defined hepatocyte growth medium hcm  (Lonza)

Journal: bioRxiv

Article Title: Cellular transformation by combined lineage conversion and oncogene expression

doi: 10.1101/525600

Figure Lengend Snippet: ( A ) Schematic outline of the cell transformation assay for making lineage-specific cancer by lentiviral expression of three lineage-specific TFs to convert HFs to induced hepatocytes (iHep) and defined oncogenic drivers to transform iHeps to proliferating and tumorigenic cells. ( B ) Comparison of TF combinations ( Du et al , 2014 , Huang et al , 2014 , Morris et al , 2014 ) for converting human fibroblasts to iHeps by detecting transcript levels for liver marker genes ( ALBUMIN , TRANSFERRIN and SERPINA1/α-1-antitrypsin ) by qRT-PCR at different time points after iHep conversion, normalized to GAPDH levels (mean ± standard error).

Article Snippet: The medium was changed to fresh fibroblast medium containing β-mercaptoethanol on day 2 and to a defined hepatocyte growth medium (HCM, Lonza) on day On day 6, the cells were passaged on plates coated with type I collagen (Sigma) in several technical replicates, and thereafter, the HCM was replenished every two–three days.

Techniques: Cell Transformation Assay, Expressing, Comparison, Marker, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Cellular transformation by combined lineage conversion and oncogene expression

doi: 10.1101/525600

Figure Lengend Snippet: ( A ) Gene set enrichment analysis (GSEA) results for CMT-iHeps and CMT+sg TP53 -iHeps compared to control fibroblasts against liver cancer signature [Subclass 2 ( Hoshida et al , 2009 )] from molecular signatures database (MSigDB). Positive normalized enrichment score (NES) reflects overrepresentation of liver cancer signature genes among the top ranked differentially expressed genes in CMT-iHep and CMT+sg TP53 -iHep conditions compared to control fibroblasts. ( B ) Differential expression levels [log2(fold change)] of marker genes for fibroblasts, hepatocytes, and liver cancer in bulk RNA-seq measurements from CMT+sg TP53 -iHeps and CMT-iHeps at p20 (∼22 weeks after oncogene transduction) as well as xenograft tumor from CMT+sg TP53 cells against control fibroblasts (mean ±standard error, n=3). ( C ) IGV snapshots for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) showing ATAC-seq enrichment from fibroblast and CMT+sg TP53 -iHeps. ( D ) Chromatin accessibility and CpG methylation of DNA measured using NaNoMe-seq. Cytosine methylation detected using Nanopore sequencing from CMT+sg TP53 -iHeps and control fibroblasts is shown for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) using a window of TSS ±1500 bp. GpCpH methylation (all GC sequences where the C is not part of a CG sequence also, top) reports on chromatin accessibility, whereas HpCpG methylation reports on endogenous methylation of cytosines in the CpG context. ( E ) CpG methylation detected using bisulfite-sequencing from primary human foreskin fibroblasts and from normal adult liver [data from the ]. IGV snapshots from the genomic loci corresponding to the MMP3 , SERPINA1 , and SAA1 promoters (same regions as indicated in ) showing methylation proportions [methylated calls / (methylated calls + unmethylated calls)] for all CpGs covered by at least 4 reads.

Article Snippet: The medium was changed to fresh fibroblast medium containing β-mercaptoethanol on day 2 and to a defined hepatocyte growth medium (HCM, Lonza) on day On day 6, the cells were passaged on plates coated with type I collagen (Sigma) in several technical replicates, and thereafter, the HCM was replenished every two–three days.

Techniques: Control, Quantitative Proteomics, Marker, RNA Sequencing, Transduction, CpG Methylation Assay, Methylation, Nanopore Sequencing, Sequencing, Methylation Sequencing