iPSC Culture and Directed Differentiation into Hepatocytes
iPSCs were maintained on matrigel-coated 6-well tissue culture plates in 2 mL of mTeSR 1 feeder-free medium (STEMCELL Technologies) with 200 ng/mL Primocin (Invitrogen) and incubated at 37°C/5% CO2. The directed differentiation from iPSCs into hepatocytes was performed as previously described (39). After D0, the cells were incubated in hypoxic conditions at 37°C. For D1-D5 of the differentiation protocol, the STEMdiff Definitive Endoderm kit (STEMCELL Technologies) was used according to manufacturer’s instructions.
Immortalized Cell Culture
Vero E6 (Cercopithecus aethiops kidney cell line, ATCC CRL-1586) and Huh-7 cells (human hepatocellular carcinoma cell line, provided by J. Alonso, Texas Biomedical Research Institute, San Antonio, TX) were maintained in Dulbecco’s Modified Eagle Media (DMEM) (Gibco), 10% fetal bovine serum (FBS), 200 nM L-glutamine in solution, and 5000 units/mL Penicillin Streptomycin solution. Cells were incubated at 37°C with 5% CO2.
Primary Hepatocyte Cell Culture
The PHH co-culture with murine embryonic fibroblasts 3T3-J2s were generated by seeding cryopreserved primary human hepatocytes purchased from BioIVT on collagen-patterned 96-well plates as described previously (33, 34). 3T3-J2s were a kind gift provided by Howard Green (Harvard Medical School). Fibroblast-depleted micropatterned PHH cultures were obtained by incorporation of 3T3-J2s expressing an inducible apoptosis switch (inducible caspase-9, Casp9). 3T3-J2s were lentivirally transduced with the pMSCV-F-del Casp9.IRES.GFP plasmid (15567, Addgene) followed by FACS purification (top 15%, FACSAria II, BD Biosciences) (47). On the day of infection, PHH co-cultures were dosed with a chemical inducer of dimerization (0.5 µM AP20187, Takarabio) to trigger fibroblast removal via apoptosis. PHH co-cultures were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Corning) supplemented with 10% fetal bovine serum (Gibco), 1% ITS (insulin/transferrin/selenous acid and linoleic acid, BD Biosciences), 7 ng/mL glucagon (Sigma-Aldrich), 40 ng/mL dexamethasone (Sigma-Aldrich), 15 mM HEPES (Gibco), and 100 µg/mL penicillin-streptomycin (Corning). Cells were kept at 37C with 5% CO2.
Virus Infection And Purification
Work with infectious EBOV was performed at Boston University’s National Emerging Infectious Diseases Laboratories, Boston, MA and at the NIH/NIAID Integrated Research Facility, Fort Detrick, MD. EBOV isolate Mayinga (GenBank accession number AF086833.2; kindly provided by H. Feldmann, NIH NIAID Rocky Mountain Laboratories, Hamilton, MT) and recombinant EBOV-GFP (Mayinga isolate; kindly provided by the World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX) expressing green fluorescence protein (GFP) from an additional transcription unit (52) were propagated in Vero E6 cells as described before (53). Cell supernatants were centrifuged at 1,000xg for 10 minutes at 4°C to remove cellular debris. To remove soluble factors, including cytokines and chemokines, clarified supernatants were purified over a 20% sucrose cushion by centrifugation at 25,000 rpm for 2 hours at 4°C and the pelleted viral particles were resuspended on PBS. Viral titers were determined by tissue culture infectious dose 50 (TCID50) assays and focus forming unit (FFU) assays as previously described (53). Recombinant vesicular stomatitis virus (VSV) expressing GFP or mCherry from an additional transcription unit (VSV-GFP and VSV-mCherry; kindly provided by J. Connor, Boston University) was propagated in Vero E6. Viral titers were determined by TCID50.
All EBOV infection studies were performed with purified virions. If not stated otherwise, cells were infected at a multiplicity of infection (MOI) of 10 for infection studies. After addition of the inoculum, virions were allowed to adsorb for 20 minutes to 1 hour at 37ºC before cells were washed and fresh cell culture medium was added.
