Name: an efficient method for directed hepatocyte-like cell induction from human embryonic stem cells
Uploaded: 2021-05-06T15:00:00
Description: Watch this Scientific Journal Video about An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells at JoVE.com

Dette arbejde blev støttet af National Natural Science Foundation of China (Nr. 81870432 og 81570567 til X.L.Z.), (Nr. 81571994 til P.N.S.); Natural Science Foundation of Guangdong-provinsen, Kina (nr. 2020A1515010054 til P.N.S.), Li Ka Shing Shantou University Foundation (nr. L1111 2008 til P.N.S.). Vi vil gerne takke professor Stanley Lin fra Shantou University Medical College for nyttige råd.

1. Vedligeholdelse af stamceller
BEMÆRK: Den cellevedligeholdelsesprotokol, der er beskrevet nedenfor, gælder for hES03-cellelinjen, der vedligeholdes i et klæbende monolag. For alle følgende protokoller i dette manuskript skal celler håndteres under et biologisk sikkerhedsskab.
<ol><li>Forbered 1x mTesR stamcellekultur medium ved at fortynde 5x supplerende medium til mTesR grundlæggende medium.</li>
	<li>Forbered 30x hESC-kvalificeret Matrigel medium ved at fortynde 5 mL hESC-kvalific...

