About the HepaRG™ cell line
HepaRG™ is a human bipotent progenitor cell line capable of differentiating into both biliary and hepatocyte lineages in the presence of DMSO. It was originally derived from a female patient diagnosed with a cholangiocarcinoma and hepatitis C. The main characteristics of the cell line are:
- Can undergo a complete programme of hepatocyte differentiation until a fully adult hepatic phenotype
- Are polarized and form bile canaliculi
- Breath aerobically, consume lactate and contain as many mitochondria as the human hepatocytes
- Has the potential to express major properties of stem cells
- High plasticity & complete transdifferentiation capacity
The cells express the major nuclear receptors referred also as xeno-sensing receptors (Pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AHR)), drug transporters (BSEP, MRP2, MRP3, MRP4, MRP5, OSTo/B, etc.) and functional levels of phase I (CYP (CYP1A1/2, CYP2B6, CYP2Cs, CYP3A4 etc.) (Figure 1) (Jinpeng Li et al. 2019) and Il (UGT1A1, GSTA1, GSTA4, GSTM1) drug metabolizing enzymes as well as key hepatic nuclear factors such as Hepatocyte Nuclear Factor 4 (HNF4) which expression overcomes repression of the hepatic phenotype in dedifferentiated hepatoma cells (Figure 2) (Sasaki et al. 2018; Tascher et al. 2019).
Furthermore, HepaRG™ cells show functional mitochondria, hepatokine secretion abilities and an adequate response to insulin (Figure 3). The HepaRG™ cell system appears as a robust surrogate for primary hepatocytes, which is versatile enough to study not only xenobiotic detoxification, but also the control of hepatic energy metabolism, secretory function and disease-related mechanisms (Tascher et al. 2019).
Last but not least, they can survive up to 34 weeks days in culture until senescence and as such be a much better suited model than their primary hepatocyte cultures counterparts (Figure 4).
Figure 1. Metabolic activity of PHH and HepaRG™ cells in 2D sandwich and 3D spheroid cultures.
The activity of CYP1A2 (A, B), CYP2B6 (C, D), and CYP3A4 (E, F) was assessed. The fold change relative to the activity in freshly thawed cells is presented. Four independent experiments were conducted, each of which had three replicate measurements. Data are presented as mean ± SEM of independent experiments. **p < 0.01 compared to freshly thawed and 2D sandwich cultures using one-way ANOVA with Tukey's multiple comparison test (Jinpeng Li et al. 2019).
Figure 2. The main features of highly differentiated HepaRG™ cells cultivated in the differentiation medium (1.7% dimethyl sulfoxide (DMSO), 10% calf serum (FCS)).
(A) Phase contrast microscopy, staining of cytoskeletal F-actin, and the immunostaining of ZO-1 junctional protein, HNF4 transcription factor, and β-catenin in HepaRG™ progenitors (Pr) and differentiated HepaRG™ (HPR116) cells at days 6, 12, and 18 (d6 to d18). (B) Mean cell size (± SD) of primary human hepatocytes (PHH) and HepaRG™ and HepG2 cells (Tascher et al. 2019).
Figure 3. Insulin metabolism in highly differentiated HepaRG™ cells.
(A) The relative difference of protein abundances in HepaRG™ (defined serum-free medium: 0.5% DMSO, growth factor supplementation) and in HepG2 cells versus PHH, as shown as heatmaps for factors of insulin signaling/resistance pathways (white boxes: proteins not seen). Non-detected proteins are colored in grey. (B) mRNA levels of SREBP1c and PEPCK in differentiated HepaRG™ cells (differentiation medium: 1.7% DMSO, 10% FCS) in response to insulin stimulation (means ± SEM of three determinations). * indicates significant differences due to insulin stimulation for a given time point (ANOVA and post hoc Tukey tests; p < 0.05). (C) Phosphorylation status of IRS2, PKB, and GSK3β in differentiated HepaRG™ cells in response to insulin stimulation (means ± SEM of three determinations). (Tascher et al. 2019)
Figure 4. Viability of PHH and HepaRG cells in 2D sandwich and 3D spheroid cultures.
Cell viability of PHH (A) and HepaRG (B) was assessed by measuring cellular ATP content. The RLUs of the luminescence signal were directly proportional to the amount of ATP, which serves as an indicator of viability/cell stress. Four independent experiments were conducted, each of which had three replicate measurements. Data are presented as mean ± SEM of independent experiments. *p < 0.05 and **p < 0.01 compared to freshly thawed and 2D sandwich cultures using one-way ANOVA with Tukey's multiple comparison test. 2D, two dimensional; 3D, three dimensional; ANOVA, analysis of variance; PHH, primary human hepatocyte; RLU, relative light unit; SEM, standard error of the mean.
The process of HepaRG differentiation
Two main stages are being identified, namely:
The proliferation stage
The proliferation stage during which the cells are doubling every 24h. Usually at day 15 they reach high confluence. At this moment the cells can be either split and re-introduced to re-start proliferation or enter the next differentiation stage.
The differentiation stage
To undergo hepatic differentiation, the cells are treated with medium enriched with 1.7% DMSO for a 14-days period. Within 3-4 days, two distinctive cell types are recognised: the one corresponding to hepatocyte colonies and the other one corresponding to primitive biliary cells. Once fully differentiated, the cells express a full array of functions, responses, and regulatory pathways of primary human hepatocytes including: Phase I and II, and transporter activities consistent with those found within a population of primary human hepatocytes including intact response elements, PXR, CAR and PPARa.
Confluent HepaRG™ cells start to commit into either hepatocyte (coloured in red) or biliary cells (coloured in grey).
After 2 weeks, a mixed population of 2 types of cells, namely hepatocyte-like colonies surrounded by clear epithelial cells corresponding to primitive biliary cells, form a confluent monolayer which can be maintained in this stable form for several weeks in the presence of 1.7% DMSO.