This work was supported by the National Institutes of Health (DK55743, DK087377, DK102716 and HG006398 to S.A.D). We would like to thank Dr. Behshad Pournasr, Dr. James Heslop and Ran Jing for their contributions.

1. Culture of Human Induced Pluripotent Stem Cells
<ol>
	<li>Coating recombinant Human E-Cadherin Fc Fusion Protein (E-cad-Fc) or other matrices suitable for hPSC culture6
	<ol>
		<li>Dilute E-cad-Fc to 15 &#956;g/mL with Dulbecco&#39;s Phosphate-Buffered Saline containing calcium and magnesium (DPBS (+)).</li>
		<li>Coat 100-mm suspension tissue culture dishes with 5 mL of diluted E-cad-Fc and incubate at 37 &#730;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.</li>
		<li>Retrieve a vial of cryopreserved iPSCs from liquid nitrogen. Thaw at 37 &#730;C until a small ice crystal remains. Gently pipette cells into a sterile 15-mL conical tube containing 4 mL of the M-medium.</li>
		<li>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 &#181;M of Y-27632, a selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Maintaining human iPSCs in culture
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#730;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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Coating 96-well tissue culture plates with reduced growth factor basement membrane matrix
	<ol>
		<li>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 &#956;L aliquots and store at -80 &#730;C until required. Chill 10 mL of DMEM/F12 on ice for 30 min. Retrieve a 250 &#956;L aliquot of 2 mg/mL matrix stock and thaw on ice.</li>
		<li>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.</li>
		<li>Transfer 100 &#956;L of diluted matrix to each well of a 96-well tissue culture plate. Incubate at 37 &#730;C, 4% O2/5% CO2 for at least 1 h. Discard the matrix and replace with 50 &#956;L of M-medium per well. Store at 37 &#176;C, 4% O2/5% CO2 until needed.</li>
	</ol>
	</li>
	<li>Plate cells for differentiation
	<ol>
		<li>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.</li>
		<li>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 &#176;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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>Evenly distribute the cells onto 96-well matrix-coated plates by pipetting 50 &#956;L 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 &#730;C, 4% O2/5% CO2. 
		Note: It is important that an equal number of cells is deposited into each well to ensure reproducible differentiations.</li>
	</ol>
	</li>
	<li>Induce differentiation of the cells
	<ol>
		<li>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 &#730;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.</li>
		<li>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 &#956;L of medium per well and incubate at 37 &#730;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.</li>
		<li>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 &#730;C, in ambient O2/5% CO2 with daily medium change for 3 days.</li>
		<li>At differentiation day 5, before replacing with fresh culture medium, treat each well with 100 &#956;L 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.</li>
		<li>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 &#730;C, 4% O2/5% CO2 for 5 days with daily medium change. This stage induces cells to differentiate into hepatic progenitor cells.</li>
		<li>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 &#730;C, 4% O2/5% CO2 for 5 days with daily medium change.</li>
		<li>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 &#730;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).</li>
	</ol>
	</li>
</ol>
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 previously5,9. 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 &#956;g/mL.
<ol>
	<li>Treatment of iPSC - derived hepatocytes with small molecules
	<ol>
		<li>Dilute the master compound library to generate plates of working stocks with compounds at a concentration of 20 &#956;g/mL in 2% DMSO and store at -20 &#730;C.</li>
		<li>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.</li>
		<li>At the completion of differentiation (day 20, see 2.3.7), replace the culture medium with 100 &#956;L of fresh HCM supplemented with 20 ng/mL of Oncostatin-M. Incubate at 37 &#730;C, 4% O2/5% CO2 for exactly 24 h. Retrieve the culture medium into a fresh 96-well plate and store at -20 &#730;C. Use this sample to calculate the level of APOB in the medium before the addition of drug.</li>
		<li>Add 90 &#956;L of fresh HCM supplemented with 20 ng/mL of Oncostatin-M into each well. Add 10 &#956;L 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 &#730;C, ambient O2/5% CO2 for exactly 24 h. Collect the culture medium and store at -20 &#730;C. Use this sample to determine the level of APOB after treatment with compounds.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Determine APOB levels by ELISA
	<ol>
		<li>Determine Apolipoprotein B (APOB) concentration from both the pre- and post-drug treated samples using a human APOB ELISA development kit following the manufacturer&#39;s protocols.</li>
		<li>Preparing the following solutions
		<ol>
			<li>Wash buffer, composed of 0.05% Tween 20 in PBS, pH 7.4. Store at 4 &#730;C until use.</li>
