This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.); the Natural Science Foundation of Guangdong Province, China (No. 2020A1515010054 to P.N.S.), The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.

1. Stem cell maintenance
NOTE: The cell maintenance protocol described below applies to the hES03 cell line maintained in an adherent monolayer. For all the following protocols in this manuscript, cells should be handled under a biological safety cabinet.
<ol>
	<li>Prepare 1x mTesR stem cell culture medium by diluting 5x supplementary medium to mTesR basic medium.</li>
	<li>Prepare 30x hESC-qualified Matrigel medium by diluting 5 mL of hESC-qualified Matrigel with 5 mL of DMEM/F12 on the ice. Store at -20 &#176;C.</li>
	<li>Prepare 1x hESC-qualified Matrigel medium by diluting 33.3 &#181;L of 30x hESC-qualified Matrigel with 1 mL of DMEM/F12.</li>
	<li>Coat sterile 6 well, tissue culture-treated plates with 1 mL of 1x hESC-qualified Matrigel in each well in advance and store at 4 &#176;C overnight. Leave at room temperature (RT) for at least 30 min before use.</li>
	<li>Thaw the cryopreserved hESCs in a 37 &#176;C water bath for 3 min without shaking. Then immediately transfer the cells by pipetting into a 15 mL centrifuge tube containing 4 mL prewarmed 37 &#176;C mTesR medium, pipet up and down gently 2 times.</li>
	<li>Centrifuge the hESCs at 200 x g for 2 min at RT.</li>
	<li>Aspirate the supernatant and gently resuspend the cells in 1 mL of mTesR medium.</li>
	<li>Aspirate the DMEM/F12 medium from the plate and seed the cells into a well of 6-well plate at a density of 1 x 105 cells in 2 mL of mTesR.</li>
	<li>Incubate in a 5% CO2 incubator and maintain the cells by replacing with preheated mTesR medium daily. Passage the cells at roughly 70-80% confluence or when the cell colonies begin to make contact.</li>
</ol>
2. Stem cell passage and differentiation
<ol>
	<li>For passaging cells, aspirate the medium and incubate the cells with 1 mL per well of enzyme solution (e.g., Accutase&#160;for 5 min at 37 &#176;C. Then transfer the cells by pipetting to a 15 mL centrifuge tube containing 4 mL of DMEM/F12 prewarmed to 37 &#176;C.</li>
	<li>Centrifuge the cells at 200 x g at RT for 2 min.</li>
	<li>Aspirate the medium and gently resuspend the cell pellet in 1 mL of mTesR medium.</li>
	<li>Prepare 24-well plates by coating with 250 &#181;L of 1x hESC-qualified Matrigel medium in advance. Seed hESCs as described above at a density of 1 - 1.5 x 105 cells per well in 500 &#181;L of mTesR medium.</li>
	<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2 incubator.</li>
</ol>
3. DE formation
<ol>
	<li>Prepare stock solutions of 100 &#181;g/mL Activin A with DPBS containing 0.2% BSA and 3 mM CHIR99021 with DMSO.</li>
	<li>Prepare Stage I differentiation basic medium by supplementing RPMI medium with 1x B27.</li>
	<li>Add Activin A to a final concentration of 100 ng/mL and CHIR99021 to a final concentration of 3 &#181;M in an appropriate volume of preheated Stage I differentiation basic medium.</li>
	<li>Aspirate mTesR medium from the cells and replace with Stage I differentiation media containing added differentiation factors (e.g., 0.5 mL per well of a 24-well plate).</li>
	<li>Allow cells to incubate for 3 days at 37 &#176;C in a CO2 incubator, replacing Stage I media daily with freshly added differentiation factors (Days 0-2). 
	&#8203;NOTE: After 3 days of differentiation, differentiated cells should express markers of DE cells, such as FOXA2, SOX17, GATA4, CRCR4, and FOXA1 (minimum 80% of SOX17 needed for proceeding).</li>
</ol>
4. Differentiation of hepatic progenitor cells
<ol>
