Name: using human induced pluripotent stem cell-derived hepatocyte-like cells for drug discovery
Uploaded: 2018-05-19T12:30:00
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Dette arbejde blev støttet af National Institutes of Health (DK55743, DK087377, DK102716 og HG006398 til sad). Vi vil gerne takke Dr. Behshad Pournasr, Dr. James Heslop og Ran Jing for deres bidrag.

1. kultur af Human induceret pluripotente stamceller
<ol><li>
Belægning rekombinant humant E-Cadherin Fc Fusion Protein (E-cad-Fc) eller andre matrixer, der er egnet til hPSC kultur 6<ol>
<li>Fortyndes E-cad-Fc til 15 μg/mL med Dulbecco's Phosphate-Buffered saltvand indeholder calcium og magnesium (DPBS (+)).</li>
		<li>Pels 100-mm suspension vævskultur retter med 5 mL fortyndet E-cad-Fc og inkuberes ved 37 ˚C for mindst 1 h. Fjern substrat og erstatte med 5 mL af medium (<e...

<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. E., 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=Feeder-independent+culture+of+human+embryonic+stem+cells.">Feeder-independent culture of human embryonic stem cells.</a> Nat Methods. 3 (8), 637-646 (2006).</li><li>International Stem Cell Initiative. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+embryonic+stem+cell+lines+by+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by International Stem Cell Initiative.</a> Nat Biotechnol. 25 (7), 803-816 (2007).</li><li>Cayo, M. 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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=Roles+of+integrins+in+human+induced+pluripotent+stem+cell+growth+on+Matrigel+and+vitronectin.">Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin.</a> Stem Cells Dev. 19 (8), 1231-1240 (2010).</li><li>Nagaoka, M., Si-Tayeb, K., Akaike, T., 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=Culture+of+human+pluripotent+stem+cells+using+completely+defined+conditions+on+a+recombinant+E-cadherin+substratum.">Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum.</a> BMC Dev Biol. 10, 60 (2010).</li><li>Hay, D. C., 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+differentiation+of+hepatocytes+from+human+embryonic+stem+cells+exhibiting+markers+recapitulating+liver+development+in+vivo.">Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.</a> Stem Cells. 26 (4), 894-902 (2008).</li><li>Mallanna, S. K., Cayo, M. A., Twaroski, K., Gundry, R. L., 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=Mapping+the+Cell-Surface+N-Glycoproteome+of+Human+Hepatocytes+Reveals+Markers+for+Selecting+a+Homogeneous+Population+of+iPSC-Derived+Hepatocytes.">Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.</a> Stem Cell Reports. 7 (3), 543-556 (2016).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4 (2), 67-73 (1999).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+score,+SSMD*,+z*+score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z score, SSMD*, z* score, and t statistic for hit selection in RNAi high-throughput screens.</a> J Biomol Screen. 16 (7), 775-785 (2011).</li><li>Lorenz, C., 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=Human+iPSC-Derived+Neural+Progenitors+Are+an+Effective+Drug+Discovery+Model+for+Neurological+mtDNA+Disorders.">Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.</a> Cell Stem Cell. 20 (5), 659-674 (2017).</li><li>Rashid, S. 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. K., 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=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. E., 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=Diverse+Population+Cohorts+of+hiPSCs+and+Derived+Hepatocyte-like+Cells+Reveal+Functional+Genetic+Variation+at+Blood+Lipid-Associated+Loci.">Diverse Population Cohorts of hiPSCs and Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid-Associated Loci.</a> Cell Stem Cell. 20 (4), 558-570 (2017).</li><li>Davidson, M. D., Ware, B. R., Khetani, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+liver+cells+for+drug+testing+and+disease+modeling.">Stem cell-derived liver cells for drug testing and disease modeling.</a> Discov Med. 19 (106), 349-358 (2015).</li><li>Choi, S. 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Target baseret drug discovery, hvor små molekyler identificeres der påvirker aktiviteten af et bestemt protein, har været genstand for mange eksisterende screening indsats. Selv om denne tilgang har givet talrige lægemidler, skærme baseret på at vende en fænotype, klassisk farmakologi, har haft større succes med at identificere i førsteklasses forbindelser, der er blevet klinisk virkningsfuldt1. En ulempe for fænotypisk drug discovery er, at det er afhængig af tilgængeligheden af passe...

