Development of Hepatocyte‐like Cell Derived from Human Induced Pluripotent Stem cell as a Host for Clinically Isolated Hepatitis C Virus

Khanit Sa‐ngiamsuntorn1, Suradej Hongeng2, Adisak Wongkajornsilp3

1 Department of Biochemistry, Faculty of Pharmacy, Mahidol University, Bangkok, 2 Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 3 Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.13
DOI:  10.1002/cpsc.35
Online Posting Date:  August, 2017
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Abstract

This unit describes protocols to develop hepatocyte‐like cells (HLCs) starting from mesenchymal stem cells (MSCs) as a natural host for hepatitis C virus (HCV). These include the preparation of MSCs from bone marrow, the reprogramming of MSCs into induced pluripotent stem cells (iPSCs), and the differentiation of iPSCs into HLCs. This unit also incorporates the characterization of the resulting cells at each stage. Another section entails the preparations of HCV. The sources of HCV are either the clinically isolated HCV (HCVser) and the conventional JFH‐1 genotype. The last section is the infection protocol coupled with the measurement of viral titer. © 2017 by John Wiley & Sons, Inc.

Keywords: cellular reprogramming; hepatitis C virus (HCV); hepatocyte‐like cell (HLC); induced pluripotent stem cell (iPSC); mesenchymal stem cell (MSC); JFH‐1; HCVcc; HCVser

     
 
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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: The Isolation of Human Mesenchymal Stem Cell from Bone Marrow
  • Basic Protocol 2: The Maintenance of Human Mesenchymal Stem Cell (MSC)
  • Basic Protocol 3: The Preparation of Lentiviral Vectors for Reprogramming MSCS to iPSCS
  • Basic Protocol 4: Reprogramming MSCs to iPSCs
  • Basic Protocol 5: The Screening of Human‐Induced Pluripotent Stem Cells
  • Basic Protocol 6: The Differentiation of iPSC to Homogeneous Hepatocyte‐Like Cells
  • Basic Protocol 7: Characterization of Hepatocyte‐Specific Markers
  • Basic Protocol 8: Production of Hepatitis C Virus Derived from Cell Culture (HCVcc), JFH‐1
  • Basic Protocol 9: Production of HCVcc from Infectious Virus
  • Basic Protocol 10: HCVcc or HCVser Infectitity Titer Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Isolation of Human Mesenchymal Stem Cell from Bone Marrow

  Materials
  • Phosphate‐buffered saline (DPBS) without calcium and magnesium
  • Ficoll (GE Healthcare Life Sciences) or IsoPrep (Robbins Scientific)
  • Trypan Blue solution, 0.85% in saline (Sigma)
  • MEM Alpha modification (α ‐MEM, Hyclone, cat. no. SH30265.02) supplemented with 10% fetal bovine serum (FBS, GE Healthcare Life Sciences)
  • Penicillin G sodium (Sigma, cat. no. P7794)
  • Streptomycin (Sigma, cat. no. S6501)
  • 50‐ and 15‐ml polypropylene centrifuge tubes
  • Centrifuge
  • Sterile Pasteur pipettes
  • Neubauer hemacytometer
  • 75‐cm2 flasks
  • Inverted microscope

Basic Protocol 2: The Maintenance of Human Mesenchymal Stem Cell (MSC)

  Materials
  • Human MSCs in 75‐cm2 flasks (see protocol 1)
  • Phosphate‐buffered saline (DPBS) without calcium and magnesium.
  • Trypsin‐EDTA, 0.25% (w/v), 1 mM EDTA in PBS
  • MEM Alpha modification (α ‐MEM, HyClone, cat. no. SH30265.02) supplemented with 10% fetal bovine serum (FBS; HyClone GE Healthcare Life Sciences)
  • Penicillin G sodium (Sigma, cat. no. P7794)
  • Streptomycin (Sigma, cat. no. S6501)
  • Trypan blue
  • Inverted microscope
  • Hemacytometer
  • Centrifuge