<ol>
	<li>Hou, Y., Hu, S., Li, X., He, W., Wu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+Acid+Metabolism+in+the+Liver:+Nutritional+and+Physiological+Significance.">Amino Acid Metabolism in the Liver: Nutritional and Physiological Significance.</a> Advances in Experimental Medicine and Biology. 1265, 21-37 (2020).</li><li>Huang, C., Li, Q., Xu, W., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+of+liver+dysfunction+in+COVID-19.">Molecular and cellular mechanisms of liver dysfunction in COVID-19.</a> Discovery Medicine. 30, 107-112 (2020).</li><li>Todorovic Vukotic, N., Dordevic, J., Pejic, S., Dordevic, N., Pajovic, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antidepressants-+and+antipsychotics-induced+hepatotoxicity.">Antidepressants- and antipsychotics-induced hepatotoxicity.</a> Archives of Toxicology. , (2021).</li><li>Thomson, J. 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Li, 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=Generation+of+qualified+clinical-grade+functional+hepatocytes+from+human+embryonic+stem+cells+in+chemically+defined+conditions.">Generation of qualified clinical-grade functional hepatocytes from human embryonic stem cells in chemically defined conditions.</a> Cell Death &amp; Disease. 10, 763 (2019).</li><li>Rassouli, H., 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=Gene+Expression+Patterns+of+Royan+Human+Embryonic+Stem+Cells+Correlate+with+Their+Propensity+and+Culture+Systems.">Gene Expression Patterns of Royan Human Embryonic Stem Cells Correlate with Their Propensity and Culture Systems.</a> Cell Journal. 21, 290-299 (2019).</li><li>Loh, K. 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+endoderm+induction+from+human+pluripotent+stem+cells+by+logically+directing+signals+controlling+lineage+bifurcations.">Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations.</a> Cell Stem Cell. 14, 237-252 (2014).</li><li>Mukherjee, S., 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=Sox17+and+beta-catenin+co-occupy+Wnt-responsive+enhancers+to+govern+the+endoderm+gene+regulatory+network.">Sox17 and beta-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network.</a> Elife. 9, (2020).</li><li>Ang, L. 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=A+Roadmap+for+Human+Liver+Differentiation+from+Pluripotent+Stem+Cells.">A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells.</a> Cell Reports. 22, 2190-2205 (2018).</li><li>Carpentier, 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=Engrafted+human+stem+cell-derived+hepatocytes+establish+an+infectious+HCV+murine+model.">Engrafted human stem cell-derived hepatocytes establish an infectious HCV murine model.</a> Journal of Clinical Investigation. 124, 4953-4964 (2014).</li><li>Gomez, G. 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=Human+neural+crest+induction+by+temporal+modulation+of+WNT+activation.">Human neural crest induction by temporal modulation of WNT activation.</a> Developmental Biology. 449, 99-106 (2019).</li><li>Lee, J., Choi, S. H., Lee, D. R., Kim, D. S., Kim, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Isthmic+Organizer-Like+Cells+from+Human+Embryonic+Stem+Cells.">Generation of Isthmic Organizer-Like Cells from Human Embryonic Stem Cells.</a> Molecules and Cells. 41, 110-118 (2018).</li><li>Matsuno, 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=Redefining+definitive+endoderm+subtypes+by+robust+induction+of+human+induced+pluripotent+stem+cells.">Redefining definitive endoderm subtypes by robust induction of human induced pluripotent stem cells.</a> Differentiation. 92, 281-290 (2016).</li><li>Mathapati, S., 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=Small-Molecule-Directed+Hepatocyte-Like+Cell+Differentiation+of+Human+Pluripotent+Stem+Cells.">Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells.</a> Current Protocols in Stem Cell Biology. 38, 1-18 (2016).</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=Highly+efficient+differentiation+of+hESCs+to+functional+hepatic+endoderm+requires+ActivinA+and+Wnt3a+signaling.">Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 12301-12306 (2008).</li><li>Siller, R., Greenhough, S., Naumovska, E., Sullivan, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule-driven+hepatocyte+differentiation+of+human+pluripotent+stem+cells.">Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells.</a> Stem Cell Reports. 4, 939-952 (2015).</li><li>Yu, J. S., 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=PI3K/mTORC2+regulates+TGF-beta/Activin+signalling+by+modulating+Smad2/3+activity+via+linker+phosphorylation.">PI3K/mTORC2 regulates TGF-beta/Activin signalling by modulating Smad2/3 activity via linker phosphorylation.</a> Nature Communications. 6, 7212 (2015).</li><li>Borowiak, 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=Small+molecules+efficiently+direct+endodermal+differentiation+of+mouse+and+human+embryonic+stem+cells.">Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.</a> Cell Stem Cell. 4, 348-358 (2009).</li><li>Tahamtani, 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=Treatment+of+human+embryonic+stem+cells+with+different+combinations+of+priming+and+inducing+factors+toward+definitive+endoderm.">Treatment of human embryonic stem cells with different combinations of priming and inducing factors toward definitive endoderm.</a> Stem Cells and Development. 22, 1419-1432 (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 Research. 19, 1233-1242 (2009).</li><li>Sullivan, G. 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=Generation+of+functional+human+hepatic+endoderm+from+human+induced+pluripotent+stem+cells.">Generation of functional human hepatic endoderm from human induced pluripotent stem cells.</a> Hepatology. 51, 329-335 (2010).</li><li>Xia, 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=Human+stem+cell-derived+hepatocytes+as+a+model+for+hepatitis+B+virus+infection,+spreading+and+virus-host+interactions.">Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions.</a> Journal of Hepatology. 66, 494-503 (2017).</li><li>Li, S., 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=Derivation+and+applications+of+human+hepatocyte-like+cells.">Derivation and applications of human hepatocyte-like cells.</a> World Journal of Stem Cells. 11, 535-547 (2019).</li><li>Ogawa, S., 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=Three-dimensional+culture+and+cAMP+signaling+promote+the+maturation+of+human+pluripotent+stem+cell-derived+hepatocytes.">Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes.</a> Development. 140, 3285-3296 (2013).</li><li>Kim, D. 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=Prediction+of+drug-induced+immune-mediated+hepatotoxicity+using+hepatocyte-like+cells+derived+from+human+embryonic+stem+cells.">Prediction of drug-induced immune-mediated hepatotoxicity using hepatocyte-like cells derived from human embryonic stem cells.</a> Toxicology. 387, 1-9 (2017).</li></ol>

Her præsenterer vi en trinvis metode, der fremkalder HLC'er fra HESC'er i tre faser. I første fase blev Activin A og CHIR99021 brugt til at differentiere HESC'er til DE. I anden fase blev KO-DMEM og DMSO brugt til at differentiere DE til lever stamceller. I tredje fase blev HZM plus HGF, OSM og hydrocortison 21-hemisuccinat natriumsalt brugt til fortsat at differentiere lever stamceller til HLC'er.
Følgende kritiske trin skal tages i betragtning, når protokollen bruges. Den oprindelige dif...

