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Systematic comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes

ABSTRACT: Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to primary human hepatocytes (PHH). Here, we systematically compared the activation of four different stress pathways in PHH, HepG2 liver cancer cells, hiPSCs and different stages within the differentiation towards HLCs (definitive endoderm, hepatoblast, immature hepatocytes and HLCs). We exposed all different cell types in a concentration response to four different compounds that specifically activate the oxidative stress response (diethyl-maleate), unfolded protein response (tunicamycin), DNA damage response (cisplatin) and inflammatory signalling (TNF). We used targeted RNA-sequencing to map concentration response transcriptional similarities and differences using bench-mark concentration modelling for the various stress responses in the different test systems. We observed that HLCs are more sensitive to oxidative stress than PHH showing activation at ten-fold lower concentrations and induce a strong anti-oxidant response. Although similar UPR gene sets were activated in HLCs compared to PHH, PHH were about ten times more sensitive. Furthermore, HLCs were highly sensitive to inflammation conditions similar to HepG2 cells and in sharp contrast to hiPSC, which showed hardly any response. On the contrary, hiPSC and HepG2 were highly sensitive to DNA damage signalling while HLCs resisted a strong p53 DNA damage response upon exposure. Overall, the data indicate that despite limitation in full hepatocyte maturation, HLCs did gain specific adaptive cellular stress response networks that mimic those of PHHs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155771 | GEO | 2021/02/18

REPOSITORIES: GEO

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Project description:Primary human hepatocytes (PHH) are a main instrument in drug metabolism research and in the prediction of drug-induced phase I/II enzyme induction in humans. The HepG2 liver-derived cell line is commonly used as a surrogate for human hepatocytes, but their use in ADME and toxicity studies can be limited because of lowered basal levels of metabolizing enzymes. Despite their widespread use, the transcriptome of HepG2 cells compared to PHH is not well characterized. In this study, microarray analysis was conducted to ascertain the differences and similarities in mRNA expression between HepG2 cells and human hepatocytes before and after exposure to a panel of fluoroquinolone compounds. Comparison of the naÃ¯ve HepG2 cell and PHH transcriptomes revealed a substantial number of basal gene expression differences. When HepG2 cells were dosed with a series of fluoroquinolones, trovafloxacin, which has been associated with human idiosyncratic hepatotoxicity, induced substantially more gene expression changes than the other quinolones, similar to previous observations with PHH. While TVX-treatment resulted in many gene expression differences between HepG2 cells and PHH, there were also a number of TVX-induced commonalities, including genes involved in RNA processing and mitochondrial function. Taken together, these results provide insight for interpretation of results from drug metabolism and toxicity studies conducted with HepG2 cells in lieu of PHH, and could provide further insight into the mechanistic evaluation of TVX-induced hepatotoxicity. Experiment Overall Design: HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in Minimum Essential Medium (Invitrogen Life Technologies, Carlsbad,CA) with 10% Fetal Bovine Serum under a humidified 5% CO2 atmosphere using T-162 plastic culture flasks. The cells were split when they reached approximately 70-85% confluence after washing with sterile phosphate buffered saline and detachment of the cells with trypsin (Invitrogen Life Technologies, Carlsbad, CA). The HepG2 cells were cultured in 6-well plastic plates upon exposure to quinolone compounds. Primary human hepatocytes, obtained from In Vitro Technologies (IVT, Baltimore, MD) in 6-well type I collagen coated plates, were cultured with 2 mL of Hepatocyte Incubation Media (IVT) at 37Â°C with 5% CO2 for 24 hours after receipt. Experiment Overall Design: For the genomic experiments, quinolone compounds, dissolved in 0.1 N KOH (Sigma Chemical Co., St. Louis, MO), were added to the wells with fresh media at levels of 100 ÂµM (HepG2) or 400 ÂµM (primary human hepatocytes) for 24 hours using at least two technical replicates. Trovafloxacin was dosed using two separate preparations of HepG2 cells and two separate donors of human hepatocytes. Vehicle control cells were dosed with an equivalent volume of 0.1N KOH as the experimental samples. For intracellular comparison (HepG2 cells vs PHH), naÃ¯ve cells were harvested using TRIzolâ?¢ reagent (Invitrogen Life Technologies, Carlsbad, CA). Experiment Overall Design: Total RNA was isolated from the TRIzolâ?¢ extracts using the standard procedure from the manufacturer. O.D. at 260 nm determined RNA concentrations. RNA quality was accessed using an Agilent Technologies bioanalyzer before proceeding to microarray sample preparation. Microarray analysis was performed using the standard protocol provided by Affymetrix Inc. (Santa Clara, CA) and as previously described, starting with 5 Âµg of total RNA (Richert et al., 2006). Experiment Overall Design: Fragmented, labeled cRNA was hybridized to an Affymetrix human genome U133A array, which contains sequences corresponding to roughly 22,200 transcripts at 45Â°C overnight. The arrays were washed, developed, and scanned.

Project description:It is well-known that embryonic stem cells (ESC) are much more sensitive to replication-induced stress than differentiated cells but the underpinning mechanisms are largely unknown. H2A.X, a minor variant of H2A, constitutes only 1-10% of the mammalian genome. H2A.X plays a well-known for role in the DNA damage response and maintaining stability in the genome, including the regions frequently experiencing replication stress, such as the fragile sites. Intriguingly, several recent studies have reported that H2A.X function is elevated in ESC; and others reported that H2A.X function is provoked during cellular reprogramming (in induced pluripotent stem cells, iPSC), indicating that increased proliferation during iPS may trigger replication stress and the H2A.X DNA damage response. However, several studies of genomic instability in iPSC led to different conclusions on this important issue. For example, frequent copy number variants (CNV) were reported at the genomic regions sensitive to replication stress, such as the fragile sites. On the other hand, another study reported the lack of genomic instability in mouse iPS clones that are able to generate “all-iPS” animals in tetraploid complementation assays (4N+ iPSC), indicative of a potential link between pluripotency and genome integrity. However, whether if high level genomic instability occurs in the 4N- iPSC iPSC clones at replication stress sensitive regions is unknown. Moreover, due to the lack of mechanistic insights on genome integrity maintenance, how pluripotency and genome integrity are connected remains elusive. Here we show that H2A.X plays unexpected roles in maintaining pluripotency and genome integrity in ESC and iPSC. In ESC, it is specially enriched at genomic regions sensitive to replication stress so that it protects genome integrity thereat. Faithful H2A.X deposition is critical for genome integrity and pluripotency in iPSC. H2A.X depositions in 4N+ iPSC clones faithfully recapitulate the ESC pattern and therefore, prevent genome instability. On the other hand, insufficient H2A.X depositions in 4N- iPSC clones at such regions lead to genome instability and defects in replication stress response and DNA repair, reminiscent of the H2A.X deficient ESC. Detect and compare different H2A.X deposition patterns in ES cells and iPS cells, with Illumina HiSeq 2000 and Illumina Genome Analyzer IIx

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Systematic comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes

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