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 ...

The overall goal of this experiment is to differentiate human induced pluripotent stem cells into hepatocytes-like cells in a manner that is suitable for high-throughput screening. Liver cells are difficult to grow in the laboratory, however they can be generated from induced pluripotent stem cells which provides a screening platform to identify treatments for liver disease. The main advantage of this technique is that unlimited numbers of hepatocytes can be generated from any patient with liver disease that recapitulate the disease in the laboratory.The procedure is relatively easy to establish, even without extensive knowledge of stem cell biology. Its reproducibility allows researchers to screen thousands of compounds without a need for complex robotics. Ray Liu, a postdoctoral fellow, and Paige Lamprecht, a graduate student, will be demonstrating the procedure today.First, dilute the fusion protein Ecad to 15 micrograms per milliliter with Dulbecco's phosphate-buffered saline, or DPBS, that contains calcium and magnesium. Coat 100 millimeter suspension tissue culture dishes with five milliliters of diluted matrix and incubate at 37 degrees Celsius for at least one hour. Following incubation, remove the substrate, and replace with five milliliters of iPSC culture medium.Retrieve a vial of cryopreserved human induced pluripotent stem cells from liquid nitrogen. Thaw at 37 degrees Celsius until a small ice crystal remains. Then, gently pipette the cells into a sterile 15 milliliter conical tube containing four milliliters of the culture medium.Centrifuge the cells at 300 G for five minutes. Remove the supernatant and gently re-suspend the cells with five milliliters of medium supplemented with ten micromolar of Y-27632, a selective inhibitor of a Rho-associated, coiled-coil-containing protein kinase. Now, remove the culture medium from the Ecad-coated ten millimeter suspension tissue culture dishes and add five milliliters of the cells.Once the iPSCs reach around 80%confluency, remove the 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 dishes. Then, incubate the cells for up to three minutes at room temperature.As soon as the cells begin to release, remove the 0.02%EDTA solution and flood the dish with ten milliliters of M medium to release the cells. Aid the detachment of iPSCs by gentle pipetting. Transfer 1/10th of the suspended cells per fresh Ecad-coated tissue culture dish containing five milliliters of M medium.Incubate the cultures at 37 degrees Celsius under 4%oxygen and 5%carbon dioxide, changing the medium daily. Retrieve one vial of 250 microliters of ice-cold matrix stock with concentration of two milligrams per milliliter and thaw it on ice. Next, combine the thawed matrix with ten milliliters of cold DMEM/F12 by mixing gently using a pre-chilled pipette to make a final concentration of 0.05 milligrams per milliliter.Transfer 100 microliters of the diluted matrix to each well of a 96-well tissue culture plate. Incubate the plate at 37 degrees Celsius under 4%oxygen and 5%carbon dioxide for at least one hour. Following incubation, discard the matrix and replace with 50 microliters of M medium per well, repeating the same coating procedure with the rest of the plates.Now, culture the iPSCs on 100 millimeter dishes until the cells approach 80%confluency. To harvest the iPSCs from each 100 millimeter plate, aspirate the culture medium and rinse with five milliliters of DPBS. Replace the rinse with three milliliters of cell detachment solution, such as Accutase.Then, incubate for around two minutes at 37 degrees Celsius under 4%oxygen and 5%carbon dioxide. As soon as the cells begin to detach, harvest by adding seven milliliters of M medium. Pipette several times to dissociate the cell colonies into small clusters of around three to six cells.Next, collect the iPSCs into a 15 milliliter sterile centrifuge tube and centrifuge at 300 G for five minutes. When finished, remove the supernatant and re-suspend the cell pellet in five milliliters of M medium. Evenly distribute the cells onto 96-well matrix-coated plates by pipetting 50 microliters of suspension cells into each well using a multi-channel pipette.Then, culture the cells overnight at 37 degrees Celsius under 4%oxygen and 5%carbon dioxide and repeat the cell seeding procedures with the rest of the plates. Examine the 96-well plates by microscopy to insure that cells cover at least 70%of the surface of each individual well. For day one to day two of differentiation, replace the culture medium with 100 microliters of RPMI-based differentiation medium per