Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

Summary

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

Abstract

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

Introduction

Success in identifying drugs that can be used to target a rare disease relies on the development of assays that can be used for screening. Hypothesis or target-based screens (reverse pharmacology) are useful, but require a detailed understanding of the molecular basis of the disease. Phenotypic screens (classical pharmacology) avoid the need for a detailed understanding of biochemical pathways, but instead rely on the development of models that accurately mirror the pathophysiology of the disease. Despite enthusiasm for target-based approaches, analyses of FDA approved first-in-class drugs reveal that phenotypic screens have been far more successful^1....

Protocol

1. Culture of Human Induced Pluripotent Stem Cells

1. Coating recombinant Human E-Cadherin Fc Fusion Protein (E-cad-Fc) or other matrices suitable for hPSC culture⁶
   1. Dilute E-cad-Fc to 15 μg/mL with Dulbecco's Phosphate-Buffered Saline containing calcium and magnesium (DPBS (+)).
   2. Coat 100-mm suspension tissue culture dishes with 5 mL of diluted E-cad-Fc and incubate at 37 ˚C for at least 1 h. Remove substrate and replace with 5 mL of medium (e.g., mTeSR1 referred as M-medium henceforth)⁷.
      Note: The culture medium used in this ....

Results

Generation of hepatocyte-like cells: Figure 1 describes the timeframe of the changes that occur during the differentiation of human iPSCs to hepatocyte-like cells. The culture of iPSCs on E-Cad-Fc provides approximately 2 mm diameter colonies that express the pluripotent marker OCT4 (Figure 1A-B). The morphology of the cells grown on an E-cadherin matrix will be .......

Discussion

Target based drug discovery, where small molecules are identified that influence the activity of a specific protein, has been the focus of many existing screening efforts. Although this approach has provided numerous pharmaceuticals, screens based on reversing a phenotype, classical pharmacology, have been more successful in identifying first-in-class compounds that have been clinically efficacious¹. A disadvantage to phenotypic drug discovery is that it relies on the availability of appropriate d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mm x 20 mm sterile tissue culture dishes	Corning	430167
100 mm x 20 mm sterile suspension culture dishes	Corning	430591
96-wells tissue culture plate	Corning	3595
Anti-human Albumin	Dako	A 0001
Anti-human FOXA2(6C12)	Novus Biological	H00003170-M12
Anti-human HNF4 alpha	Santa Cruz	SC-6556
Anti-human Oct-3/4 antibody	Santa Cruz	SC-9081
Anti-human SOX17	R&D	AF1924
Anti-human TRA-1-60 FITC conjugated	Millipore	FCMAB115F
Activin A Recombinant Human Protein	Invitrogen	PHC9563
B-27 Supplement, minus insulin	Invitrogen	0050129SA
B-27 Supplement, serum free	Invitrogen	17504044
BMP4 Recombinant Human Protein	Invitrogen	PHC9533
Cell Dissociation Reagent StemPro  Accutase	Invitrogen	A1110501
CellTiter-Glo Luminescent Cell Viability Assay	Promega	7572
DPBS+(calcium, magnesium)	Invitrogen	14040-133
DPBS-(no calcium, no magnesium)	Invitrogen	14190-144
DMEM/F-12, HEPES	Invitrogen	11330057
ELISA human APOB ELISA development kit	Mabtech	3715-1H-20
Fibroblast Growth Factor 2 (FGF2)	Invitrogen	PHG0023
Hepatocyte Culture Medium (HCM Bullet Kit)	Lonza	CC-3198
Hepatocyte Growth Factor  (HGF)	Invitrogen	PHC0321
L-Glutamine	Invitrogen	25030081
MEM Non-Essential Amino Acids Solution	Invitrogen	11140076
Oncostatin M (OSM) Recombinant Human Protein	Invitrogen	PHC5015
Penicillin-Streptomycin	Invitrogen	15140163
Feeder free pluripotent stem cell medium: mTesR1	STEMCELL technologies	5850
Reduced Growth Factor Basement Membrane Matrix	Invitrogen	A1413301
RPMI 1640 Medium, HEPES	Invitrogen	22400105
StemAdhere Defined Matrix for hPSC (E-cad-Fc)	Primorigen Biosciences	S2071
TMB-ELISA Substrate Solution	Thermo Scientific	34022
Anti-TRA-1-60 FITC conjugated	Millipore	FCMAB115F
Versene (EDTA) 0.02%	Lonza	17-711E
Y-27632 ROCK inhibitor	STEMCELL Technologies	72302