Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Summary

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Abstract

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

Protocol

1. Initial preparation of all chemical stocks and coating of culture plasticware

All steps to be carried out in a tissue culture hood under aseptic conditions.

1. Preparation of human basic Fibroblast Growth Factor (hbFGF)
   1. Prepare 10% BSA solution in PBS and filter through a 0.22 μm filter.
   2. From the 10% BSA solution prepare a 0.2% BSA solution.
   3. Add 10 mL 0.2% BSA solution/100 μg hbFGF.
   4. Pre-wet a 0.22 μm filter by filtering 5 mL 10% BSA solution through the filter. Discard the 10 mL BSA wash.
   5. Filter the hbFGF through the pre-washed filter.
   6. Aliquot the hbFGF in sterile ....

Discussion

We have developed a simple, homogeneous and highly reproducible in vitro model to generate scalable levels of human HLCs. Our model has been validated by a number of external collaborating laboratories. We routinely characterise stem cell derived HLCs using our in house tool box of developmental markers and liver specific functional assays (most of which are commercially available). The critical stages in our process are: the maintenance of stem cell pluripotency; the ability to direct stem cell differentiation .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel coating plates and flasks Matrigel (10 mL, BD Biosciences, UK); store at -20°C. KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4°C. Tissue culture plates (6 well, 12 well, Corning, UK) Tissue culture flask (25 cm2 vented, Corning, UK) hESC Maintenance Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R & D Systems, USA); store at -20°C. BSA solution (50 mL, Sigma Aldrich, UK); store at 4°C. Human basic fibroblast growth factor (100 μg, Peprotech, USA); store at -20°C. Passaging hESCs with collagenase Confluent well or flask of hESCs. Matrigel coated wells or flasks as appropriate. Phospate buffered saline (-MgCl2, -CaCl2) (500 mL, Gibco, Invitrogen, UK); store at room temperature. Collagenase IV (1 g, Gibco, Invitrogen, UK); store at 4°C. Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R & D Systems, USA). Human basic fibroblast growth factor (100 μg, Peprotech, USA). Differentiation of hESCs to hepatic endoderm RPMI 1640 (500 mL, Gibco, Invitrogen, UK); store at 4°C. B27 Supplement (10 mL, Gibco, Invitrogen, UK); store at -20°C. Activin A (2 μg, Peprotech, USA); store at -20°C. Recombinant mouse Wnt3a (2 μg, R & D Systems, USA); store at -20°C. KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4°C. KO-SR (500 mL, Gibco, Invitrogen, UK); store at -20°C. Non-essential amino acids (100 mL, Gibco, Invitrogen, UK); store at 4°C. β-Mercapt–thanol (10 mL, Gibco, Invitrogen, UK); store at 4°C. DMSO (Sigma Aldrich, UK); store at room temperature Leibovitz L-15 culture medium (500 mL, Sigma Aldrich, UK); store at 4°C. Tryptose phosphate broth (100 mL, Sigma Aldrich, UK); store at 4°C. F–tal bovine serum, heat inactivated (500 mL, Gibco, Invitrogen, UK); store at -20°C. Hydrocortisone 21-hemisuccinate (100 mg, Sigma Aldrich, UK); store at -20°C. Insulin (bovine pancreas) (100 mg, Sigma Aldrich, UK); store at -20°C. L-Glutamine (100 mL, Gibco, Invitrogen, UK); store at -20°C. Ascorbic acid (25 g, Sigma Aldrich, UK); store at -20°C. Human HGF (10 μg, Peprotech, USA); store at -20°C. Recombinant Human Oncostatin M (OSM) (50 μg, R & D Systems, USA); store at -20°C. Syringe driven filter unit 0.22 μm (Millipore, UK) Characterisation of hESC derived Hepatic Endoderm Immunostaining Phosphate buffer saline (-MgCl2, -CaCl2) (500 mL, Gibco, Invitrogen, UK); store at room temperature. PBST, PBS made up with 0.1% TWEEN 20 (Sigma-Aldrich, UK). Paraformaldehyde (PFA) (Sigma-Aldrich, UK) is made up in PBS, store -20°C. Glycerol (Sigma-Aldrich, UK), store at room temperature. Tris Base (Sigma-Aldrich, UK), store at room temperature. Ethanol Serum (AbD Serotech, UK), store at -20°C. Secondary Antibody, Alexa Fluorophores (Molecular Probes, Invitrogen, UK). MOWIOL 4-88 (Polysciences Inc, USA) is made up in Tris HCL and glycerol as per manufacturers instructions. DAPI (Pierce, Thermo Fisher Scientific, UK) is added to the MOWIOL solution at a 1:1000 dilution. Primary antibodies Antigen* Type Supplier Dilution ALB Mouse Monoclonal Sigma Aldrich 1/500 E-Cadherin Mouse Monoclonal Millipore 1/100 α-fetoprotein Mouse Monoclonal Sigma 1/500 SSEA-4 FITC Mouse Monoclonal Biolegend 1/100 IgG Mouse Monoclonal DAKO 1/500 Secondary antibodies Anti-mouse FITC conjugate Goat Monoclonal Invitrogen 1/400 Table 2. The antibodies used for hESC derived hepatic endoderm immunostaining, the concentrations used, the species developed in and the companies they are purchased from. Functional Analysis of Hepatic Endoderm and Normalisation (per mg protein) Cytochrome P450 Assays p4-GLO CYP3A4, CYP1A2, Kits and luminometer (Promega, USA). White flat bottom 96 well assay plate (BD Biosciences, UK). BCA Assay Kit (Pierce, Thermo Fisher Scientific, UK). Transparent 96 well assay plate (IWAKI, UK)