Basic Protocol 3: The Preparation of Lentiviral Vectors for Reprogramming MSCS to iPSCS

  Materials
  • Lentiviral plasmids:
    • pRRL.PPT.SF.hOKSM.idTomato.preFRT (polycistronic OSKM‐dTomato or equivalence)
    • pMDLg/pRRE (packaging plasmid; contains Gag and Pol; Addgene plasmid # 12251)
    • pRSV‐REV (Rev, Addgene plasmid # 12253)
    • pMD2.G (VSV‐G, Addgene plasmid # 12259)
  • NucleoBond Xtra Midi Kit (MN, cat. no. 740410.10).
  • Transformed E. coli culture
  • Luria broth medium (see recipe)
  • HEK293T cell (CRL‐3216, ATCC)
  • DMEM/high glucose (HyClone, cat. no. SH30243.02)
  • Fetal bovine serum (FBS; GE Healthcare Life Sciences)
  • Penicillin G sodium (Sigma, cat. no. P7794).
  • Streptomycin (Sigma, cat. no. S6501)
  • Opti‐MEM Reduced Serum Medium (Thermo Fisher Scientific, cat. no. 31985088)
  • X‐tremeGENE HP DNA Transfection Reagent (Roche Diagnostics)
  • Lenti‐X concentrator (Takara Bio)
  • Phosphate‐buffered saline (DPBS) without calcium and magnesium
  • 10‐cm culture dishes
  • 1.5‐ml microcentrifuge tubes
  • Pipettes and micropipettes
  • 37°C, 5% CO2 incubator
  • 20‐ml syringes
  • Sterile syringe filter (0.45 μm Sartorius)
  • Centrifuge

Basic Protocol 4: Reprogramming MSCs to iPSCs

  Materials
  • Human mesenchymal stem cell (MSC) passage 3rd – 5th
  • Lentivirus
  • MSC growth medium (see recipe)
  • Hexadimethrine bromide or polybrene (Sigma, cat. no. H9268)
  • Sodium butyrate (Sigma, cat. no. B5887)
  • Inactivated MEF (mouse embryonic fibroblast, PMEF‐CFX, Merck Millipore)
  • iPSC medium [DMEM/F12, 20% Knockout Serum Replacement (KO‐SR), 110 mM 2‐mercaptoethanol, 1× non‐essential amino acid (NEAA), 1× GlutaMAX, 1× penicillin, streptomycin, and 10 ng/ml basic fibroblast growth factor, bFGF]
  • DMEM/F12 (HyClone, cat. no. SH30261.02)
  • KnockOut Serum Replacement (Thermo Fisher Scientific, cat. no. 10828028)
  • 2‐Mercaptoethanol (Thermo Fisher Scientific, cat. no. 21985023)
  • MEM Non‐Essential Amino Acids (100×; NEAA; Thermo Fisher Scientific, cat. no. 11140050)
  • GlutaMAX Supplement (Thermo Fisher Scientific, cat. no. 35050061)
  • Penicillin G sodium (Sigma, cat. no. P7794)
  • Streptomycin (Sigma, cat. no. S6501)
  • Recombinant Human FGF‐basic (bFGF; Peprotech, cat. no. 100‐18B)
  • TRA‐1‐60 Antibody (SC‐21705, SantaCruz)
  • Phosphate‐buffered saline (DPBS) without calcium and magnesium
  • Goat anti‐Mouse IgG/IgM (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A‐10684)
  • Geltrex LDEV‐Free, hESC‐Qualified (Thermo Fisher Scientific, cat. no. A1413301)
  • Essential 8 Medium (Thermo Fisher Scientific, cat. no. A1517001)
  • mTeSR 1 (Stem Cell Technologies, cat. no. 05850)
  • Y‐27632 (Cell Guidance Systems, cat. no. SM2)
  • Versene solution (0.48 mM EDTA; Thermo Fisher Scientific, cat. no. 15040066)
  • Fetal bovine serum (FBS; GE Healthcare Life Sciences)
  • EmbryoMax 0.1% Gelatin Solution, Merck Millipore, cat. no. ES‐006‐B)
  • 6‐well plates
  • 10‐cm cell culture dishes
  • Stereomicroscope
  • 3.5‐cm dishes