	<li>Prepare Stage II differentiation basic media by dissolving knockout serum replacement (KOSR) to a final concentration of 20% in Knock Out DMEM.</li>
	<li>Prepare Stage II differentiation medium containing 1% DMSO, 1x GlutaMAX, 1x non-essential amino acid (NEAA) solution, 100 &#181;M 2-mercaptoethanol and 1x penicillin/streptomycin (P/S) to the appropriate volume of KOSR/knockout DMEM.</li>
	<li>On Day 3 of differentiation, aspirate the medium and replace with Stage II medium (e.g., 0.5 mL per well of a 24 well plate). Then incubate for 24 h.</li>
	<li>On Day 4 of differentiation, aspirate the medium and replace with Stage II medium (e.g., 1 mL per well of a 24 well plate).</li>
	<li>Change the medium every other day (replace medium on Day 6). 
	&#8203;NOTE: After 8 days of differentiation, differentiated cells should express appropriate markers of hepatic progenitor cells, such as HNF4&#945;, AFP, TBX3, TTR, ALB, NTCP, CEBPA (minimum 80% of HNF4&#945; needed for proceeding).</li>
</ol>
5. Hepatocyte differentiation
<ol>
	<li>Prepare 10 &#181;g/mL hepatocyte growth factor (HGF), 20 &#181;g/mL Oncostatin (OSM) stock solutions with DBPS (containing 0.2% BSA) and filter with a 0.22 &#181;m filter membrane.</li>
	<li>Prepare Stage III differentiation basic medium (HepatoZYME-SFM (HZM) medium supplemented with 10 &#181;M hydrocortisone-21-hemisuccinate and 1x P/S).</li>
	<li>Add HGF to a final concentration of 10 ng/mL and OSM to a final concentration of 20 ng/mL to the appropriate volume of preheated Stage III differentiation medium.</li>
	<li>On Day 8 of differentiation, aspirate Stage II medium from cells and replace with Stage III medium (e.g., 1 mL per well of a 24 well plate).</li>
	<li>Allow cells to incubate for 10 days at 37 &#176;C in a CO2 incubator, changing Stage III media every other day with freshly added differentiation factors (Days 8-18). 
	&#8203;NOTE: After 18 days of differentiation, differentiated cells should express characteristic markers of hepatocytes, such as AAT, ALB, TTR, HNF4&#945;, NTCP, ASGR1, CYP3A4. The percentage of Albumin-positive cells usually is more than 90%.</li>
</ol>
6. RNA isolation, cDNA synthesis and RT-qPCR analysis
<ol>
	<li>Aspirate medium from cells and add the proper amount of RNAiso Plus reagent, (e.g., 0.3 mL per well of a 24-well plate). Collect the supernatant in a 1.5 mL tube and let stand at RT for 5 min.</li>
	<li>Add 60 &#181;L of chloroform reagent and leave it at RT for 5 min. Then centrifuge at 12,000 x g for 15 min.</li>
	<li>Collect the 125 &#181;L supernatant and transfer it to another centrifuge tube. Add 160 &#181;L of isopropanol, mix well, and stand at RT for 10 min.</li>
	<li>Centrifuge at 12,000 x g for 10 min.</li>
	<li>Remove the supernatant, add 75% ethanol to the precipitated, and centrifuge at 7,500 x g for 5 min.</li>
	<li>After drying at RT, add 40 &#181;L of DEPC-treated water to dissolve. For qPCR, reverse transcribe 2 &#181;g of total RNA with a reverse transcription kit according to the manufacturer&#39;s protocol.</li>
	<li>Perform RT-qPCR using a commercial kit according to the manufacturer&#39;s protocol.</li>
	<li>Normalize gene expression relative to non-induced stem cells and determine fold variation following normalization to GAPDH.</li>
</ol>
7. Immunofluorescence Validation of Differentiation
<ol>
	<li>Prepare 1x PBST washing buffer by adding 0.1% Tween-20 to PBS.</li>
	<li>Aspirate medium from adherent cells and wash briefly with PBS at RT on a shaker.</li>
	<li>Aspirate PBS and incubate cells with 500 &#181;L of ice-cold methanol per well of a 24-well plate to fix the cells at -20 &#176;C overnight.</li>