well.Then, incubate the cells at 37 degrees Celsius in ambient oxygen and 5%carbon dioxide for two days with a daily medium change. For day three to day five of differentiation, replace the culture medium with 100 microliters of RPMI-based differentiation medium supplemented with 2%B27 and 100 nanograms per milliliter of active NA.Culture the cells at 37 degrees Celsius in ambient oxygen and 5%carbon dioxide for three days with a daily medium change. At differentiation day five, treat each well with 100 microliters of 0.02%EDTA solution for 30 seconds and remove the solution immediately.Add 100 microliters of fresh RPMI-based differentiation medium and repeat the same procedure for the rest of the plates. For differentiation between days six to ten, replace the culture medium with 100 microliters of RPMI-based differentiation medium. Incubate the cells at 37 degrees Celsius under 4%oxygen and 5%carbon dioxide for five days with a daily medium change.For differentiation between days 11 to 15, replace the culture medium with 100 microliters of RPMI-based differentiation medium, supplemented with 2%B27 and 20 nanograms per milliliter of HGF. Incubate the cells at 37 degrees Celsius under 4%oxygen and 5%carbon dioxide for five days with a daily medium change. For differentiation between days 16 to 20, replace the culture medium with a hepatocyte cell culture medium supplemented with 20 nanograms per milliliter of oncostatin-M.Incubate the cells at 37 degrees Celsius in ambient oxygen and 5%carbon dioxide for five days with a daily medium change. At the completion of differentiation on day 20, replace the culture medium with 100 microliters of fresh hepatocyte cell culture medium supplemented with 20 nanograms per milliliter of oncostatin-M. After incubating for 24 hours, retrieve the culture medium into a fresh 96-well plate and store at 20 degrees Celsius.Repeat the same collection procedure for the rest of the plates. Add 90 microliters of fresh hepatocyte cell culture medium supplemented with oncostatin-M into each well of the 96-well plate. Add ten microliters of each chosen compound from a previously prepared working stock solution.Add DMSO to 0.2%to the control wells to match the final concentration in the treated samples. After incubating for 24 hours, collect the culture medium into a fresh 96-well plate and store at 20 degrees Celsius. Repeat the same collection procedure for the rest of the plates.After five days of treatment, the cells express proteins that are characteristically expressed in the Definitive Endoderm. After five more days of differentiation, the Endoderm converts to a hepatic fate, and the cells express FOXA2 and HNF 4a. After HGF addition, the cells express proteins found in fetal hepatocytes, and at completion of differentiation, the cells adopt a cuboidal morphology with a nucleus containing prominent nucleoli and a large cytoplasm with multiple lipid droplets.Immunostaining to detect HNF 4a and albumin at day 20 of differentiation shows that the distribution of these hepatocyte proteins is similar across all wells. iPSCs were used to screen for APOB-lowering compounds to provide an example of using iPSC-derived hepatocytes for small molecule screening. The z-factor of the APOB assay is 0.73, which confirms the suitability of using the platform for drug discovery.The post-drug, pre-drug ratio of APOB was determined for each compound. An identification of hits was achieved by z-score analysis. 24 potential hits with an APOB ratio from 0.86 to 0.44 were identified.After excluding compounds that had a negative impact on cell viability, two compounds were found to be reproducible for lowering APOB. Once mastered, this technique can be done in 25 days if it is performed properly. While attempting this procedure, it is important to remember to prepare fresh medium daily.Incorrect concentration of components due to degradation will change the ability to cells to differentiate. After its development, this technique paved the way for researchers in the field of drug discovery to explore therapeutic treatment for liver disease using human induced pluripotent stem cells. After watching this video, you should have a good understanding of how to culture and maintain human induced pluripotent stem cells and to perform hepatic differentiation in a small surface pressed form for high-throughput screening.Don't forget that working with human induced pluripotent stem cells can be extremely hazardous and the personal protective equipment, such as gloves and a lab coat, should always be worn while performing this procedure.

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