Hepatocytes: Methods and Protocols (Methods in Molecular Biology, 2544)

Description

This volume details experimental protocols to study structure and function of hepatocytes in vivo and in vitro, and for characterizing heterogeneity and plasticity of hepatocytes in healthy and in injured liver. Chapters guide readers through separating hepatocyte subfractions, characterizing the heterogeneity, novel culture systems, hepatocyte-like cells from human pluripotent stem cells, analyzing drug metabolism, monitoring protein sorting, manipulating gene expression, and methods for expanding primary hepatoblastoma and identifying hepatocellular carcinoma subtypes. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.   Authoritative and cutting-edge, Hepatocytes: Methods and Protocols aims to provide techniques to further understand molecular mechanisms involved in the regulating hepatocyte functions and underlying liver diseases. Preface Contents Contributors Chapter 1: Long-Term Expansion of Murine Primary Hepatocyte Organoids 1 Introduction 2 Materials 2.1 Hepatocyte Isolation 2.2 Establishment and Cryopreservation of Hepatocyte Organoids 2.3 Gene Expression Analysis by qRT-PCR 2.4 Whole-Mount Immunofluorescence Staining 3 Methods 3.1 Liver Perfusion 3.2 Hepatocyte Purification 3.3 Organoid Establishment 3.4 Passaging Organoids 3.5 Cryopreservation and Thawing of Cells 3.6 Hepatocyte Maturation 3.7 Gene Expression Analysis by qRT-PCR 3.8 Whole-Mount Organoid Immunofluorescence Staining 4 Notes References Chapter 2: Generation of In Vivo Traceable Hepatocyte-Like Cells from Human iPSCs 1 Introduction 1.1 hiPSCs as an Alternative Source of Hepatocytes for Cell Therapy 1.2 The Three-Step Process for Generating HLCs 1.3 Use of Radionuclide Reporter Genes for HLC Tracking In Vivo 1.4 Choice of Lentiviral Genetic Engineering for iPSC-Derived Progeny 2 Materials 2.1 Plasmid Production 2.2 Virus Production 2.3 Maintenance and Differentiation of iPSCs to HLCs 2.3.1 Tissue Culture Incubator Conditions 2.3.2 Preparation of Coated Well Plates and Dishes 2.3.3 Growth Factors for Differentiation 2.3.4 hiPSC and HLC Differentiation Media 2.4 HLC Transplantation and In Vivo Imaging 3 Methods 3.1 Preparation of Plasmid Stocks 3.1.1 Transformation of Plasmid DNA into E. coli 3.1.2 Isolation of Plasmid DNA from Liquid Cultures 3.2 Lentiviral Particle Production 3.2.1 HEK293T Multi-layer Flask Preparation for Lentiviral Particle Production 3.2.2 Lentiviral Particle Production from Pre-seeded Multi-layer Flasks by PEI Transfection 3.2.3 Quantification of Viral Titer 3.3 Maintenance and Passaging of hiPSCs 3.4 Differentiation of hiPSCs to HLCs and Lentiviral Transduction for In Vivo Tracking 3.4.1 Induction of Endodermal Differentiation 3.4.2 Hepatic Progenitor Cell Differentiation 3.4.3 Maturation of Hepatic Progenitors 3.4.4 Lentiviral Transduction of HLCs 3.4.5 Passaging of Immature Transgenic HLCs 3.5 Radiolabeling Traceable HLCs In Vitro for In Vivo Transplantation 3.5.1 Testing Reporter Gene Function of HLCs In Vitro 3.5.2 Calculating Radiotracer Uptake in Pre-labeled/Transplanted HLCs 3.6 Tracking Transplanted HLCs In Vivo by NanoSPECT/CT Imaging 3.6.1 Intraliver HLC Transplantation Preparation Surgery and Cell Injection 3.6.2 Animal Preparation for Radionuclide Imaging and Radiotracer Administration 3.6.3 Imaging by NanoSPECT/CT 3.6.4 Ex Vivo Confirmation of In Vivo Tracking Data 4 Notes References Chapter 3: Generation of Hepatocyte Organoids from Human iPS Cells 1 Introduction 2 Materials 2.1 Cell-Repellent Microwell Arrays Preparation 2.2 Human Embryoid Bodies (hEBs) Generation 2.3 Hepatocyte Organoids Generation 2.4 Fluorescent Antibodies Immunostaining Evaluation 2.5 