Flow Cytometry Analysis
2.5x105 cells D0 and D5 cells were stained for flow cytometry analysis of the endoderm markers cKit and CXCR4. Cells were harvested by centrifugation at 300xg at 4°C for 5 minutes. Cells were resuspended in PBS and stained with APC-conjugated cKit (BioLegend) and PE-conjugated CXCR4 (Invitrogen) primary antibodies for 30 minutes. Cells were washed with FACS buffer and filtered through a 35 µm filter into a 5 mL polystyrene FACS tube and analyzed on a Stratadigm Flow Cytometry machine or a FACS Calibur Flow Cytometry machine. Analysis of the flow plots was performed using FlowJo.
qRT-PCR Analysis
Cells were harvested for RNA analysis by using either the RNeasy Kit (Qiagen) or TRIzol Reagent (ThermoFisher) according to manufacturer’s instructions. For hepatic cellular markers, the TaqMan qRT-PCR system (ThermoFisher) was used. cDNA libraries of the harvested cellular RNA were prepared using SuperScript III Reverse Transcriptase (ThermoFisher). 500 ng of the cDNA library was added to a TaqMan probe reaction master mix containing 10 µL of the TaqMan Universal PCR Master Mix 2x Buffer, 1 µL of TaqMan gene probe, and nuclease-free water to a final volume of 20 µL per reaction. For qRT-PCR of IFNβ, 1x106 or 4x105 iPSC-derived hepatocytes and Huh7 cells were harvested 1 day post-VSV infection by using TRIzol Reagent (ThermoFisher) according to manufacturer’s instructions. The QuantiTect SYBR Green RT-PCR kit was used (Qiagen) for qRT-PCR analysis. 50 ng of purified cellular RNA was added to a SYBR Green reaction master mix containing 12.5 µL of SYBR master mix, 2.5 µL of QuantiTect gene probe, 0.25 µL of QuantiTect Reverse Transcriptase mix, and nuclease-free water to a final volume of 25 µL per reaction. For both systems, samples were run on a StepOnePlus Real Time PCR machine (ThermoFisher). CT values were plotted using Prism GraphPad Software. Statistical significance was determined by paired t-tests with comparisons of interest plotted on each graph.
Periodic Acid-Schiff (PAS) Staining
PAS staining was performed on 1x106 iPSC-derived hepatocytes or Huh7 cells using the Periodic Acid-Schiff (PAS) kit (Sigma-Aldrich) according to manufacturer’s instructions. Cells were fixed with 1 mL of 4% paraformaldehyde (PFA) solution for 10 minutes at room temperature prior to PAS staining. PBS was added to the cells for imaging on an inverted microscope.
LDL-Uptake Assay
1x106 D0 cells, iPSC-derived hepatocytes, and Huh7 cells were analyzed using the LDL Uptake Assay Kit (Cell-Based) (Abcam) according to manufacturer’s instructions. The LDL-DyLight reagent was diluted in the appropriate cell culture medium for each cell type. The cells were incubated with the LDL reagents at 37°C for 4 hours. Cells were washed one time with 1 mL of PBS, and 2 mL of PBS was added to the cells for visualization of LDL update on an inverted microscope with a filter capable of capturing excitation and emission wavelengths of 540 and 570 nm.
CYP3A4 P450-Glo Assay
The quantification of CYP3A4 in 1x106 iPSCs, iPSC-derived hepatocytes, and Huh7 cells was performed using the P450-Glo Assay kit (Promega) according to manufacturer’s instructions. One day prior to the assay, Huh7 cells were treated with cell culture media containing 100 nM Dexamethasone. One the day of the assay, the Luciferin Glo substrate was added to the appropriate cell culture medium for each cell type. Cells were incubated with luciferin reagents at 37°C for 4 hours. Supernatant was clarified at 300xg for 5 minutes at room temperature. Clarified supernatant and detection reagent were incubated at room temperature for 20 minutes. Luminescence was measured on a LUMIStar Omega Luminometer (BMG Labtech).
Immunofluorescence Analysis
1x106 or 4x105 cells were fixed with either 4% PFA in DMEM for 10 minutes at room temperature or 10% formalin for at least 6 hours at 4°C. Cells were washed in PBS, permeabilized with a solution of acetone-methanol in a 1:1 volume ratio for 5 minutes at -20°C, and incubated in blocking buffer for at least 20 minutes at room temperature. Due to cross-reactivity with the BSA in the blocking buffer, the cells were not incubated in blocking buffer when stained for albumin. Cells were incubated with primary antibody overnight at 4°C, and primary antibody names and dilutions are listed in Table 2. Cells were incubated with secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI) for one hour at room temperature (dilutions listed in Table 2).
Table 2
Antibodies used for immunofluorescence analysis.
