Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Summary

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Abstract

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Introduction

The liver performs diverse metabolic functions, such as regulating the availability of readily usable energy substrates like glucose and ketone bodies, as well as detoxifying xenobiotic compounds. In recent years, there has been a significant increase in the prevalence of liver diseases, largely attributed to non-alcoholic steatohepatitis (NASH), which, if untreated, can advance to cirrhosis or cancer¹. Therefore, it is imperative to understand the metabolic functions of the liver and its related diseases to facilitate the development of effective treatments^2,3.

Protocol

1. Management of hiPSC

NOTE: hiPSCs (CS03iCTR-n3 cell line) were commercially purchased. The appropriate management of the extracellular matrix gel coating and the hiPSC medium is key for attaching the hiPSCs to the plates and feeding them. Here, the volumes needed for one 6-well plate were described. The remaining hiPSCs from the 6-well plate, which will not differentiate into organoids, may be stored in liquid nitrogen for long-term storage.

1. Dispensing extracellu.......

Discussion

The current protocol offers various methodological details on how to handle hiPSCs and the subsequent 3D organoid cultures. This includes the two major critical steps: (i) the detaching of the 2D cultures and then their development into 3D hepatic organoids after 10 days, as well as (ii) the delicate dissociation of a 3D structure into single cells. Based on available information, this is the first report of a 3D HHO model to study thyroid hormone action, demonstrating a peak expression of DIO2 in hepatoblast-like cells<.......

Materials

Name	Company	Catalog Number	Comments
10 µL Universal Pipette Tips Filtered, Low rentention, Pre-sterile	VWR	613-6462	All procedures
1000 µL Universal Pipette Tips Filtered, Low rentention, Pre-sterile	VWR	613-6470	All procedures
15 mL Polypropilene Conical Tube	Falcon (Corning)	352097	Dissociation Hepatic Organoids
200 µL Universal Pipette Tips Filtered, Low rentention, Pre-sterile	VWR	613-6465	All procedures
3,3',5-Triiodo-L-thyronine	Sigma	T2877-100	Hepatic Organoid differentiation
40 μm Cell Strainer	Corning	431750	Dissociation Hepatic Organoids
50 mL tube	Falcon (Corning)	352070	All procedures
6 well-plate Nunc Cell-Culture Treated Multidishes	Thermo fisher scientific	140675	hiPSC maintenance
A83-01	R&D Systems	2939/10	Hepatic Organoid differentiation
Advanced DMEM/F12	Gibco	12634010	Hepatic Organoid differentiation
ART Wide Bore Filtered Pipette Tips	ART	2069GPK	All procedures
B27 supplement	Gibco	17504044	Hepatic Organoid differentiation
BMP7	R&D Systems	354-BP-010/CF	Hepatic Organoid differentiation
Bovine Albumin Fraction V (7.5% solution)	Gibco	15260037	Dissociation Hepatic Organoids
BSA, Fraction V, Fatty Acid Free for Tissue Culture	GoldBio	A-421-100	Dissociation Hepatic Organoids
CHIR99021	R&D Systems	4423/10	Hepatic Organoid differentiation
Corning 96-well Clear Flat Bottom Ultra-Low Attachment	Corning	3474	Hepatic Organoid differentiation
Costar 6-well Clear Flat Bottom Ultra-Low Attachment	Corning	3471	Hepatic Organoid differentiation
CS03iCTR-n3 human induced Pluripotent Stem Cell line	Cedar-sinai		hiPSC maintenance
DAPT	R&D Systems	2634/10	Hepatic Organoid differentiation
dbCAMP	Millipore Sigma	D0627-100MG	Hepatic Organoid differentiation
Dexamethasone	R&D Systems	1126/100	Hepatic Organoid differentiation
DMEM/F12	Gibco	11320033	hiPSC maintenance
DNAse I, RNase-free, HC	Thermo Fisher scientific	EN0523	Dissociation Hepatic Organoids
Falcon 10 mL Serological Pipet, Polystyrene, 0.1 Increments, Individually Packed, Sterile	Corning	357551	All procedures
Falcon 5 mL Serological Pipet, Polystyrene, 0.1 Increments, Individually Packed, Sterile	Corning	357543	All procedures
Falcon 50 mL Serological pipet, Polystyrene, 1.0 Increments, Individually Packed, Sterile	Corning	357550	All procedures
Gentle Cell Dissociation Reagent (GCDR)	Stemcell Technologies	100-0485	Hepatic Organoid differentiation
Glutamax supplement	Gibco	35050061	Hepatic Organoid differentiation
L-Thyroxine	Sigma	T1775-1G	Hepatic Organoid differentiation
Matrigel hESC-Qualified Matrix, LDEV-free, 5 mL	Corning	354277	Extracellular matrix gel
mFreSR	Stemcell Technologies	5855	hiPSC cryopreservation medium
mTeSR 5x Supplement	Stemcell Technologies	100-0276	hiPSC medium
mTeSR Plus	Stemcell Technologies	100-0276	hiPSC medium
Multi Platform Shaker	Fisherbrand (Thermo Fisher technologies)	88861021	Hepatic Organoid differentiation
N2 supplement	Gibco	17502048	Hepatic Organoid differentiation
Nicotinamide	R&D Systems	4106/50	Hepatic Organoid differentiation
PBS, pH 7.4	Gibco	10010023	hiPSC maintenance
Recombinant human BMP4	R&D Systems	314-BP-010/CF	Hepatic Organoid differentiation
Recombinant human EGF	R&D Systems	236-EG-200	Hepatic Organoid differentiation
Recombinant human FGF basic/FGF2/bFGF	R&D Systems	233-FB-010/CF	Hepatic Organoid differentiation
Recombinant human FGF19	R&D Systems	959-FG-025/CF	Hepatic Organoid differentiation
Recombinant human HGF	R&D Systems	294-HG-005/CF	Hepatic Organoid differentiation
Recombinant Human Jagged-1 Fc Chimera	R&D Systems	1277-JG-050	Hepatic Organoid differentiation
Recombinant human KGF/FGF7	R&D Systems	251-KG-010/CF	Hepatic Organoid differentiation
ReLeSR	Stemcell Technologies	100-0483	hiPSC detaching medium
RNAse Inhibitor Ambion, cloned, 40 U/μL	Invitrogen	AM2682	Dissociation Hepatic Organoids
RNase Zap	Invitrogen	AM9780	Dissociation Hepatic Organoids
Sorvall  Legend XT/XF Centrifuge Series	Thermo Fisher Scientific	75004539	All procedures
STEMdiff Definitive Endoderm Kit	Stemcell Technologies	5110	Hepatic Organoid differentiation
Trypan Blue solution (0.4%)	Gibco	15250061	Dye solution
TrypLE Express Enzyme	Gibco	12604013	Cell Dissociation enzyme
Trypsin 0.5% - EDTA (10X)	Gibco	15400054	Dissociation Hepatic Organoids
Valproic acid, sodium salt	R&D Systems	2815/100	Hepatic Organoid differentiation
Vari-Mix Platform Rocker	Thermo Fisher scientific	M79735Q	Dissociation Hepatic Organoids
Y-27632 dihydrochloride	R&D Systems	1254	Hepatic Organoid differentiation