Name: using human induced pluripotent stem cell-derived hepatocyte-like cells for drug discovery
Uploaded: 2018-05-19T12:30:00
Description: Watch this Scientific Journal Video about Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery at JoVE.com

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 me...

<ol>
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A., 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=A+Drug+Screen+using+Human+iPSC-Derived+Hepatocyte-like+Cells+Reveals+Cardiac+Glycosides+as+a+Potential+Treatment+for+Hypercholesterolemia.">A Drug Screen using Human iPSC-Derived Hepatocyte-like Cells Reveals Cardiac Glycosides as a Potential Treatment for Hypercholesterolemia.</a> Cell Stem Cell. 20 (4), 478-489 (2017).</li><li>Nagaoka, 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=E-cadherin-coated+plates+maintain+pluripotent+ES+cells+without+colony+formation.">E-cadherin-coated plates maintain pluripotent ES cells without colony formation.</a> PLoS One. 1, e15 (2006).</li><li>Ludwig, T. 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T., 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=Modeling+inherited+metabolic+disorders+of+the+liver+using+human+induced+pluripotent+stem+cells.">Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells.</a> J Clin Invest. 120 (9), 3127-3136 (2010).</li><li>Lu, W. Y., 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=Hepatic+progenitor+cells+of+biliary+origin+with+liver+repopulation+capacity.">Hepatic progenitor cells of biliary origin with liver repopulation capacity.</a> Nat Cell Biol. 17 (8), 971-983 (2015).</li><li>Ogawa, 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=Directed+differentiation+of+cholangiocytes+from+human+pluripotent+stem+cells.">Directed differentiation of cholangiocytes from human pluripotent stem cells.</a> Nat Biotechnol. 33 (8), 853-861 (2015).</li><li>Sampaziotis, F., 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=Cholangiocytes+derived+from+human+induced+pluripotent+stem+cells+for+disease+modeling+and+drug+validation.">Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.</a> Nat Biotechnol. 33 (8), 845-852 (2015).</li><li>Mallanna, S. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+hepatocytes+from+pluripotent+stem+cells.">Differentiation of hepatocytes from pluripotent stem cells.</a> Curr Protoc Stem Cell Biol. 26 (Unit 1G 4), (2013).</li><li>Song, Z., 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+generation+of+hepatocyte-like+cells+from+human+induced+pluripotent+stem+cells.">Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.</a> Cell Res. 19 (11), 1233-1242 (2009).</li><li>Cai, J., 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=Directed+differentiation+of+human+embryonic+stem+cells+into+functional+hepatic+cells.">Directed differentiation of human embryonic stem cells into functional hepatic cells.</a> Hepatology. 45 (5), 1229-1239 (2007).</li><li>Si-Tayeb, K., 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=Highly+efficient+generation+of+human+hepatocyte-like+cells+from+induced+pluripotent+stem+cells.">Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.</a> Hepatology. 51 (1), 297-305 (2010).</li><li>Pashos, E. 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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. 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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 border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:588px;">100 mm x 20 mm sterile tissue culture dishes&#160;</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">430167</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm x 20 mm sterile suspension culture dishes</td>
			<td>Corning</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-wells tissue culture plate&#160;</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human Albumin</td>
			<td>Dako</td>
			<td>A 0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human FOXA2(6C12)</td>
			<td>Novus Biological</td>
			<td>H00003170-M12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human HNF4 alpha</td>
			<td>Santa Cruz</td>
			<td>SC-6556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human Oct-3/4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human SOX17</td>
			<td>R&#38;D</td>
			<td>AF1924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human TRA-1-60 FITC conjugated</td>
			<td>Millipore</td>
			<td>FCMAB115F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Activin A Recombinant Human Protein&#160;</td>
			<td>Invitrogen</td>
			<td>PHC9563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement, minus insulin&#160;</td>
			<td>Invitrogen</td>
			<td>0050129SA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement, serum free&#160;</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BMP4 Recombinant Human Protein&#160;</td>
			<td>Invitrogen</td>
			<td>PHC9533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Dissociation Reagent StemPro&#160; Accutase&#160;</td>
			<td>Invitrogen</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTiter-Glo Luminescent Cell Viability Assay&#160;</td>
			<td>Promega</td>
			<td>7572</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS+(calcium, magnesium)</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS-(no calcium, no magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12, HEPES&#160;</td>
			<td>Invitrogen</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ELISA human APOB ELISA development kit</td>
			<td>Mabtech</td>
			<td>3715-1H-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor 2 (FGF2)</td>
			<td>Invitrogen</td>
			<td>PHG0023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepatocyte Culture Medium (HCM Bullet Kit)&#160;</td>
			<td>Lonza</td>
			<td>CC-3198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepatocyte Growth Factor&#160; (HGF)</td>
			<td>Invitrogen</td>
			<td>PHC0321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine&#160;</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Invitrogen</td>
			<td>11140076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oncostatin M (OSM) Recombinant Human Protein&#160;</td>
			<td>Invitrogen</td>
			<td>PHC5015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin&#160;</td>
			<td>Invitrogen</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Feeder free pluripotent stem cell medium: mTesR1&#160;</td>
			<td>STEMCELL technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reduced Growth Factor Basement Membrane Matrix&#160;</td>
			<td>Invitrogen</td>
			<td>A1413301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium, HEPES&#160;</td>
			<td>Invitrogen</td>
			<td>22400105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemAdhere Defined Matrix for hPSC (E-cad-Fc)</td>
			<td>Primorigen Biosciences</td>
			<td>S2071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMB-ELISA Substrate Solution</td>
			<td>Thermo Scientific&#160;</td>
			<td>34022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TRA-1-60 FITC conjugated</td>
			<td>Millipore</td>
			<td>FCMAB115F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versene (EDTA) 0.02%&#160;</td>
			<td>Lonza</td>
			<td>17-711E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">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 ...

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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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Developmental Biology

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