Antigen
|
Company
|
Species
|
Dilution
|
Albumin
|
Dako
|
Rabbit
|
1:100
|
Alexa Fluor α-mouse 594
|
Thermo Fisher
|
Donkey
|
1:200
|
Alexa Fluor α- rabbit 488
|
Thermo Fisher
|
Goat
|
1:200
|
Alexa Fluor α-goat 488
|
Thermo Fisher
|
Donkey
|
1:200
|
CK18, human
|
Abcam
|
Rabbit
|
1:100
|
EBOV NP
|
Gift from G. Olinger, USAMRIID
|
Mouse
|
1:200
|
EBOV VP35
|
Antagene, custom
|
Goat
|
1:1000
|
EBOV VP40
|
BEI
|
Mouse
|
1:200
|
FOXA1
|
Abcam
|
Rabbit
|
1:100
|
FOXA2
|
Abcam
|
Rabbit
|
1:100
|
HNF4α
|
Abcam
|
Mouse
|
1:50
|
NFκB p65 (A)
|
Santa Cruz
|
Rabbit
|
1:200
|
Transferrin
|
ThermoFisher
|
Rabbit
|
1:50
|
VSV Bioassay
4x105 iPSC-derived hepatocytes from 3 donors and triplicate samples of Huh7 cells were infected with VSV-GFP or VSV-mCherry at an MOI of 10. 1 dpi, cells were imaged for GFP expression and cellular RNA was purified for qRT-PCR analysis.
LPS stimulation for NFκB Activation
4x105 iPSC-derived hepatocytes from three donors were treated with 300 ng of Ultrapure LPS (Invitrogen) diluted in D19 medium for 1 hour at 37°C and 5%CO2. Cells were fixed and used for immunofluorescence analysis.
Electron Microscopy
For conventional thin-section microscopic evaluation, 4x105 EBOV-infected iPSC-derived hepatocytes were inactivated and preserved in 2.5% glutaraldehyde (E.M. Sciences, Warrington, PA), in Millonig’s sodium phosphate buffer (Tousimis Research, Rockville, MD), for 24 hours. After fixation was complete, the cells were washed in Millonig’s buffer, and incubated for two hours in 1.0% osmium tetroxide, in the same buffer. Following rinsing steps in ultrapure water and en bloc staining with 2.0% uranyl acetate, the samples were dehydrated in a series of graded ethanol washes and infiltrated and embedded in Spurr’s plastic resin (E.M. Sciences). Embedded blocks were sectioned using a Leica UC7 Ultramicrotome. 70–80 nm sections were collected on 200 mesh copper grids, and post-stained with Reynold’s lead citrate. Samples were examined in a FEI Tecnai Spirit Twin transmission electron microscope, operating at 80 kV.
SmartSeq2 Sequencing
1x106 iPSC-derived hepatocytes or Huh7 cells were seeded in 6-well tissue culture plates, and 1x104 PHHs were seeded in 96-well glass-bottom plates. The cells were infected with EBOV at an MOI of 10 or left uninfected and lysed 1 dpi. Cellular RNA was isolated using TRIzol reagent (ThermoFisher) according to the manufacturer’s instructions. 50 ng of purified RNA was used for sequencing by diluting to a final concentration of 5 ng/µL in a final volume of 10 µL. Samples were pipetted into a twin.tec® PCR 96-well plate (Eppendorf) and sent out for sequencing on dry ice. Sequencing was performed at the Broad Institute Genomics Services Core. The quality of the raw data was assessed using FastQC v.0.11.7. The sequence reads were aligned to a combination of the human genome reference (GRCh38) and the Ebola virus reference (NC_002549.1) using STAR v.2.6.0c. Counts per gene were summarized using the featureCounts function from the subread package v.1.6.2. The matrix of counts per gene per sample was then analyzed using the limma/voom normalization (limma v. 3.39.19, edgeR v.3.25.10). After exploratory data analysis (Glimma v. 1.11.1), contrasts for differential expression testing were done for each EBOV-infected sample compared to mock-infected samples (controls) at each time point separately. Functional predictions were performed using WebGestalt for gene set analysis (59).
Illumina Sequencing
1x106 iPSC-derived hepatocytes were mock-infected or infected with EBOV at an MOI of 10. Cells were harvested for RNA analysis at 1, 2, 3, and 7 dpi using TRIzol reagent (ThermoFisher). RNA was isolated according to manufacturer’s instructions. 1 µg of purified cellular RNA was diluted to a concentration of 50 ng/µL in a final volume of 20 µL and shipped to Novogene on dry ice for library preparation and Illumina sequencing. The quality of the raw data was assessed using FastQC v.0.11.7. The sequence reads were aligned to a combination of the human genome reference (GRCh38) and the EBOV reference sequence (NC_002549.1) using STAR v.2.6.0c. Counts per gene were summarized using the featureCounts function from the subread package v.1.6.2. The matrix of counts per gene per sample was then analyzed using the limma/voom normalization (limma v. 3.39.19, edgeR v.3.25.10). After exploratory data analysis (Glimma v. 1.11.1), contrasts for differential expression testing were done for each EBOV-infected sample compared to the mock control at each time point separately. Statistical significance of the logFC for each time point contrast was determined by a paired t-test and reported as a Benjamini-Hochberg false discovery rate adjusted p-value. Gene Set Enrichment Analysis (GSEA) was performed using all genes in the dataset, ranked on logFC, using WebGestalt to analyze Biological Processes in GO databases or the Reactome in Pathway databases with the following parameters: minimum number of genes in a category = 15; false discovery rate (FDR) ≤ 0.01; permutations = 1000 (59).