Basic Protocol 5: The Screening of Human‐Induced Pluripotent Stem Cells

  Materials
  • iPSC line (see protocol 4)
  • Alkaline Phosphatase Detection Kit (Merck Millipore, cat no. SCR004) containing:
    • Naphthol/Fast Red Violet Solution
    • 1× Rinse Buffer
  • 4% paraformaldehyde (PFA) fixing solution (see recipe)
  • Blocking solution (see recipe)
  • Primary antibodies: NANOG, SOX2, SSEA4, OCT4, TRA‐1‐60, TRA‐1‐81, alpha fetoprotein (AFP), alpha‐smooth muscle actin, and β‐III tubulin)
  • Tween‐20
  • Goat anti‐Mouse IgG/IgM (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Scientific, cat. no. A‐10684)
  • Goat anti‐Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Scientific, cat. no. A‐11008)
  • Hoechst 33342 (Thermo Scientific, cat. no. H3570)
  • Fluorescence Mounting Medium (Dako, cat. no. S302380)
  • illustra RNAspin Mini Kit (GE Healthcare Life Sciences, cat. no. 25‐0500‐71)
  • ImProm‐II reverse transcription system (Promega, cat. no. A3800)
  • KAPA2G Fast HotStart ReadyMix PCR Kit (Kapa Biosystems, cat. no. KK5603)
  • Gel electrophoresis apparatus and reagents including:
    • Molecular‐biology‐grade agarose (Vivantis, cat. no. PC0701)
    • 10× Tris‐Acetate‐EDTA (TAE) Buffer (Vivantis, cat. no. PB0940)
    • RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, cat. no. 21141)
    • 6× Loading Dye with SDS (Vivantis cat. no. NM0416)
    • DNA size ladder (Vivantis, cat. no. NL1401)
  • 24‐well plate
  • Phase‐contrast inverted microscope
  • Fluorescence microscope
  • Thermal cycler

Basic Protocol 6: The Differentiation of iPSC to Homogeneous Hepatocyte‐Like Cells

  Materials
  • iPSC line (see protocol 4)
  • Geltrex LDEV‐Free, hESC‐Qualified (Thermo Fisher Scientific, cat. no. A1413301)
  • Essential 8 Medium (Thermo Fisher Scientific, cat. no. A1517001)
  • Endoderm differentiation basal medium (see recipe)
  • Endoderm commitment medium (see recipe)
  • RPMI 1640 medium (HyClone, cat. no. SH30255.01)
  • B‐27 Supplements (Thermo Fisher Scientific, cat. no. 17504044)
  • Activin A (Cell Guidance Systems. cat. no. GFH6AF)
  • Recombinant Human FGF‐10 (Peprotech, cat. no. 100‐26)
  • Recombinant Human BMP‐4 (Peprotech, cat. no. 120‐05ET)
  • Recombinant Human Oncostatin M (Cell Guidance Systems, cat. no. GFH356)
  • Recombinant Human HGF (Peprotech, cat. no. 100‐39H)
  • Williams' Medium E (Thermo Fisher Scientific, cat. no. 12551032)
  • Dimethyl sulfoxide (Sigma, cat. no. D2650)
  • HBM Basal Medium (Lonza, cat. no. CC‐3199)
  • 6‐well plates

