDF-T (GSEA). Filled bars: NES > 0, enriched in PD high . Filled bars: NES < 0, enriched in PD low . Red line marks FDR = 0.05. Note that HOXA9, JNK and PDGF-ERK gene sets contain genes up and downregulated after silencing of the procarcinogenic genes in malignant cell lines

Article Snippet: For pathway analysis, normalized data from RNAseq was filtered for duplicate symbols and analyzed using GSEA software (Broad Institute) according to published methods [ ].

Techniques: Cell Culture, Software

Journal: Stem Cell Research & Therapy

Article Title: A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

doi: 10.1186/s13287-023-03311-w

Figure Lengend Snippet: PD high diHLC-T gene expression resemble human hepatocytes. iHDF-T were reprogrammed to diHLC-T by incubation in HMM media containing 250 ng/mL Dox for 12 days. Control cells were cultured in parallel without Dox (iHDF-T HMM ). A Representative phase contrast images of iHDF-T HMM and diHLC-T after 12 days culture. White bar equals 100 μm. B mRNA levels of multiple hepatic gens in diHLC-T. Values correspond to the average plus standard deviation of two different experiments (four samples each) determined by RT-qPCR and expressed relative to adult human liver (pooled RNA from 3 donors). Fetal liver values were obtained by comparison with values extracted from GSE15238 . Statistical significance between diHLC-T and fetal liver is included.* p < 0.05; ** p < 0.01; *** p < 0.005. C RNAseq extracted heatmaps depicting expression of genes within Aizarani_hepatocyte single-cell , Zabulica and archetypal HDF genes (Additional file : Table S3). Enrichment plots (GSEA) are shown on the right. D Table containing the most significant (FDR > 0.05) signature gene sets determined by GSEA using C8 MSigDB cell types signature gene sets ( https://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=C8 ). E Bar graph depicting the most significant (FWER) signature gene sets determined by GSEA using all liver 31 single-cell signature gene sets reported by Aizarani . Orange: NES > 0, enriched in diHLC-T. Blue: NES < 0, enriched in iHDF-T. Red line marks FDR = 0.05. As expected, stellate cells, a quiescent fibroblast cell present in the liver, correlate with iHDF-T

Article Snippet: For pathway analysis, normalized data from RNAseq was filtered for duplicate symbols and analyzed using GSEA software (Broad Institute) according to published methods [ ].

Techniques: Gene Expression, Incubation, Control, Cell Culture, Standard Deviation, Quantitative RT-PCR, Comparison, Expressing