Quantitative Polymerase Chain Reaction (PCR) Analysis 2.6 Enzyme-Linked Immunosorbent Assay (ELISA) 2.7 Assessment of Liver Metabolic Functions 2.8 Equipment 3 Methods 3.1 Cell-Repellent Microwell Array Preparation 3.2 Human Embryoid Bodies (hEBs) Generation 3.3 Hepatocyte Organoids Generation 3.4 Morphological Evaluation of the Hepatocyte Organoids 3.5 Fluorescent Antibodies Immunostaining Evaluation 3.6 Quantitative Polymerase Chain Reaction (PCR) Analysis 3.7 Enzyme-Linked Immunosorbent Assay (ELISA) for Hepatic and Coagulation Factor Markers 3.8 Assessment of Liver Metabolic Functions 4 Notes References Chapter 4: Induction of Bile Canaliculi-Forming Hepatocytes from Human Pluripotent Stem Cells 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Immunostaining 3 Methods 3.1 Differentiation of PSC to Hepatocytes 3.1.1 Differentiation of PSCs to Definitive Endoderm Cells 3.1.2 Hepatic Lineage Specification 3.1.3 Differentiation to Hepatic Progenitor Cells 3.1.4 Differentiation to Hepatocytes 3.2 Monitoring the Different Steps in Hepatocyte Differentiation 3.3 Monitoring Bile Canaliculi-Forming Capacity 3.3.1 Fixed Cells 3.3.2 Living Cells 4 Notes References Chapter 5: Human Hepatocyte Transduction with Adeno-Associated Virus Vector 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Reagents 2.3 Equipment 2.4 Mice 3 Methods 3.1 Cell Culture of Primary Human Hepatocytes 3.2 rAAV Vectors Preparation by Triple Plasmids Transfection 3.3 AAV Infection for Human Primary Hepatocytes 3.4 AAV Infection in Humanized Chimeric FRG Mice (see Note 6). 3.5 Post-mortem Analysis of AAV Transduction in Chimeric Mice by Immunofluorescence Staining 3.6 Analyze AAV Transduction of Human Hepatocytes by Flow Cytometry: 4 Notes References Chapter 6: Hepatocyte-Directed Delivery of Lipid-Encapsulated Small Interfering RNA 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Materials for Hepatocyte Isolation 2.3 Solutions for Hepatocyte Isolation 2.4 Materials for Generation of LNP (see Notes 1 and 2) 2.5 Materials for Lipoplex-based Transfection 3 Methods 3.1 Cell Culture 3.1.1 Cell Thawing 3.1.2 Cell Seeding 3.2 Isolation of Hepatocytes 3.3 Testing of siRNA Sets 3.4 Liver Specificity to the LNP-based Nanoparticles 4 Notes References Chapter 7: Co-culture Model for Hepatitis B Virus Infection Using iPSC-Derived Liver Progenitor Cells and Liver Sinusoidal End... 1 Introduction 2 Materials 2.1 Induction of LSEC 2.2 Induction of LPCs 2.3 HBV Infection 3 Methods 3.1 Co-culture of Hepatocytes and iPSC-Derived LSECs 3.1.1 Induction of LSECs from Human iPSCs 3.1.2 Induction of LPCs from Human iPSCs 3.1.3 Co-culture of LPCs with LSECs in Trans-well Plate 3.2 Infection of HBV 3.2.1 Preparation of Infectious HBV 3.2.2 HBV Attachment Assay (see Fig. 3) 3.2.3 HBV Internalization Assay 3.2.4 HBV Replication Assay 3.2.5 Preparation of HBV DNA 3.2.6 Quantitative (q-) PCR for the Detection of HBV DNA 3.2.7 q-PCR for the Detection of HBV cccDNA 3.2.8 Quantification of Hepatitis B Surface Antigen 4 Notes References Chapter 8: In Vitro Assay System to Detect Drug-Induced Bile Acid-Dependent Cytotoxicity Using Hepatocytes 1 Introduction 2 Materials 2.1 Preparation of Bile Acid Mixture Stock Solution (see Note 1) 2.2 Preparation of Drug Solution (see Note 2) 2.3 Preparation of Collagen-coated Plate (Under Sterile Condition, see Note 3) 2.4 Hepatocyte Thawing, Seeding, and Culture (Under Sterile Conditions) 2.5 Cell Death Assay (see Note 4) 3 Methods 3.1 Thawing Cryopreserved Hepatocytes (Under Sterile Conditions) 3.2 Hepatocyte Seeding (Under Sterile Conditions) 3.3 Sandwich Configuration (Under Sterile Conditions) 3.4 Bile Acid Concentration-dependent Toxicity Assessment (Under Sterile Conditions