Basic Protocol 7: Characterization of Hepatocyte‐Specific Markers

  Materials
  • Hepatocyte‐like cell (HLCs; see protocol 6)
  • Human mesenchymal stem cells (MSCs; see protocol 2)
  • 95% ethanol
  • 5% formaldehyde
  • Periodic Acid‐Schiff (PAS) Kit (Sigma, cat. no. 395B) containing:
    • Periodic acid solution
    • Schiff's reagent
    • Hematoxylin solution
  • dH 2O
  • HepaRG cell line (Thermo Fisher Scientific, cat. no. HPRGC10)
  • illustra RNAspin mini Kit (GE Healthcare Life Sciences, cat. no. 25‐0500‐71)
  • ImProm‐II reverse transcription system (Promega, cat. no. A3800).
  • Real‐time PCR primer sets (1st BASE)
  • Gene‐specific primers (see Table 4.13.2) KAPA SYBR FAST qPCR Kit Master Mix (2×) (Kapa Biosystems, cat. no. KK4600)
  • Primary human hepatocyte (Thermo Fisher Scientific, cat. no. HMCPTS)
  • Rifampicin (Sigma, cat. no. R3501)
  • Omeprazole (Sigma, cat. no. O104)
  • Phenobarbital (Sigma, cat. no. P5178)
  • Ethanol
  • P450‐Glo 1A1, 2B6, 2C9 and 3A4 assay kits (V8751, V8321, V8791, V9001; Promega,)
  • Williams' Medium E (Thermo Fisher Scientific, cat. no. 12551032)
  • Luciferin‐CEE, Luciferin‐2B6, Luciferin‐H, and Luciferin‐IPA (P450‐Glo assay kit, Promega)
  • Luceferin detection reagent
  • Fetal bovine serum (FBS; GE Healthcare Life Sciences)
  • Fluorescent‐conjugated antibodies
  • Mayer's hematoxylin (Sigma, cat. no. MHS1‐100ML)
  • 35‐mm dishes
  • 4‐ or 8‐well plates, optional
  • Light microscope
  • Vector NTI (version 11.5; Invitrogen)
  • Mix3005P qPCR system (Agilent Technologies)
  • Collagen type IV‐coated 6‐cm dish (IWAKI)
  • 96‐well opaque white luminometer plate (Nunc)
  • Spectrofluorometer
  • 24‐well plates
Table 4.1.2   MaterialsPrimer Sets and Conditions Used in Quantitative Real‐Time PCR (qPCR)