	<li>Remove the methanol and wash the cells 3 times with PBS on a shaker for 10 min each time at RT.</li>
	<li>Block cells with 500 &#181;L of 5% BSA prepared in PBS for 1-2 h at RT on a shaker.</li>
	<li>Dilute primary antibody at 1: 200 in the same solution used for blocking.</li>
	<li>Incubate the fixed cells in 300 &#181;L primary antibody solution overnight at 4 &#176;C on a shaker.</li>
	<li>After overnight incubation, wash cells 3 times with PBST for 10 min each time at RT on a shaker.</li>
	<li>Prepare secondary antibody solution in blocking solution at 1:1000.</li>
	<li>Incubate in 300 &#181;L of secondary antibody solution protected from light for 1 h at RT on a shaker.</li>
	<li>Aspirate secondary antibody solution and wash cells 3 times with PBST for 10 min each time at RT on a shaker.</li>
	<li>Incubate cells with 300 &#181;L of 1 &#181;g/mL DAPI for 3-5 min, and then wash twice with PBST.</li>
	<li>After aspirating PBST, add an appropriate amount of PBS for taking pictures under a fluorescence microscope.</li>
</ol>
8. Western blot analysis
<ol>
	<li>Prepare 1x TBST washing buffer by adding 0.1% Tween-20 to Tris-buffered saline (TBS).</li>
	<li>Prepare SDS-PAGE electrophoresis buffer and western transfer buffer by using a commercial kit according to the manufacturer&#39;s protocol.</li>
	<li>Resolve total cell protein (40 &#181;g) on a 10% sodium dodecyl sulfate polyacrylamide gel and run in SDS-PAGE electrophoresis buffer at 15 mA constant current for about 2 h.</li>
	<li>Transfer the gel to a polyvinylidene difluoride (PVDF) membrane at 300 mA constant current for 2 h at low temperature. 
	NOTE: The running time and transfer time may vary depending on the equipment used and the type and percentage of the gel.</li>
	<li>Block the membrane with 3% BSA prepared in TBST for 1 h at RT on a shaker.</li>
	<li>Incubate in primary antibody solution (1:1000 dilution) overnight at 4 &#176;C on a shaker.</li>
	<li>After overnight incubation, wash blotting membrane 3 times with TBST for 15 min per time at RT on a shaker.</li>
	<li>Incubate in secondary antibody solution (1:2000 dilution) for 2 h at RT on a shaker.</li>
	<li>Discard secondary antibody solution and wash the membrane 3 times with TBST, for 15 min each time, at RT on a shaker.</li>
	<li>Visualize immunoreactive bands using a chemiluminescence reagent followed by autoradiography. Use &#946;-actin as the loading control.</li>
</ol>
9. Indocyanine green uptake
<ol>
	<li>Prepare 200 mg/mL indocyanine green stock solution in DMSO.</li>
	<li>Prepare a working solution by supplementing Stage III differentiation medium with indocyanine green solution to a final concentration of 1 mg/mL.</li>
	<li>Incubate in a 37 &#176;C, 5% CO2 incubator for 1-2 h.</li>
	<li>Aspirate medium and wash the cells 3 times with PBS.</li>
	<li>Photograph under a microscope.</li>
</ol>
10. Periodic Acid-Schiff (PAS) staining and Hematoxylin-Eosin (H&#38;E) staining
<ol>
	<li>Aspirate medium from adherent cells and briefly wash twice with PBS.</li>
	<li>For cell fixation, aspirate PBS and incubate cells with ice-cold methanol at -20 &#176;C overnight.</li>
	<li>Visualize glycogen storage by PAS staining and observe binucleated cells by H &#38; E staining using a kit following the manufacturer&#39;s instructions.</li>
	<li>Take images with a fluorescence microscope.</li>
</ol>
11. Assay for CYP Activity
<ol>
	<li>Measure CYP450 activity using commercially available cell-based assays (P450-Glo Assays) according to the manufacturer&#39;s instructions.</li>
	<li>Use three independent repeats for testing. Data are represented as the mean &#177; SD.</li>
</ol>