Leveren er et meget metabolisk organ, der spiller flere roller, herunder deoxidation, opbevaring af glykogen og sekretion og syntese af proteiner1. Forskellige patogener, stoffer og arvelighed kan forårsage patologiske ændringer i leveren og påvirke dens funktioner2,3. Hepatocytter, som den vigtigste funktionelle enhed i leveren, spiller en vigtig rolle i kunstig leverstøtte systemer og narkotika toksicitet elimination. Men, ressourcen af primære menneskelige hepatocytter er begrænset i celle-baseret behandling, samt i leversygdom forskning. Derfor er udvikling af nye kilder til funktionelle menneskelige hepatocytter en vigtig forskningsretning inden for regenerativ medicin. Siden 1998, hvor hESC'er er blevet etableret4, hESC'er er blevet bredt overvejet af forskere på grund af deres overlegne differentieringspotentiale (de kan differentiere sig til forskellige væv i et passende miljø) og høj grad af selvfornyelse og dermed give ideelle kildeceller til bioartificial lever, hepatocyttransplantation og endda levervævsteknik5.
I øjeblikket kan den hepatisk differentieringseffektivitet øges betydeligt ved at berige endoderm6. I differentieringsdifferentiering af stamceller til endoderm er niveauer af den transformerende vækstfaktor β (TGF-β) signalering og WNT-signalveje de vigtigste faktorer i lyden i endodermdannelsesfasen. Aktivering af et højt niveau af TGF-β og WNT signalering kan fremme udviklingen af endoderm7,8. Activin A er en cytokin, der tilhører TGF-β superfamilien. Derfor anvendes Activin A i vid udstrækning til endoderminduktion af menneskeskabte pluripotente stamceller (hiPSC'er) og HESC'er9,10. GSK3 er en serine-threonine protein kinase. Forskere har konstateret, at CHIR99021, en specifik hæmmer af GSK3β, kan stimulere typiske WNT-signaler og kan fremme stamcelledifferentiering under visse betingelser, hvilket tyder på, at CHIR99021 har potentiale til at inducere stamcelledifferentiering i endoderm 11,12,13.
Her rapporterer vi om en effektiv og reproducerbar metode til effektivt at reducere differentieringen af HESC'er til funktionelle HLC'er. Den sekventielle tilføjelse af Activin A og CHIR99021 producerede omkring 89,7±0,8% SOX17 (DE-markør)-positive celler. Efter at være blevet yderligere modnet in vitro, udtrykte disse celler leverspecifikke markører og udøvede hepatocytlignende morfologi (baseret på hematoxylin-eosin farvning (H &E)) og funktioner, såsom optagelse af indocyaningrøn (ICG), glykogenlagring og CYP3-aktivitet. Resultaterne viser, at HESC'er med succes kan differentieres til modne funktionelle HLC'er ved denne metode og kan danne grundlag for leversygdomsrelateret forskning og in vitro-lægemiddelscreening.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>488 labeled goat against mouse IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZF-0512</td>
			<td>For IF,second antibody</td>
		</tr>
		<tr>
			<td>488 labeled goat against Rabbit IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZF-0516</td>
			<td>For IF,second antibody</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell Technologies</td>
			<td>7920</td>
			<td>For cell passage</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>peproTech</td>
			<td>120-14E</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>Anti - Albumin (ALB)</td>
			<td>Sigma-Aldrich</td>
			<td>A6684</td>
			<td>For IF and WB, primary antibody</td>
		</tr>
		<tr>
			<td>Anti - Human Oct4</td>
			<td>Abcam</td>
			<td>Ab19587</td>
			<td>For IF, primary antibody</td>
		</tr>
		<tr>
			<td>Anti - &#945;-Fetoprotein (AFP)</td>