Gene Genbank Accession Sense primer ‘5’ → 3’ (Tm°C) Antisense primer 3’ → 5’ (Tm°C) Amplicon size (bp) Annealing temp. (°C) Putative function
ALB NM_000477 TGAGAAAACGCCA GTAAGTGAC (56.5) TGCGAAATCATCC ATAACAGC (54.7) 265 60 Albumin
AFP NM_001134 GCTTGGTGGTGG ATGAAACA (57.2) TCCTCTGTTATTT GTGGCTTTTG (54.6) 157 60 α‐fetoprotein
CK18 X12881 GAGATCGAGGC TCTCAAGGA (57.9) CAAGCTGGCC TTCAGATTTC (55.8) 357 60 Cytokeration 18
G6PD U01120 GCTGGAGTCCTGTC AGGCATTGC (58.1) TAGAGCTGAGGCG GAATGGGAG (63.1) 349 60 Glucose‐6‐phosphate dehydrogenase
HNF‐4α AY680696 GCCTACCTCAAA GCCATCAT (56.4) GACCCTCCCAG CAGCATCTC (62.9) 256 60 Hepatocyte nuclear factor 4α
TAT NM_000353 TGAGCAGTCTGTCC ACTGCCT (62.3) ATGTGAATGAGGAGG ATCTGAG (54.9) 338 60 Tyrosine aminotransferase
CYP2B6 NM_000767 ATGGGGCACTG AAAAAGACTGA (58.0) AGAGGCGGGGA CACTGAATGAC (63.5) 283 60 CYP2B6
CYP2D6 NM_000106 CTAAGGGAACG ACACTCATCAC (56.6) GTCACCAGGAAAG CAAAGACAC (58.1) 289 60 CYP2D6
CYP2C9 NM_000771 CCTCTGGGGCA TTATCCATC (57.1) ATATTTGCACAGT GAAACATAGGA (52.9) 137 60 CYP2C9
CYP2C19 NM_000769 TTCATGCCT TTCTCAGCAGG (56.8) ACAGATAGTG AAATTTGGAC (47.9) 277 60 CYP2C19
CYP3A4 AK298451 GCCTGGTGCT CCTCTATCTA (57.6) GGCTGTTGACC ATCATAAAAGC (56.0) 187 60 CYP3A4
CYP1A2 AF182274 ACCCCAGCTG CCCTACTTG (61.8) GCGTTGTGT CCCTTGTTGT (57.4) 101 60 CYP1A2
CYP2E1 NM_000773 ACCTGCCCCATG AAGCAACC (62.8) GAAACAACTCC ATGCGAGCC (58.9) 246 60 CYP2E1
UGT1A1 BC128414 GGAGCAAAAGG CGCCATGGC (65.6) GTCCCCTCTGC TGCAGCTGC (66.6) 178 60 Uridine diphosphate glucuronyltransferase 1A1
OATP2 AJ132573 GCCCACGC GTCCGACT (63.8) ACAGAGCTGCCAA GAACATCT (57.6) 277 60 Organic anion transporting polypeptide 2
Claudin‐1 NM_021101 GTGGAGGATTTA CTCCTATGCCG (59.1) ATCAAGGCAC GGGTTGCTT (59.1) 165 60 Claudin‐1
Occludin NM_001205255 ACAGGCCTGA TGAATTGCCA (58.5) GTGAAGGCACG TCCTGTGT (59.3) 218 60 Occludin
SR‐B1 NM_005505 TGCACTATGCCC AGTACGTC (58.7) TAGGCCTGAAT GGCCTCCTT (60.3) 148 60 Scavenger receptor class B type I
CD81 NM_004356 ACCTCCTGTATCTG GAGCTGG (60.0) TTGGCGATCTGGT CCTTGTTG (59.4) 235 60 Cluster of Differentiation 81
ApoE XM_005258867 CGCTTTTGGGA TTACCTGCG (58.8) GGGGTCAGTTG TTCCTCCAG (59.8) 158 60 Apolipoprotein E
miR‐122 NR_029667.1 ACACTCCAGC TGGGTGGAGTGT GACAATCC (65.7) TGGTGTCGTGG AGTCG (48.5) 66 60 MiroRNA 122
SEC14L2 NM_012429 GGGATCCTTTA AGAGGCGGG (55.9) GTCATCTGGA TTCGGCAGGG (55.9) 262 60 SEC14‐like protein 2
GAPDH NG_009349.4 GAAATCCCATCACC ATCTTCC (55.0) AAATGAGCCCCAG CCTTCTC (59.6) 124 60 Glyceraldehyde‐3‐p dehydrogenase

Basic Protocol 8: Production of Hepatitis C Virus Derived from Cell Culture (HCVcc), JFH‐1

  Materials
  • JFH‐1 plasmid propagated in E. coli
  • Luria broth (LB; see recipe)
  • Ampicillin (Sigma, cat. no. A1593)
  • NucleoBond Xtra Midi Kit (MN, cat. no. 740410.10)
  • FastDigest XbaI (Thermo Fisher Scientific, cat. no. FD0684)
  • Buffered phenol, pH 7 to 8
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol
  • Chloroform (Merck Millipore)
  • 3 M sodium acetate, pH 7.0
  • Isopropanol (Merck Millipore)
  • UltraPure DNase/RNase‐Free Distilled Water (Thermo Fisher Scientific, cat. no. 10977015)
  • 75% (v/v) ethanol
  • 100% (v/v) ethanol (Merck Millipore)
  • TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, cat. no. K0441) containing:
    • 5× TranscriptAid Reaction Buffer
    • ATP/CTP/GTP/UTP mix
    • TranscriptAid enzyme mix
  • Hepatocyte‐like cell (HLCs)
  • Huh7 cell
  • DMEM/F12 (HyClone, cat. no. SH30261.02)
  • Fetal bovine serum (FBS; GE Healthcare Life Sciences)
  • Penicillin G sodium (Sigma, cat. no. P7794)
  • Streptomycin (Sigma, cat. no. S6501)
  • Opti‐MEM Reduced Serum Medium (Thermo Fisher Scientific, cat. no. 31985088)
  • X‐tremeGENE HP DNA Transfection Reagent (Roche Diagnostics)
  • Sucrose (Sigma. cat. no. S0389)
  • Centrifuge
  • Pipettes
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Nanodrop spectrophotometer
  • 200‐μl PCR tubes
  • 10‐cm cell culture dishes
  • Micropipettes
  • 37°C, 5% CO 2 incubator
  • 0.45‐μm sterile syringe filter (Sartorius)