			<li>Incubation buffer, composed of 0.05% Tween 20 and 0.1% bovine serum albumin (BSA) in PBS, pH 7.4. Store at 4 &#730;C until use.</li>
		</ol>
		</li>
		<li>Prepare APOB (mAB 20/17) antibody by diluting to 2 &#181;g/mL in PBS, pH 7.4. Add 100 &#956;L of the diluted antibody per well and incubate at 4 &#730;C overnight.</li>
		<li>Remove the antibody from the assay plates and wash each well twice with 200 &#956;L of PBS, pH 7.4.</li>
		<li>Add 200 &#956;L/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.</li>
		<li>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.</li>
		<li>Dilute the test samples 1:4 with 0.05% Tween 20 and 0.1% BSA in PBS, pH 7.4.</li>
		<li>After 1 h of blocking with incubation buffer, discard the buffer and wash the assay plates five times with 200 &#956;L of 0.05% Tween 20 in PBS, pH 7.4.</li>
		<li>Completely remove any residual washing buffer and transfer 90 &#956;L of the diluted samples and the APOB standards to the assay plates. Incubate at room temperature on a shaker for 1 h.</li>
		<li>Discard the sample and wash with 200 &#956;L 0.05% Tween 20 in PBS, pH 7.4 per well. Repeat the washes a total of 5 times. Add 100 &#956;L 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.</li>
		<li>Discard the reagent and wash with 200 &#956;L per well washing buffer 5 times. Add 100 &#956;L 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.</li>
		<li>Discard the reagent and wash with 200 &#956;L 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 &#956;L per well of Tetramethylbenzidin (TMB) substrate solution, and incubate at room temperature for 8 min.</li>
		<li>Without removing the TMB substrate, directly add 100 &#956;L per well of stop solution (1N HCl).</li>
		<li>Determine the absorbance at 450 nm using a plate reader.</li>
		<li>A standard curve can be established, using the Four Parameter Logistic Regression method, then used to define the concentration of APOB in each sample10.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+drug+screening+and+gene+correction+for+treating+liver+disease+using+patient-specific+stem+cells.">Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells.</a> Hepatology. 57 (6), 2458-2468 (2013).</li><li>Tafaleng, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+model+personalized+variations+in+liver+disease+resulting+from+alpha1-antitrypsin+deficiency.">Induced pluripotent stem cells model personalized variations in liver disease resulting from alpha1-antitrypsin deficiency.</a> Hepatology. 62 (1), 147-157 (2015).</li><li>Jing, R., Duncan, C. B., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+screen+reveals+that+HSP90beta+promotes+the+conversion+of+induced+pluripotent+stem+cell-derived+endoderm+to+a+hepatic+fate+and+regulates+HNF4A+turnover.">A small-molecule screen reveals that HSP90beta promotes the conversion of induced pluripotent stem cell-derived endoderm to a hepatic fate and regulates HNF4A turnover.</a> Development. 144 (10), 1764-1774 (2017).</li></ol>

The authors declare that they have no competing financial interests.

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...

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.

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

using human induced pluripotent stem cell-derived hepatocyte-like cells for drug discovery

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.

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 ...

Watch this Scientific Journal Video about Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery at JoVE.com

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

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.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

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JoVE Journal

Developmental Biology

10.2K Views.  Medical University of South Carolina. 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.

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

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

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

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.

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

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Biology

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

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.

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

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

Immunology and Infection

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

A Physiologically-Relevant All-Human 2D+ Hepatic System for Drug Discovery

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

Author Spotlight: Developing a Simple and Robust Hepatic Model for Pharmacological and Toxicological Applications 

Recent technical advances enabled the scaled production of an in vitro platform for drug metabolism and toxicity applications. An all-human 2D+ hepatic system (TV2D+) provides physiologically relevant results using traditional two-dimensional culture methods. This protocol will support end-users in the setup, maintenance, and application of the system.

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...