Stem cell-derived polarized hepatocytes

Abstract

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Fig. 1. Stem cell-based differentiation on transwells to generate polarized hepatocyte-like cells (pol-HLCs).

a Schematic depicting the cellular organization of non-polarized, columnar polarized, and hepatocyte/multi-polarized cells. Blue boxes are tight junctions separating apical and basal membranes. b Stem cell differentiation protocol on transwells to generate pol-HLCs. c Representative immunofluorescence images of hESCs (day 0), definitive endoderm (day 5), hepatic progenitor (day 9 and 13), immature HLCs (day 16), and pol-HLCs (day 21). Cells were stained for immature hepatocyte markers AFP (green) and FoxA2 (magenta) or for mature hepatocyte markers ALB (green) and HNF4α (magenta). Scale bars = 500 μm/250 μm. d–f RT-qPCR analysis of the indicated genes along the pol-HLC differentiation protocol relative to D0 (n = biological replicates). g Pol-HLCs metabolize carboxyfluorescein diacetate (CFDA) and indocyanine green (ICG), and store glycogen as evidenced by periodic acid-schiff staining (PAS) staining. Scale bars = 250 μm h Paracellular permeability of polarized HLCs incubated O/N with 4 kDa FITC-dextran or 70 kDa RITC-dextran in the absence or presence of EDTA, plotted relative to diffusion across empty filters (n = biological replicates). i Rate of albumin (left panel) and urea (right panel) secretion into either the top or bottom compartment during pol-HLC differentiation (n = biological replicates). Statistical analysis was performed using a two-tailed unpaired t test with Bonferroni adjustment for multiple comparisons.

Fig. 2. Structural polarization and organization of polarized HLCs.

a Transmission electron microscopy, cross-sectional view of nonpol- and pol-HLCs. No, nucleolus; Nu, nucleus; Mi, mitochondria; ER, endoplasmic reticulum; Lys, lysosome; TJ, tight junctions. Scale bar in insets = 2 μm. b xy images of pol-HLCs stained for the tight-junction marker ZO-1 (green), breast cancer resistance protein (BCRP, green), multi-drug resistance protein 2 (MRP2, green), or scavenger receptor-B1 (SR-BI, green), and DAPI (blue). Scale bars = 200 μm c Transferrin-conjugate binding to pol-HLC. Pol-HLCs were incubated with 25 µg/mL Transferrin-594 (green) for 10 min at 37 °C prior to washing and staining with anti-ZO-1 (magenta). Bottom panels: xz images from cross sections indicated by the dashed line in the corresponding xy-images above. Scale bars = 15 μm. d Cross-sectional views (xz) of nonpol- and pol-HLCs stained for indicated marker. Yellow arrows = basolateral membrane of pol-HLCs. * = autofluorescence of transwell pore. Images are representative of three independent differentiations.

Fig. 3. Vectorial hepatic cargo secretion from polarized HLCs.

a Heatmap of Z score-normalized CPM values for enzyme and transporter genes involved in lipoprotein metabolism in nonpol- compared with pol-HLCs (n = biological replicates). b Density distribution of ApoB100 and ApoE secreted from nonpol- and pol-HLCs, labeled metabolically for 4 h with [³⁵S] methionine/cysteine. Supernatants from labeled cells were subjected to density gradient centrifugation followed by ApoB100 or ApoE immunoprecipitation of each fraction, separation by SDS-PAGE, and detection by [³⁵S] fluorography. Results are representative of three independent differentiations. c Nonpol- and pol-HLCs were treated with indicated concentrations of the MTP inhibitor Lomitapide, prior to metabolic labeling, ApoB100-immunoprecipitation and detection. Results are representative of three independent differentiations. d Heatmap for Z score-normalized CPM values of enzyme and transporter genes involved in bile acid metabolism in nonpol- (Non) compared with pol-HLCs (Pol) (n = biological replicates). e Total bile acid release from nonpol- and pol-HLCs treated for 24 h with 10 μg/ml high-density lipoprotein (HDL) or 10 μm cyclosporine A (CsA), as indicated (n = biological replicates). f–g LC-MS analysis of primary and conjugated cholic acid (f) and chenodeoxcycholic acid (g) released from pol-HLCs treated with DMSO or 10 μm 7α-hydroxylcholesterol (7α-CHO) for 4 h (n = biological replicates). Statistical analysis was performed using a two-tailed unpaired t test with Bonferroni adjustment for multiple comparisons.

Fig. 4. Modeling polarized infection of hepatitis A and E viruses.

a Newly secreted focus-forming infectious particles (FFU) from HEV infected pol-HLCs released in either apical or basolateral compartment were titered on hepatoma cells. Pol-HLCs were treated with DMSO or 10 μm of the HEV replication inhibitor Sofosbuvir (n = biological replicates). b HEV particles released from HEV infected pol-HLCs 7 d post infection were also measured by qRT-PCR quantification of viral RNA copies or ORF2 capsid ELISA (n = biological replicates). c Newly secreted infectious HEV particles from infected pol-HLCs were treated with 1:100 anti-HEV capsid ORF2 antibody and titered on hepatoma cells (n = biological replicates). d Relative infectivity of HEV particles recovered from the lysate or released in the supernatant of hepatoma cells transfected with HEV RNA from strain Kernow-C1 P6, 7 d post-transfection. Harvested HEV particles were treated with 1:100 anti-ORF2 antibody, IgG-control antibody and/or 0.1% sodium deoxycholate (DOC) for 30 min at RT. HEV infectivity was determined by titration on hepatoma cells (n = biological replicates). e Intra- and extracellular HEV particles from HEV P6 RNA-transfected hepatoma cells were mixed with either apical or basal supernatant from pol-HLCs and/or 1:100 anti-ORF2 antibody prior to titration on hepatoma cells (n = biological replicates). f Western blot analysis of cell lysates from shCYP8B1-inducible, H9-derived pol-HLCs (H9/shCYB8B1). Cells were treated or untreated for 48 h with 3 μg/ml doxycycline (DOX) to induce shRNA expression. Shown are representative images of n = 2. g Apical total bile acid release from H9/shCYP8B1-derived pol-HLCs treated with 3 μg/ml DOX as indicated (n = biological replicates). h Extracellular HEV particles from HEV P6 RNA-transfected S10-3 cells were mixed with apical supernatant from H9/shCYP8B1-derived pol-HLCs treated with or without 3 μg/ml DOX (n = biological replicates). i HEV secretion model from non-polarized and polarized HLCs. j Pol-HLCs were infected with HAV strain HM175/18 f. 48 h post infection, newly released particles were harvested and titered on hepatoma cells (n = biological replicates). Statistical analysis was performed using a two-tailed unpaired t test with Bonferroni adjustment for multiple comparisons.

Fig. 5. Polarized HLCs for drug–drug interaction, absorption, and secretion studies.

Relative mRNA levels quantified by RT-qPCR of (a) basolateral and (b) apical transporters in DE, Huh7.5, nonpol-, and pol-HLCs. Statistical analysis was performed using a two-tailed unpaired t test (n = biological replicates). c Modeling stribild disposition in pol-HLCs. Pol-HLCs were incubated with 10 μm elvitegravir (EVT), 13 μm emtricitabine (FTC), 20 μm tenofovir disoproxil fumarate (TDF) ± 10 μm cobicistat (COBI) from the basolateral compartment. At the indicated time points, compounds and the TDF metabolite tenofovir (TF) were quantified by LC-MS in apical and basolateral compartments (n = biological replicates).