			<td>Sigma-Aldrich</td>
			<td>A8452</td>
			<td>For IF and WB, primary antibody</td>
		</tr>
		<tr>
			<td>Anti -SOX17</td>
			<td>Abcam</td>
			<td>ab224637</td>
			<td>For IF, primary antibody</td>
		</tr>
		<tr>
			<td>Anti-Hepatocyte Nuclear Factor 4 alpha (HNF4&#945;)</td>
			<td>Sigma-Aldrich</td>
			<td>SAB1412164</td>
			<td>For IF, primary antibody</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beyotime</td>
			<td>ST023-200g</td>
			<td>For cell blocking</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Sigma-Aldrich</td>
			<td>SML1046</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>C1006</td>
			<td>For nuclear staining</td>
		</tr>
		<tr>
			<td>DEPC-water</td>
			<td>Beyotime</td>
			<td>R0021</td>
			<td>For RNA dissolution</td>
		</tr>
		<tr>
			<td>DM3189</td>
			<td>MCE</td>
			<td>HY-12071</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>H&#38;E staining kit</td>
			<td>Beyotime</td>
			<td>C0105S</td>
			<td>For H&#38;E staining</td>
		</tr>
		<tr>
			<td>Hepatocyte growth factor (HGF)</td>
			<td>peproTech</td>
			<td>100-39</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>HepatoZYME-SFM (HZM)</td>
			<td>Gibco</td>
			<td>17705-021</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>Hydrocortisone-21-hemisuccinate</td>
			<td>Sigma-Aldrich</td>
			<td>H4881</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>Indocyanine</td>
			<td>Sangon Biotech</td>
			<td>A606326</td>
			<td>For Indocyanine staining</td>
		</tr>
		<tr>
			<td>Knock Out DMEM</td>
			<td>Gibco</td>
			<td>10829-018</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>Knock Out SR Multi-Species</td>
			<td>Gibco</td>
			<td>A31815-02</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified</td>
			<td>Corning</td>
			<td>354277</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>MEM NeAA</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>For hepatic progenitor differentiation</td>
		</tr>
		<tr>
			<td>mTesR 5X Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>mTesR Basal Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Oncostatin (OSM)</td>
			<td>peproTech</td>
			<td>300-10</td>
			<td>For hepatocyte differentiation</td>
		</tr>
		<tr>
			<td>P450 - CYP3A4 (Luciferin - PFBE)</td>
			<td>Promega</td>
			<td>V8901</td>
			<td>For CYP450 activity</td>
		</tr>
		<tr>
			<td>PAS staining kit</td>
			<td>Solarbio</td>
			<td>G1281</td>
			<td>For PAS staining</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>For cell differentiation</td>
		</tr>
		<tr>
			<td>Peroxidase-Conjugated Goat anti-Mouse IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZB-2305</td>
			<td>For WB,second antibody</td>
		</tr>
		<tr>
			<td>Primary Antibody Dilution Buffer for&#160;Western Blot</td>
			<td>Beyotime</td>
			<td>P0256</td>
			<td>For primary antibody dilution</td>
		</tr>
		<tr>
			<td>ReverTraAce qPCR RT Kit</td>
			<td>TOYOBO</td>
			<td>FSQ-101</td>
			<td>For cDNA Synthesis</td>
		</tr>
		<tr>
			<td>RNAiso Plus</td>
			<td>TaKaRa</td>
			<td>9109</td>
			<td>For RNA Isolation</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td>For second antibody preparation</td>
		</tr>
		<tr>
			<td>SYBR Green Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25742</td>
			<td>For RT-PCR Analysis</td>
		</tr>
		<tr>
			<td>Torin2</td>
			<td>MCE</td>
			<td>HY-13002</td>
			<td>For definitive endoderm formation</td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>Sigma-Aldrich</td>
			<td>WXBB7485V</td>
			<td>For washing buffer preparation</td>
		</tr>
	</tbody>
</table>

