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 successful¹. The overall goal of this method is to establish a platform for high throughput screening that can be used to identify small molecules for the treatment of metabolic liver disease. Several in vitro models have been described including primary hepatocytes, hepatoma cells, and liver progenitor cells². However, most of these models have limitations, and there is a need for new models that can accurately recapitulate the pathophysiology of metabolic liver deficiencies in culture. Recently, human pluripotent stem cells combined with gene editing have offered an opportunity to model even the rarest of rare diseases in culture without the need to access patients directly³. While the use of patient-specific iPSCs as a tool to discover small molecules for the treatment of rare liver diseases is conceptually reasonable, there are only a few reports demonstrating the feasibility of this approach⁴. However, we have recently established a platform that used iPSC-derived hepatocytes to successfully identify drugs that can be repurposed for the treatment of deficiencies in liver metabolism⁵.

This protocol explains the process of differentiating human iPSCs to hepatocyte-like cells in 96-well plates and using them to screen a library of small molecules. It also describes the endpoint analysis using hypercholesterolemia as an example of metabolic liver disease. This approach should be useful to study the role and application of small molecules in the context of infectious liver disease, metabolic liver disease, drug toxicity, and other liver disorders.

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 protocol is prepared following published protocols⁷. However, several other media preparations have been described, or are available commercially, that are likely compatible with the procedure.
   3. Retrieve a vial of cryopreserved iPSCs from liquid nitrogen. Thaw at 37 ˚C until a small ice crystal remains. Gently pipette cells into a sterile 15-mL conical tube containing 4 mL of the M-medium.
   4. Centrifuge at 300 x g for 5 min. Remove the supernatant and gently re-suspend cells with 5 mL of the medium supplemented with 10 µM of Y-27632, a selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK).
   5. Remove the M-medium from step 1.1.2, and transfer 5 mL of cells from step 1.1.4 to E-cad-Fc-coated 100-mm suspension tissue culture dishes.
2. Maintaining human iPSCs in culture
   1. Once iPSCs reach around 80% confluency, remove culture medium and wash once with calcium and magnesium free DPBS (-). Aspirate the DPBS (-) and add a sufficient amount of 0.02% EDTA solution to cover the plates, then incubate for up to 3 min at room temperature.
      Note: At 80% confluence, the plate should contain around 2 x 10⁷ cells. It is important not to overgrow the cells, and to ensure that the cells retain an undifferentiated morphology. The pluripotency of the cells can be determined by staining with stem cell marker Tra-1-60 or equivalent⁸.
   2. As soon as the cells begin to release, remove the 0.02% EDTA solution and flood the dish with 10 mL of the M-medium to release the cells. Aid the detachment of iPSCs by gently pipetting.
      Note: The average incubation time for cells to detach is around 3 min, but this needs to be determined empirically for each iPSC line. The cells should be released as small clusters containing around 5 - 10 iPSCs per cluster.
   3. Transfer 1/10 of the suspended cells per fresh E-cad-Fc coated 100 mm suspension tissue culture dish containing 5 mL of the M-medium. Incubate the cultures at 37 ˚C under 4% O₂/5% CO₂ and change the medium daily.
      Note: Although iPSCs are routinely cultured under physiological oxygen conditions to promote pluripotency, iPSCs can also be grown using ambient oxygen.

2. Differentiation of Human iPSCs to Hepatocyte-like Cells on 96-Well Plates

Note: This section describes the differentiation of iPSCs in a format that is compatible with screening. Using this approach, it is possible to induce human iPSCs to differentiate and form hepatocyte-like cells in 20 days. While this is suitable for most assays, the length of culture can be extended to improve the maturity of the cells.