Basic Protocol 9: Production of HCVcc from Infectious Virus

  Materials
  • Hepatocyte‐like cell (HLCs; see protocol 6)
  • Huh7 cell line
  • DMEM/F12 (HyClone, cat. no. SH30261.02)
  • Fetal bovine serum (FBS; GE Healthcare Life Sciences)
  • Penicillin G sodium (Sigma, cat. no. P7794)
  • Streptomycin (Sigma, cat. no. S6501)
  • Hepatitis C virus derived from cell culture with titer >105 ffu/ml (JFH‐1 HCVcc) (see protocol 8)
  • Phosphate‐buffered saline (DPBS) without calcium and magnesium
  • Trypsin‐EDTA, 0.05% w/v, 1 mM EDTA in PBS
  • Sucrose (Sigma. cat. no. S0389)
  • 10‐cm cell culture dishes
  • 50‐ml conical tubes
  • 0.45‐μm sterile syringe filter (Sartorius)

Basic Protocol 10: HCVcc or HCVser Infectitity Titer Analysis

  Materials
  • Hepatocyte‐like cell (HLCs; see protocol 6) (From iPSCs differentiation protocol)
  • Huh7 cell line
  • DMEM/F12 (HyClone, cat. no. SH30261.02)
  • Fetal bovine serum (FBS; GE Healthcare Life Sciences)
  • Penicillin G sodium (Sigma. cat. no. P7794)
  • Streptomycin (Sigma, cat. no. S6501)
  • Hepatitis C virus derived from cell culture with titer >105 ffu/ml (JFH‐1 HCVcc)
  • Hepatitis C virus derived from patient serum with titer >105 ffu/ml (HCVser)
  • Ice
  • Methylcellulose overlay (see recipe)
  • Paraformaldehyde fixing solution (see recipe)
  • Phosphate‐buffered saline (DPBS) without calcium and magnesium.
  • Triton X‐100 (Sigma, cat. no. X100)
  • Bovine serum albumin (HyClone, GE Healthcare Life Sciences, cat. no. SH30574.02)
  • Normal goat serum (Thermo Fisher Scientific, cat. no. 31872)
  • Primary antibodies: HCV core antigen, NS3, NS5A, NS5B (Santa Cruz)Tween‐20 (Sigma. cat. no. P9416)
  • Goat anti‐Mouse IgG/IgM (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A‐10684)
  • Goat anti‐Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A‐11008)
  • Hoechst 33342 (Thermo Fisher Scientific, cat. no. H3570)
  • Fluorescence mounting medium (Dako, cat. no. S302380)
  • 50% (v/v) glycerol
  • Williams' Medium E (Thermo Fisher Scientific, cat. no. 12551032)
  • α‐tocopherol (Merck Millipore, cat. no. 613420)
  • Sucrose (Sigma, cat. no. S0389)
  • 8‐well chamber slide (Thermo Fisher Scientific, cat no. 154534)
  • 37°C water or dry bath
  • Pipettes
  • Fluorescence microscope
  • Parafilm
  • 6‐well plates
  • 50‐ml conical tubes
  • 0.45‐μm sterile syringe filter (Sartorius)
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Literature Cited

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