an efficient method for directed hepatocyte-like cell induction from human embryonic stem cells

De potentielle funktioner i hepatocytlignende celler (HLC'er) fra menneskelige embryonale stamceller (HESC'er) rummer store løfter om sygdomsmodellering og lægemiddelscreeningsapplikationer. Forudsat her er en effektiv og reproducerbar metode til differentiering af HESCs i funktionelle HLCs. Etableringen af en endoderm afstamning er et vigtigt skridt i differentieringen til HLCs. Ved vores metode regulerer vi de vigtigste signalveje ved løbende at supplere Activin A og CHIR99021 under hESC-differentiering til endelig endoderm (DE), efterfulgt af generering af lever stamceller og endelig HLCs med typisk hepatocytmorfologi i en trinvis metode med helt definerede reagenser. De hESC-afledte NPC'er, der produceres ved denne metode, udtrykker scenespecifikke markører (herunder albumin, HNF4α nuclear receptor og natrium taurocholat cotransporterende polypeptid (NTCP)) og udviser særlige egenskaber i forbindelse med modne og funktionelle hepatocytter (herunder indokyaningrøn farvning, glykogenlagring, hæmatoboxyl-eosin farvning og CYP3-aktivitet) og kan udgøre en platform for udvikling af HLC-baserede anvendelser i undersøgelsen af leversygdomme.

Det skematiske diagram over HLC-induktion fra HESC'er og repræsentative lysfeltsbilleder af hvert differentieringsstadium er vist i figur 1. I fase I blev Activin A og CHIR99021 tilføjet i 3 dage for at få stamceller til at danne endodermceller. I fase II differentierede endodermcellerne sig til lever stamceller efter at være blevet behandlet med differentieringsmedium i 5 dage. I fase III var tidlige hepatocytter modnet og differentieret til HLC'er efter 10 dage i HGF og OSM (<strong cl...

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

Dette manuskript beskriver en detaljeret protokol for differentiering af humane embryonale stamceller (hESC'er) til funktionelle hepatocytlignende celler (HLC' er) ved løbende at supplere Activin A og CHIR99021 under hESC-differentiering til endelig endoderm (DE).

En effektiv metode til rettet Hepatocyte-Lignende Cell Induktion fra Human Embryonale stamceller