1. Coating 96-well tissue culture plates with reduced growth factor basement membrane matrix
   1. Prepare the stock by diluting the matrix to 2 mg/mL with ice-cold sterile DMEM/F12 tissue culture medium. Divide the matrix into 250 μL aliquots and store at -80 ˚C until required. Chill 10 mL of DMEM/F12 on ice for 30 min. Retrieve a 250 μL aliquot of 2 mg/mL matrix stock and thaw on ice.
   2. Combine the matrix with 10 mL of cold DMEM/F12 by mixing gently using a pre-chilled pipette to make final concentration of 0.05 mg/mL.
   3. Transfer 100 μL of diluted matrix to each well of a 96-well tissue culture plate. Incubate at 37 ˚C, 4% O₂/5% CO₂ for at least 1 h. Discard the matrix and replace with 50 μL of M-medium per well. Store at 37 °C, 4% O₂/5% CO₂ until needed.
2. Plate cells for differentiation
   1. Culture the iPSCs on 100-mm plates until the cells approach 80% confluence. Ensure that the plate contains around 2 x 10⁷ cells. Determine cell numbers by cell flow cytometer or hemocytometer.
      Note: With experience, the quality of the iPSCs cells can be judged by microscopy; however, close to 98% of the cells should express pluripotency markers such as the TRA-1-60 epitope when measured by FACS or immunostaining. It is important that the cells remain actively dividing and are not overgrown.
   2. To harvest the iPSCs from each 100-mm plate, aspirate the culture medium and rinse with 5 mL of DPBS (-). Replace the rinse with 3 mL of cell detachment solution (e.g., accutase) and incubate for around 2 min at 37 °C, 4% O₂/5% CO₂.
      Note: It is important to follow the detachment of cells by microscopy. Do not over digest with accutase. The goal is to release the cells as small clusters with minimal treatment.
   3. As soon as the cells begin to detach, harvest by adding 7 mL of the M-medium. Pipette several times to dissociate the cell colonies into small clusters of around 3 - 6 cells (Figure 1A).
   4. Collect the iPSCs into a 15-mL sterile centrifuge tube and centrifuge at 300 x g for 5 min. Remove the supernatant and re-suspend the cell pellet in 5 mL M-medium.
   5. Evenly distribute the cells onto 96-well matrix-coated plates by pipetting 50 μL of suspension cells into each well. To set up each 96-well plate, use one 100-mm plate of iPSCs with cells at approximately 80% confluency which contains around 5 x 10⁶ cells to setup the differentiation. Use a multichannel pipette to speed up this process. Culture the cells overnight at 37 ˚C, 4% O₂/5% CO₂.
      Note: It is important that an equal number of cells is deposited into each well to ensure reproducible differentiations.
3. Induce differentiation of the cells
   1. Examine the 96-well plates by microscopy to ensure that cells cover at least 70% of the surface of each individual well. If the coverage is less than 70%, replace the medium with fresh medium and incubate for an additional 24 h at 37 ˚C, 4% O₂/5% CO₂.
      Note: Incubating cells for more than 48 h after plating without inducing differentiation commonly reduces the efficiency of differentiation. Differentiation will also occur using ambient oxygen, although the efficiency may be affected.
   2. For Day 1 to Day 2 of differentiation, replace the culture medium with RPMI supplemented with 2% B27 (without insulin), 100 ng/mL Activin A, 10 ng/mL BMP4, and 20 ng/mL FGF2. Add 100 μL of medium per well and incubate at 37 ˚C, in ambient O₂/5% CO₂ with a daily medium change for 2 days.
      Note: Dealing with large numbers of 96-well plates require the use of an assisted pipette or robot. 12-channel and 96-channel pipettes are ideal for daily medium change.
   3. For Day 3 to Day 5 of differentiation, replace the culture medium with RPMI supplemented with 2% B27 (without insulin) and 100 ng/mL Activin A. Culture cells at 37 ˚C, in ambient O₂/5% CO₂ with daily medium change for 3 days.
   4. At differentiation day 5, before replacing with fresh culture medium, treat each well with 100 μL 0.02% EDTA solution for 30 s, then remove and replace with fresh culture medium.
      Note: If the differentiation to endoderm is efficient, the monolayer of differentiating cells is prone to peeling from the plate. A brief treatment with 0.02% EDTA solution helps alleviate cell detachment. This treatment does not impact the differentiation to hepatocytes.
   5. For differentiation between Days 6 to 10, replace the culture medium with RPMI/ 2% B27 (with insulin) supplemented with 20 ng/mL BMP4 and 10 ng/mL FGF2 and incubate at 37 ˚C, 4% O₂/5% CO₂ for 5 days with daily medium change. This stage induces cells to differentiate into hepatic progenitor cells.
   6. For differentiation between Days 11 to 15, replace the culture medium with RPMI/ 2% B27 (with insulin) supplemented with 20 ng/mL HGF. Incubate at 37 ˚C, 4% O₂/5% CO₂ for 5 days with daily medium change.
   7. For differentiation between Days 16 to 20, replace the culture medium with a hepatocyte cell culture medium (HCM) supplemented with 20 ng/mL of Oncostatin-M (OSM). Incubate the cells at 37 ˚C, in ambient O₂/5% CO₂ with daily medium change for 5 days.
      Note: The reproducibility of differentiation between individual wells can efficiently be determined by measuring the relative level of Albumin, AFP, or APOB secreted into the medium by ELISA (see 3.2).

3. Small Molecule Screening

Note: The following describes the procedure used to identify compounds that can be used to lower the level of APOB in the medium of hepatocyte-like cells derived from familial hypercholesterolemia iPSCs. A description of the model and its use in the identification of cholesterol lowering drugs has been described in detail previously^5,9. In the current example, 1,000 compounds from the South Carolina Compound Collection (SC³) library were screened. Each compound was tested at a concentration of 2 μg/mL.

