MOESM1 of Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector

Additional file 1: Figure S1. Cell sorting strategy using BDFACSAriaIII™ cell sorter. Figure S2. Validation of the polycistroniclentiviral vector in HDF

MOESM2 of Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector

Additional file 2: Table S1. Primers used for qRT-PCR

Additional file 1: of Silencing of hepatic fate-conversion factors induce tumorigenesis in reprogrammed hepatic progenitor-like cells

Supplementary information. (PDF 923 kb

Functional and transcriptional comparison of IPSC-Hep 3D cultures plated as single cells or clumps.

<p>(<b>A</b>) Secreted albumin, alpha-fetoprotein, and alpha-1-antitrypsin levels as evaluated by immunoassays (mean ± s.d.; n = 3 biological replicates). (<b>B</b>) qPCR heatmap of 39 hepatic genes comparing the two 3D culture conditions to adult and fetal hepatocytes (range of expression shown as sample extrema for each gene; quantitative values shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086372#pone.0086372.s002" target="_blank">Figures S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086372#pone.0086372.s004" target="_blank">S4</a></b>). (<b>C</b>) Confocal micrograph highlighting the loss of detectable albumin in 3D single cell cultures and the spontaneous polarization of IPSC-Heps within 3D clump cultures (scalebar = 100 microns).</p

A Combination of Transcriptomics and Metabolomics Uncovers Enhanced Bile Acid Biosynthesis in HepG2 Cells Expressing CCAAT/Enhancer-Binding Protein β (C/EBPβ), Hepatocyte Nuclear Factor 4α (HNF4α), and Constitutive Androstane Receptor (CAR)

The development of hepatoma-based in vitro models to study hepatocyte physiology is an invaluable tool for both industry and academia. Here, we develop an in vitro model based on the HepG2 cell line that produces chenodeoxycholic acid, the main bile acid in humans, in amounts comparable to human hepatocytes. A combination of adenoviral transfections for CCAAT/enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) decreased intracellular glutamate, succinate, leucine, and valine levels in HepG2 cells, suggestive of a switch to catabolism to increase lipogenic acetyl CoA and increased anaplerosis to replenish the tricarboxylic acid cycle. Transcripts of key genes involved in bile acid synthesis were significantly induced by approximately 160-fold. Consistently, chenodeoxycholic acid production rate was increased by more than 20-fold. Comparison between mRNA and bile acid levels suggest that 12-alpha hydroxylation of 7-alpha-hydroxy-4-cholesten-3-one is the limiting step in cholic acid synthesis in HepG2 cells. These data reveal that introduction of three hepatocyte-related transcription factors enhance anabolic reactions in HepG2 cells and provide a suitable model to study bile acid biosynthesis under pathophysiological conditions

Functional comparison of IPSC-Hep 3D clump culture versus traditional 2D culture.

<p>(<b>A</b>) Oil red O and periodic acid staining demonstrating lipid storage and glycogen synthesis in both 2D and 3D clump cultures. (<b>B</b>) qPCR analysis of select phase I and phase II enzymes, hepatic transporters, and other hepatic markers demonstrating a shift towards a more mature phenotype in the 3D clump cultures (fold expression to undifferentiated IPSCs; mean ± s.d.; n = 3 biological replicates). (<b>C</b>) Confocal micrographs comparing the presence and localization of hepatic markers within the two culture systems (scale bar = 100 microns). (<b>D</b>) CYP3A4 activity of the two culture conditions measured over a period of 75 days (mean ± s.d.; n = 3 biological replicates).</p

Dysfunctional mitochondrial fission impairs cell reprogramming

We have recently shown that mitochondrial fission is induced early in reprogramming in a Drp1-dependent manner; however, the identity of the factors controlling Drp1 recruitment to mitochondria was unexplored. To investigate this, we used a panel of RNAi targeting factors involved in the regulation of mitochondrial dynamics and we observed that MiD51, Gdap1 and, to a lesser extent, Mff were found to play key roles in this process. Cells derived from Gdap1-null mice were used to further explore the role of this factor in cell reprogramming. Microarray data revealed a prominent down-regulation of cell cycle pathways in Gdap1-null cells early in reprogramming and cell cycle profiling uncovered a G2/M growth arrest in Gdap1-null cells undergoing reprogramming. High-Content analysis showed that this growth arrest was DNA damage-independent. We propose that lack of efficient mitochondrial fission impairs cell reprogramming by interfering with cell cycle progression in a DNA damage-independent manner.</p