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 arbeidet ble støttet av National Institutes of Health (DK55743, DK087377, DK102716 og HG006398 til S.A.D). Vi vil gjerne takke Dr. Behshad Pournasr, Dr. James Heslop og Ran Jing for deres bidrag.

1. kultur av menneskelige indusert Pluripotent stamceller
<ol><li>
Belegg rekombinant menneskelige E-Cadherin Fc Fusion Protein (E-cad-Fc) eller andre matriser egnet for hPSC kultur 6<ol>
<li>Fortynn E-cad-Fc til 15 μg/mL med Dulbecco's Phosphate-Buffered Saline inneholder kalsium og magnesium (DPBS (+)).</li>
		<li>Pels 100 mm suspensjon vev kultur retter med 5 mL utvannet E-cad-Fc og ruge på 37 grader for minst 1 h. Fjern substrat og erstatte med 5 mL av medium (f.eks </...

<ol>
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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=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. 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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. 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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Mål basert stoffet funnet, hvor små molekyler identifiseres som påvirker aktiviteten til et bestemt protein, har vært fokus for mange eksisterende screening innsats. Selv om denne tilnærmingen har gitt mange legemidler, skjermer basert på snu en fenotype, klassisk farmakologi, har vært mer vellykket i å identifisere første i klassen forbindelser som har vært klinisk effektiv1. En ulempe til fenotypiske medisiner er at den er avhengig av tilgjengelighet av riktig sykdom modeller. Dette ka...

Suksess i å identifisere legemidler som kan brukes til å målrette en sjelden sykdom er avhengig av utviklingen av analyser som kan brukes for screening. Hypotesen eller mål-baserte skjermer (omvendt farmakologi) er nyttig, men krever en detaljert forståelse av molekylære grunnlaget for sykdommen. Fenotypiske skjermer (klassisk farmakologi) unngå behovet for en grundig forståelse av biokjemiske, men i stedet stole på utvikling av modeller som nøyaktig speil i patofysiologien av sykdom. Til tross for entusiasme for mål-baserte tilnærminger avslører analyser av FDA godkjente første i klassen stoffer at fenotypiske skjermer er langt mer vellykket1. Det overordnede målet med denne metoden er å etablere en plattform for høy gjennomstrømning screening som kan brukes til å identifisere små molekyler for behandling av metabolske leversykdom. Flere i vitro modeller har blitt beskrevet som primære hepatocytter, hepatoma celler og leveren stamfar celler2. Men de fleste av disse modellene har begrensninger, og det er behov for nye modeller som kan nøyaktig recapitulate i Patofysiologien ved metabolske leveren mangler i kultur. Nylig menneskelige pluripotent stilk celler kombinert med gene redigering har tilbudt en mulighet å modellere selv de sjeldneste av sjeldne sykdommer i kultur uten å måtte tilgang pasienter direkte3. Mens bruk av pasient-spesifikke iPSCs som et verktøy for å oppdage små molekyler for behandling av sjeldne leversykdommer er konseptuelt rimelig, er det bare noen rapporter demonstrere muligheten for denne tilnærmingen4. Imidlertid har vi nylig etablert en plattform som brukes iPSC-avledet hepatocytter kunne identifisere legemidler som kan gjenbrukes i behandling av mangler i leveren metabolismen5.
Denne protokollen forklarer prosessen med å skille menneskelig iPSCs hepatocyte-lignende celler i 96-brønnen plater og bruke dem til skjermen et bibliotek av små molekyler. Den beskriver også endepunktet analysen med hyperkolesterolemi som eksempel på metabolske leversykdom. Denne tilnærmingen bør være nyttig å studere rolle og anvendelse av små molekyler i forbindelse med smittsomme leversykdom, metabolske leversykdom, drug giftighet og andre leversykdommer lidelser.

<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

Muligheten til å skille menneskelig indusert pluripotent stamceller (iPSCs) i hepatocyte-lignende celler (HLCs) gir nye muligheter til å studere medfødt feil i hepatic metabolisme. Men for å gi en plattform som støtter identifikasjon av små molekyler som kan brukes til å behandle leversykdom, krever prosedyren et kultur-format som er kompatibelt med screening tusenvis av forbindelser. Her beskriver vi en helt definert kultur vilkår, som tillater reproduserbar differensiering av menneskelig iPSCs hepatocyte-lignende celler i 96-brønnen vev kultur plater-protokoll. Vi tilbyr også et eksempel på bruk av plattformen til skjermen forbindelser for sin evne til å senke Apolipoprotein B (APOB) produsert fra iPSC-avledet hepatocytter generert fra en familiær hyperkolesterolemi pasient. Tilgjengeligheten av en plattform som er kompatibel med medisiner bør tillate forskere å identifisere romanen legemiddelselskap for sykdommer som påvirker leveren.

Generering av hepatocyte - liker celler: Figur 1 beskriver tidsrammen endringene som oppstår under differensiering av menneskelig iPSCs hepatocyte-lignende celler. Kulturen i iPSCs på E-Cad-Fc gir omlag 2 mm diameter kolonier som uttrykker pluripotent markøren OCT4 (figur 1A-B). Morfologi av celler dyrket på en matrise-E-cadherin vil være litt forskjellige f...

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

Bruker Human indusert Pluripotent Stamcelle-avledet Hepatocyte-lignende celler for medisiner

Protokollen presenteres her beskriver en plattform for å identifisere små molekyler for behandling av leversykdommer. En detaljert beskrivelse vises detaljering hvordan skille iPSCs i celler med hepatocyte egenskaper i 96-brønnen plater, og bruke cellene til skjermen for små molekyler med potensielle terapeutisk aktivitet.

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

Research

JoVE Journal

Developmental Biology

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

Generasjon Integrerings- fritt menneskeskapte Pluripotent stamceller Bruke Hair-avledet Keratinocytter

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

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Generering av Induced Pluripotent stamceller fra humane melanom Tumor-infiltrerende lymfocytter

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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

Generasjon Integrerings--frie Utførte Pluripotent stamceller fra menneskelige perifere mononukleære blodceller Bruke 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 stamcelle Avledet hjerteceller for Hjerteinfarkt 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

Definert og Scalable Generation of hepatocytter lignende celler fra menneskelige Pluripotent 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

Integreringsfri avledning av humane inducerte pluripotente stamceller ved bruk av 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

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent 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 hyperkolesterolemi menneskelige leveren Chimeric musemodell med indusert Pluripotent Stamcelle-avledet 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-automatisert produksjon av Hepatocyte som celler fra Pluripotent 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

Microfluidic analysen for vurdering av leukocytter vedheft til menneskelig indusert Pluripotent Stamcelle-avledet endotelceller (hiPSC-ECs)

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