1. Treatment of iPSC - derived hepatocytes with small molecules
   1. Dilute the master compound library to generate plates of working stocks with compounds at a concentration of 20 μg/mL in 2% DMSO and store at -20 ˚C.
   2. Prepare enough 96-well plates of iPSC-derived hepatocytes to allow each compound to be tested.
      Note: The number of compounds to be screened will dictate the number of 96-well plates that are required. In general, a single well should be prepared for each compound, plus additional wells for any control samples. The outermost wells of the plate are commonly avoided to reduce the possibility of edge effects that can corrupt interpretation. Hits can be identified using the z- score method without the need to use replicates. If replicates are included, it is more appropriate to use the t test.
   3. At the completion of differentiation (day 20, see 2.3.7), replace the culture medium with 100 μL of fresh HCM supplemented with 20 ng/mL of Oncostatin-M. Incubate at 37 ˚C, 4% O₂/5% CO₂ for exactly 24 h. Retrieve the culture medium into a fresh 96-well plate and store at -20 ˚C. Use this sample to calculate the level of APOB in the medium before the addition of drug.
   4. Add 90 μL of fresh HCM supplemented with 20 ng/mL of Oncostatin-M into each well. Add 10 μL of each compound from the working stock after mixing by pipetting. Add DMSO to 0.2% to control wells to match the final concentration in the treated samples and incubate at 37 ˚C, ambient O₂/5% CO₂ for exactly 24 h. Collect the culture medium and store at -20 ˚C. Use this sample to determine the level of APOB after treatment with compounds.
   5. Optional - the remaining cells can be processed for immunostaining, cell viability, or analyses of mRNA levels using standard approaches.
      Note: This screen was designed to identify drugs that had a rapid effect on APOB levels. However, depending on the disease model, downstream assays, or mechanism of drug action, it may be beneficial to treat the sample for multiple days.
2. Determine APOB levels by ELISA
   1. Determine Apolipoprotein B (APOB) concentration from both the pre- and post-drug treated samples using a human APOB ELISA development kit following the manufacturer's protocols.
   2. Preparing the following solutions
      1. Wash buffer, composed of 0.05% Tween 20 in PBS, pH 7.4. Store at 4 ˚C until use.
      2. Incubation buffer, composed of 0.05% Tween 20 and 0.1% bovine serum albumin (BSA) in PBS, pH 7.4. Store at 4 ˚C until use.
   3. Prepare APOB (mAB 20/17) antibody by diluting to 2 µg/mL in PBS, pH 7.4. Add 100 μL of the diluted antibody per well and incubate at 4 ˚C overnight.
   4. Remove the antibody from the assay plates and wash each well twice with 200 μL of PBS, pH 7.4.
   5. Add 200 μL/well of 0.05% Tween 20, 0.1% bovine serum albumin (BSA) in PBS, pH 7.4 and incubate on a shaking platform for 1 h at room temperature.
   6. Prepare an 8-point standard curve using 0.05% Tween 20 and 0.1% BSA in PBS, pH 7.4 with the following concentrations of APOB: 300 ng/mL, 200 ng/mL, 175 ng/mL, 150 ng/mL, 125 ng/mL, 100 ng/mL, 75 ng/mL, and 50 ng/mL.
   7. Dilute the test samples 1:4 with 0.05% Tween 20 and 0.1% BSA in PBS, pH 7.4.
   8. After 1 h of blocking with incubation buffer, discard the buffer and wash the assay plates five times with 200 μL of 0.05% Tween 20 in PBS, pH 7.4.
   9. Completely remove any residual washing buffer and transfer 90 μL of the diluted samples and the APOB standards to the assay plates. Incubate at room temperature on a shaker for 1 h.
   10. Discard the sample and wash with 200 μL 0.05% Tween 20 in PBS, pH 7.4 per well. Repeat the washes a total of 5 times. Add 100 μL per well of mAB LDL 11-biotin diluted 1:1,000 in 0.05% Tween 20 and 0.1% BSA in PBS, pH 7.4. Incubate at room temperature on a shaking platform for 1 h.
   11. Discard the reagent and wash with 200 μL per well washing buffer 5 times. Add 100 μL per well of Streptavidin-HRP antibody (1:1,000 dilution using incubation buffer), then incubate at room temperature on a shaking platform for 1 h.
   12. Discard the reagent and wash with 200 μL 0.05% Tween 20 in PBS, pH 7.4 per well. Repeat the washes a total of 5 times. Completely remove the residual washing buffer from the assay plates and add 100 μL per well of Tetramethylbenzidin (TMB) substrate solution, and incubate at room temperature for 8 min.
   13. Without removing the TMB substrate, directly add 100 μL per well of stop solution (1N HCl).
   14. Determine the absorbance at 450 nm using a plate reader.
   15. A standard curve can be established, using the Four Parameter Logistic Regression method, then used to define the concentration of APOB in each sample¹⁰.
   16. Examine the pre-drug: post-drug ratio of APOB in each sample to identify compounds with the potential to lower APOB levels in the medium.

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