Except for Cell Death Assay) 3.5 Drug and Bile Acid Co-treatment Assay (Under Sterile Conditions Except for Cell Death Assay) (see Note 6) 3.6 Evaluation of Contributing Metabolite-dependent Bile Acid Toxicity (Under Sterile Conditions Except for Cell Death Assay) 4 Notes References Chapter 9: Evaluation of Urea Cycle Activity by Metabolic Flux Analysis Using Mass Spectrometry 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment 3 Methods 3.1 Extraction of Lysate from Cultured Hepatocytes 3.2 Extraction of the Lysate from hiPSC-Liver Organoids 3.3 Extraction of the Lysate from the Liver 3.4 Deproteinization of the Lysate 3.5 Deproteinization of Urine and Serum 3.6 Induction of In Vitro Substrate Decomposition (Preparation of Samples for Metabolic Flux Analysis) 3.7 Mass Spectrometry 4 Notes References Chapter 10: Live-cell Imaging of Biosynthetic Protein Transport in Hepatocytes 1 Introduction 2 Materials 2.1 Cell Culture and Adenoviruses 2.2 Solutions 2.3 Antibodies 2.4 Imaging 3 Methods 3.1 Cell Culture and Virus Transduction 3.1.1 Culture of Primary Hepatocytes 3.1.2 Collagen Overlay 3.1.3 Virus Transduction 3.2 Temperature Block 3.3 Live-Cell Imaging 3.3.1 TGN-to-Surface 3.3.2 TGN Exit 3.4 Immunofluorescence 4 Notes References Chapter 11: Collagen Sandwich Culture of Primary Hepatocytes for Image-Based Investigations 1 Introduction 2 Materials 2.1 Preparing Collagen Solution 2.2 Coating a Glass Bottom Dish with Collagen 2.3 Plating Hepatocytes into a Glass Bottom Dish 2.4 Overlay Collagen 3 Methods 3.1 Preparing Collagen Solution (1.5 mg/mL) Using Type 1 Collagen from Rat Tail 3.2 Coating a Glass Bottom Dish with Collagen Solution 3.3 Plating Hepatocytes into the Collagen-coated Glass Bottom Dish 3.4 Overlay Collagen (see Note 23) 4 Notes References Chapter 12: Assessment of Hepatocyte Ploidy by Flow Cytometry 1 Introduction 2 Materials 2.1 Liver Tissue Dissociation via Perfusion 2.2 Hepatocyte Isolation 2.3 Nuclei Isolation 2.4 Fixation of Hepatocytes or Hepatocyte Nuclei 2.5 DNA Staining of Fixed Hepatocytes or Hepatocyte Nuclei 2.6 Flow Cytometry 3 Methods 3.1 Liver Tissue Dissociation 3.2 Hepatocyte Isolation 3.3 Nuclei Isolation from Mouse Liver 3.4 Fixation of Hepatocytes or Hepatocytes Nuclei 3.5 DNA Staining of Fixed Hepatocytes or Hepatocytes Nuclei 3.6 Flow Cytometry 4 Notes References Chapter 13: Isolation of Small Hepatocyte-Like Progenitor Cells from Retrorsine/Partial Hepatectomy Rat Livers by Laser Microd... 1 Introduction 2 Materials 2.1 Preparation of the Retrorsine/Partial Hepatectomy Rat Model 2.2 Materials for LMD 2.3 Materials for RNA Isolation 3 Methods 3.1 Preparation of Ret/PH Rat Models 3.1.1 Administration of Ret-Solution (30 mg/kg Body Weight) to Rats 3.1.2 Perform 2/3 PH 2 Weeks After the Second Injection 3.2 Preparation of Liver Sections 3.3 Isolation of SHPCs by LMD 3.4 Isolation of RNA 4 Notes References Chapter 14: Application of EdU-Based DNA Synthesis Assay to Measure Hepatocyte Proliferation In Situ During Liver Regeneration 1 Introduction 2 Materials 2.1 Partial Hepatectomy and EdU Labeling 2.2 Immunofluorescence Staining 2.3 Imaging 3 Methods 3.1 Partial Hepatectomy 3.2 EdU Labeling and Tissue Processing 3.3 Liver Tissue Processing for Paraffin Block Preparation 3.4 Immunostaining Procedure with Ki67 and EdU 4 Notes References Chapter 15: Quantification of Clonal Expansion of Hepatocytes in Normal and Injured Liver 1 Introduction 2 Materials 2.1 Reagents for Labeling Individual Hepatocyte Clones 2.2 Reagents for Fixing and Processing Mouse Liver Tissue 2.3 Software for Analysis of Hepatocyte Clonal Expansion 3 Methods 3.1 Labeling Individual Hepatocyte Clones 3.2 Mouse Liver Collection and Sectioning 3.3 