Succes i at identificere stoffer, der kan bruges til at målrette en sjælden sygdom er afhængig af udviklingen af assays, der kan bruges til screening. Hypotese eller target-baserede skærme (reverse farmakologi) er nyttige, men kræver en detaljeret forståelse af det molekylære grundlag af sygdommen. Fænotypisk skærme (klassisk farmakologi) undgå behovet for en detaljeret forståelse af biokemiske veje, men i stedet stole på udviklingen af modeller, der præcist afspejler Patofysiologi af sygdommen. Trods begejstringen for mål-baserede tilgange afsløre analyser af FDA godkendt i førsteklasses narkotika at fænotypiske skærme har været langt mere vellykket1. Det overordnede mål med denne metode er at etablere en platform for high throughput screening, kan bruges til at identificere små molekyler til behandling af metaboliske leversygdom. Flere in vitro- modeller har været beskrevet herunder primære hepatocytter, hepatoma celler og lever stamfader celler2. Men de fleste af disse modeller har begrænsninger, og der er behov for nye modeller, der kan præcist sammenfatte Patofysiologi af metaboliske leversygdom mangler i kultur. For nylig, humane pluripotente stamceller kombineret med gen redigering har tilbudt mulighed for at modellere selv de sjældneste af sjældne sygdomme i kultur uden savn hen til adgang patienter direkte3. Mens brugen af patient-specifikke iPSCs som et redskab til at opdage små molekyler til behandling af sjældne leversygdomme er begrebsmæssigt rimelig, er der kun få rapporter påviser gennemførligheden af denne metode4. Dog har vi for nylig etableret en platform, der bruges iPSC-afledte hepatocytter til at kunne identificere lægemidler, der kan repurposed til behandling af mangler i lever metabolisme5.
Denne protokol forklarer processen med at skelne menneskelige iPSCs til hepatocyt-lignende celler i 96-brønd plader og bruge dem til at screene et bibliotek af små molekyler. Det beskriver også slutpunkt analyse ved hjælp af hyperkolesterolæmi som et eksempel på metabolisk leversygdom. Denne tilgang bør være nyttige at studere den rolle og anvendelsen af små molekyler i forbindelse med smitsomme sygdomme i leveren, metabolisk leversygdom, lægemiddeltoksicitet og andre leversygdomme.

<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

Evnen til at skelne menneskelig inducerede pluripotente stamceller (iPSCs) i hepatocyt-lignende celler (HLCs) giver nye muligheder for at studere medfødte fejl i hepatisk metabolisme. Men for at skabe en platform, der understøtter identifikation af små molekyler, der potentielt kan bruges til at behandle sygdomme i leveren, proceduren kræver en kultur-format, der er kompatibel med screening tusindvis af forbindelser. Her, beskriver vi en protokol bruger helt defineret kultur betingelser, som tillader den reproducerbare differentiering af menneskelige iPSCs til hepatocyt-lignende celler i 96-brønd vævskultur plader. Vi leverer også et eksempel på brugen af platform skærmen forbindelser for deres evne til at sænke Apolipoprotein B (APOB) produceret fra iPSC-afledte hepatocytter genereret fra en familiær hyperkolesterolæmi patient. Tilgængeligheden af en platform, der er kompatibel med drug discovery bør tillade forskere til at identificere nye lægemidler til sygdomme, der påvirker leveren.

Generation af hepatocyt - indkvartering celler: Figur 1 beskriver tidsrammen for de ændringer, der opstår under differentiering af menneskelige iPSCs til hepatocyt-lignende celler. Kulturen af iPSCs på E-Cad-Fc giver ca. 2 mm diameter kolonier, der udtrykker pluripotente markør OCT4 (figur 1A-B). Morfologi af celler dyrkes på en E-cadherin matrix vil være l...

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

Ved hjælp af menneskelige induceret pluripotente stamceller-afledt hepatocyt-lignende celler for Drug Discovery

Protokollen præsenteres her beskriver en platform for at identificere små molekyler for behandlingen af leversygdom. En trinvis beskrivelse præsenteres beskriver hvordan at differentiere iPSCs i celler med hepatocyt egenskaber i 96-brønd plader og bruger cellerne til at screene for små molekyler med potentielle terapeutiske aktivitet.

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.

Research

JoVE Journal

Developmental Biology

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Generation af Integration-fri Menneskelige induceret pluripotente stamceller Brug Hair-afledte Keratinocytter

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Generation af Integration-fri induceret pluripotente stamceller fra humane perifere mononucleære blodceller Brug episomale vektorer

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Pluripotente Stem Cell Afledt hjerteceller til Myokardie Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

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

Defineret og skalerbar Generation af hepatocyt-lignende celler fra menneskelige pluripotente stamceller

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

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Integrationsfri afledning af humane inducerede pluripotente stamceller ved anvendelse af Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

En familiær hyperkolesterolæmi menneskelige lever kimære musen Model ved hjælp af induceret pluripotente stamceller-afledt hepatocytter

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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

Semi-automatiseret produktion af hepatocyt som celler fra pluripotente stamceller

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

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

Mikrofluid analyse til vurdering af leukocyt adhæsion til menneskelige induceret pluripotente stamceller-afledt endotelceller (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Generation af 3D hud Organoid fra ledningen blod-afledte induceret pluripotente stamceller

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...