Fluorescent In-situ Hybridization Assay
Fluroescent in-situ hybridization (RNA-FISH) was performed using the Advanced Cell Diagnostics RNAscope v2 kit according to manufacturer’s instructions (ACD Bio). 5x105 iPSC-derived hepatocytes were seeded onto 2 well glass chamber slides, and 1x105 Huh7 cells were seeded onto 8 well glass chamber slides. Both cell types were infected at an MOI of 10 as described above. Cells were fixed with 10% formalin for a minimum of 6 hours. After fixation, cells were dehydrated with ethanol washes before being stored in 100% ethanol at -20ºC until staining. On the day of the assay, cells were rehydrated with ethanol. Cells were pre-treated with hydrogen peroxidase and Protease III according to the manufacturer’s instructions. Cells were probed with RNAscope probes for 2 hours at 40ºC in the ACD hybridization oven. The following probes (all from ACD Bio) were used: ebolavirus Zaire VP35- C1 (undiluted), human IL6- C2 (1:50), human IFNβ- C3 (1:50), human CXCL10- C3 (1:50). After hybridization, the cells were treated with the amplification and development reagents according to manufacturer’s instructions. An additional blocking and washing step was added following amplification of channels containing viral RNA probes to prevent cross-reaction due to viral gene abundance. Fluorophores used for detection (Perkin Elmer) include Opal 520 (1:3000), Opal 570 (1:3000), and Opal 690 (1:1500). Samples were visualized on a Nikon Eclipse Ti2 inverted microscope using Photometrics Prime BSI camera, and quantification of gene-expressing cells was performed in QuPath (cite) software (https://doi.org/10.1038/s41598-017-17204-50) using the cell detection feature and manual identification of positive cells.
Macrophage Differentiation From Peripheral Mononuclear Blood Cells
MDMs were generated from leukopaks (NY Biologics Inc.) or apheresed peripheral blood mononuclear cells (PBMCs) using Ficoll separation (GE Healthcare). Whole blood was diluted to a final volume of 30 mL with PBS. 15 mL of Ficoll was added to a 50 mL conical tube, and the diluted whole blood was overlayed onto the Ficoll. Blood was centrifuged for 30 minutes at 450xg with the brake turned off of the centrifuge. The blood separated into three layers: the off-white plasma layer, a thin white buffy coat containing leukocytes, and the pelleted erythrocytes. The plasma layer was removed, and the buffy coat was isolated into a fresh 50 mL conical tube. The buffy coat was diluted to a final volume of 50 mL using 37ºC PBS. Cells were centrifuged at 350xg for 10 minutes with the centrifuge brake on. The cell pellet was then washed three times with 50 mL of warm PBS and centrifuged at 350xg for 10 minutes. After the final wash, cells were resuspended in 1 mL of warm PBS and counted. 1x107 cells were plated in a T75 flask with RPMI medium for one hour at 37ºC and 5% CO2, non-adhered cells were removed, and 10 ml RPMI medium with 10% FBS, 1% L-glutamine, 10 mM HEPES, 10ng/mL GM-CSF, and 50 U/mL penicillin with 50 mg/mL streptomycin (MDM medium) was added to the cells. Medium was changed every 2 to 3 days for a minimum of 6 days before differentiated macrophages were split into 6-well TC plates for infection experiments.
Co-Culture of iPSC-Derived Hepatocytes and Macrophages
On the day prior to infection, MDMs seeded in a T75 flask were split by incubating cells with 0.1% Trypsin-EDTA solution for 15 minutes at 37°C and 5%CO2. Cells were centrifuged at 300xg for 5 minutes at 4°C and resuspended in 1 mL of MDM medium. Cells were counted and 1x106 cells were seeded into 6-well low-attachment plates (Corning). On the day of infection, macrophages were infected with EBOV-GFP at an MOI of 10. At 1 dpi, MDMs were incubated with 1 mL of Gentle Cell Dissociation buffer (GCD; Gibco) for 2 minutes at 37°C and 5%CO2. Cells were centrifuged at 300xg for 5 minutes at 4°C. GCD was removed, and cell pellets were washed three times in 1 mL of PBS. After the third wash, cells were resuspended in 1 mL of the appropriate medium and seeded in 6- or 12-well tissue culture plates alone or onto D20 iPSC-derived hepatocytes at a 1:1 ratio.