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

Humane embryonale stamcelle-afledte hepatocyt-lignende celler er nyttige i sygdom modellering og narkotika screening. Dette er en effektiv og reproducerbar metode til effektivt at fremkalde differentieringen af menneskelige embryonale stamceller i funktionelle hepatocytlignende celler. En udifferentieret status og passende sammenløb af menneskelige embryonale stamceller er afgørende for en vellykket differentiering til hepatocyt-lignende celler.Til vedligeholdelse af stamceller skal du belægge sterile seks-brønds vævskulturbehandlede plader med en milliliter 1X humant embryonalt stamcellekvalificeret Matrigel pr. brønd og opbevare dem ved fire grader Celsius natten over. Lad pladerne stå ved stuetemperatur i 30 minutter før brug. Tø kryo-bevarede HESEs i en 37 grader Celsius vandbad i tre minutter uden at ryste, derefter straks overføre stamceller ved pipetter dem i en 15-milliliter centrifuge rør indeholder fire milliliter forvarmet, 37 grader Celsius mTESR medium.Centrifuge HESEs og aspirere supernatant, derefter forsigtigt igen suspendere cellerne i en milliliter mTESR medium. Indsug DMEM F-12-mediet fra pladen. Frø cellerne i en seks-brønds plade ved en tæthed på 1 x 10 til 5. celler i to milliliter mTESR, og inkubat i en 5%kuldioxid inkubator.Vedligehold cellerne ved at erstatte mediet med forvarmet mTESR-medium dagligt. Passage cellerne på omkring 70 til 80% sammenløb, eller når cellen kolonier begynder at komme i kontakt. For celler passaging, aspirere mediet og inkubere cellerne med en milliliter per brønd af enzymopløsning i fem minutter ved 37 grader Celsius.Overfør cellerne ved at pipettere dem til et 15-milliliter centrifugerør, der indeholder fire milliliter DMEM F-12 forvarmet til 37 grader Celsius. Forbered 24-brønds plader ved at belægge dem med 250 mikroliter af 1X hESC-kvalificeret Matrigel medium. Frø hESC'er med en tæthed på 1 til 1,5 gange 10 til 5. celler pr. brønd i 500 mikroliter mTESR-medium.Der tilsættes både aktiv A til en endelig koncentration på 100 nanogram pr. milliliter og CHIR99021 til en endelig koncentration på tre mikromolar i et passende volumen af forvarmet fase 1 differentieringsmetopionens grundmedium. Efter tre dages differentiering bør differentierede celler udtrykke markører for endelige endodermceller, såsom FOXA2, SOX17, GATA4, CXCR4 og FOXA1. På dag tre af differentiering, aspirere mediet, erstatte det med fase to medium, og inkubere i 24 timer.Skift mediet hver anden dag på dag fire til otte. Efter otte dages differentiering bør differentierede celler udtrykke passende markører for lever stamceller, såsom HNF4 alpha, AFP, TBX3, TTR, ALB, NTCP, CEBPA. På dag otte, indsug fase to medium fra cellerne, og erstatte det med fase tre medium.Lad cellerne inkubere i 10 dage ved 37 grader Celsius i en kuldioxidinkubator, og skift medierne hver anden dag med frisktilsatte differentieringsfaktorer. Efter 18 dages differentiering bør differentierede celler udtrykke karakteristiske markører for hepatocytter, såsom AAT, ALB, TTR, HNF4 alpha, NTCP, ASGR1, CYP3A4. Det skematiske diagram over hepatocytlignende celler fra HESC'er og lyse feltbilleder af hvert differentieringsstadie vises.I første fase blev der tilføjet aktivin A og CHIR99021 i tre dage for at få stamceller til at danne endodermceller. I fase to differentierede endodermcellerne sig til lever stamceller efter at være blevet behandlet med et differentieringsmedium i fem dage. I tredje fase, efter 10 dage, havde tidlige hepatocytter modnet og differentieret i hepatocyt-lignende celler i hepatocyt vækstfaktor og oncostatin.I den sidste fase af differentiering viste celler en typisk hepatocyt fænotype. RT-PCR, immunfluorescens farvning, og vestlige blotting blev brugt til at opdage markører for endoderm celler, leverprækursor celler, og modne hepatocytter. De differentierede celler viste høje ekspressionsniveauer af differentieringsrelaterede gener og proteiner i hvert trin, såsom endodermmarkører SOX17 på dag tre, hepatisk prækursormarkør HNF4 alpha på dag otte, AFP på dag 14 og moden hepatocytmarkør ALB på dag 18.HLCs udstillet grøn farvning og omfattende cytoplasmisk syre skift farvning. Hepatocytterne viste typisk bi-nukleeret morfologi. CYP3A4's enzymatiske aktivitet, den essentielle metaboliske CYP i leveren i HLC'er, blev bekræftet med en enzymanalyse.Det er af afgørende betydning, at menneskelige embryonale stamceller placeres med en passende tæthed og begynder at differentiere på følgende dato. I første fase skal du røre ved medierne forsigtigt, fordi cellerne er tilbøjelige til at flyde op. Kombinationen af activin A og andre små molekyler kan yderligere forbedre differentieringseffektiviteten og producere mere modne hepatocytlignende celler i denne procedure.Denne teknik kan give en platform til at udforske hepatocyt transplantation, levervæv engineering, bioartificial lever, og sygdom modellering.

Research

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