Clearing Mouse Liver Sections and Imaging 3.4 Quantification and Analysis 4 Notes References Chapter 16: Evaluation of SOX9-Positive Hepatocytes in Human Liver Specimens and Mature Mouse Hepatocytes 1 Introduction 2 Materials 2.1 SOX9 Chromogenic Staining 2.2 Immunofluore-scence Detection of SOX9 2.3 Induction of SOX9 in Primary Mouse Hepatocytes 2.4 Induction of SOX9 in a Mature Mouse Hepatocyte Cell Line 3 Method 3.1 SOX9 Chromogenic Staining Protocol on Paraffin-Embedded Tissue Sections 3.2 Immunofluore-scence Detection Protocol of SOX9 with Hepatocyte Markers on Paraffin-Embedded Tissue Sections 3.3 Induction of SOX9 Protein Using Mouse IL-8 Homolog in Primary Mouse Hepatocytes 3.4 Induction of SOX9 Protein Using Mouse IL-8 Homologs in a Mature Mouse Hepatocyte Cell Line 4 Notes References Chapter 17: Identification of Genes Regulating Hepatocyte Injury by a Genome-Wide CRISPR-Cas9 Screen 1 Introduction 2 Materials 2.1 Plasmid DNA 2.2 GeCKOv2 sgRNA Library and Companion Plasmid DNA Amplification 2.3 Cell Lines and Primary Hepatocytes 2.4 Tissue Culture 2.5 Lentivirus Production and qRT-PCR Titration 2.6 Transduction and Clonal Cell Line Generation 2.7 Protein Isolation and Western Blotting 2.8 Drug Kill Curve 2.9 Drug Screen and Sample Collection 2.10 Library Preparation and Sequencing 2.11 Sequencing Deconvolution and Downstream Analysis 2.12 Comparisons with Public Data and Publications 2.13 Functional Validations 3 Methods 3.1 Study Design Overview 3.2 GeCKO v2 Library Amplification and Plasmid Purification 3.3 HEK-293FT Tissue Culture 3.4 HuH-7 Tissue Culture 3.5 Lentivirus Production and Purification 3.6 Cell Transduction with Cas9 Lentivirus 3.7 Cell Transduction with the GeCKOv2 Library Lentivirus and Controls 3.8 Drug Kill Curve 3.9 Drug Screen and Sample Collection 3.10 Next-Generation Sequencing 3.11 GeCKOv2 Screen Deconvolution and Statistical Analysis 3.12 Pathway Analysis 3.13 Statistical Analysis of GEO Datasets 3.14 Drug-Gene Interaction Analysis 3.15 Gene Candidate Prioritization 3.16 Functional Validation of Gene Knockdown in Cultured Mouse Hepatocytes 4 Notes References Chapter 18: Identification of Subtypes of HCC Using Bioinformatics and the Hepatocyte Differentiation Model 1 Introduction 2 Materials 2.1 hESCs Culture 2.2 Definitive Endoderm Induction 2.3 Hepatocyte Induction 2.4 Quantitative Real-Time PCR 2.5 Clinical HCC Samples 3 Methods 3.1 Culture of Human Embryonic Stem Cells (hESCs) 3.2 Differentiation of hESCs to Hepatocytes 3.3 Characterization of Representative Markers Using Quantitative Real-Time PCR 3.4 Hierarchical Clustering Analysis Reveals Gene Expression Pattern in Tissue Samples 3.5 Topological Data Analysis (TDA) Distinguishes Signature Genes in Diseased Group Samples 4 Notes References Chapter 19: 3D Culture of Primary Patient-Derived Hepatoblastoma Tumoroids 1 Introduction 2 Materials 2.1 Isolation of Hepatoblastoma Tumoroids from Primary Patient Specimens 2.2 Cryopreservation, Passaging, and Single-Cell Isolation of Tumoroids 2.3 Preparation of Tumoroids for Whole-Mount and Cross-Sectional Imaging 2.4 Lentiviral Transduction of Tumoroids 3 Methods 3.1 Isolation of Hepatoblastoma Tumoroids from Primary Patient Specimens 3.2 Cryopreservation and Passaging of Tumoroids 3.3 Isolation of Single Cells for Cell Counting, Flow Cytometry, Colony Formation Assays 3.4 Preparation of Tumoroids for Whole-Mount and Cross-Sectional Imaging 3.5 Lentiviral Transduction of Tumoroids 4 Notes References Chapter 20: Preparation of Functional Human Hepatocytes Ex Vivo 1 Introduction 2 The Expansion of Primary Hepatocytes 3 Somatic Cell to Hepatocyte Differentiation 4 iPSC-Derived Hepatocyte-Like Cells 5 Perspective References Index