<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>

The authors have nothing to disclose.

Here, we present a stepwise method that induces HLCs from hESCs&#160;in three stages. In the first stage, Activin A and CHIR99021 were used to differentiate hESCs into DE. In the second stage, KO-DMEM and DMSO were used to differentiate DE into hepatic progenitor cells. In the third stage, HZM plus HGF, OSM and hydrocortisone 21-hemisuccinate sodium salt were used to continue to differentiate hepatic progenitor cells into HLCs.
The following critical steps need to be taken into consideration w...

The liver is a highly metabolic organ that plays several roles, including deoxidation, storing glycogen, and secretion and synthesis of proteins1. Various pathogens, drugs and heredity can cause pathological changes in the liver and affect its functions2,3. Hepatocytes, as the main functional unit of the liver, play an important role in artificial liver support systems and drug toxicity elimination. However, the resource of primary human hepatocytes is limited in cell-based therapy, as well as in liver disease research. Therefore, developing new sources of functional human hepatocytes is an important research direction in the field of regenerative medicine. Since 1998 when hESCs have been established4, hESCs have been widely considered by researchers because of their superior differentiation potential (they can differentiate into various tissues in a suitable environment) and high degree of self-renewability, and thus provide ideal source cells for bioartificial livers, hepatocyte transplantation and even liver tissue engineering5.
Currently, the hepatic differentiation efficiency can be greatly increased by enriching the endoderm6. In the lineage differentiation of stem cells into endoderm, levels of the transforming growth factor&#160;&#946;&#160;(TGF-&#946;)&#160;signaling and WNT signaling pathways are the key factors in the node of the endoderm formation stage. Activation of a high-level of TGF-&#946;&#160;and WNT signaling can promote the development of endoderm7,8. Activin A is a cytokine belonging to the TGF-&#946;&#160;superfamily. Therefore, Activin A is widely used in endoderm induction of human induced pluripotent stem cells (hiPSCs) and hESCs9,10. GSK3 is a serine-threonine protein kinase. Researchers have found that CHIR99021, a specific inhibitor of GSK3&#946;, can stimulate typical WNT signals, and can promote stem cell differentiation under certain conditions, suggesting that CHIR99021 has potential for inducing stem cell differentiation into endoderm 11,12,13.
Here we report an efficient and reproducible method for effectively inducing the differentiation of hESCs into functional HLCs. The sequential addition of Activin A and CHIR99021 produced about 89.7&#177;0.8% SOX17 (DE marker)-positive cells. After being further maturated in vitro, these cells expressed hepatic specific markers and exerted hepatocyte-like morphology (based on hematoxylin-eosin staining (H &#38; E)) and functions, such as uptake of indocyanine green (ICG), glycogen storage and CYP3 activity. The results show that hESCs can be successfully differentiated into mature functional HLCs by this method and can provide a basis for liver disease-related research and in vitro drug screening.

<table><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 - &amp;alpha;-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&amp;alpha;)</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&amp;amp;E staining kit</td><td>Beyotime</td><td>C0105S</td><td>For H&amp;amp;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&amp;nbsp;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

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

The schematic diagram of HLC induction from hESCs and representative bright-field images of each differentiation stage are shown in Figure 1. In Stage I, Activin A and CHIR99021 were added for 3 days to induce stem cells to form endoderm cells. In Stage II, the endoderm cells differentiated into hepatic progenitor cells after being treated with differentiation medium for 5 days. In Stage III, early hepatocytes had matured and differentiated into HLCs after 10 days in HGF and OSM (<strong cla...

Watch this Scientific Journal Video about An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells at JoVE.com

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

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

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

an-efficient-method-for-directed-hepatocyte-like-cell-induction-from

Research

JoVE Journal

Immunology and Infection

3.5K Views.  Shantou University Medical College. Menselijke embryonale stamcel-afgeleide hepatocyt-achtige cellen zijn nuttig bij ziektemodellering en geneesmiddelenscreening. Dit is een efficiënte en reproduceerbare methode om de differentiatie van menselijke embryonale stamcellen in functionele hepatocyt-achtige cellen effectief te induceren. Een ongedifferentieerde status en een geschikte samenvloeiing van menselijke embryonale stamcellen zijn van cruciaal belang voor een succesvolle differentiatie van hepatocytenachtige cellen.V...

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

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

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

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

Developmental Biology

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.

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 ...

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

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Biology

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

Generation of Functional Endodermal Hepatic Organoids

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The remarkable advances in organoid models have recently opened a new experimental era for various applications in disease modeling, developmental biology, and drug discovery. Adult stem cells or induced&#160;pluripotent stem cell (iPSC)-derived liver organoids govern the generation of hepatocytes to use for diverse applications. Here, we present a robust and reproducible protocol for generating hepatic organoids from pluripotent stem cells. This protocol is applicable to healthy and patient-derived cells. To achieve 3D endoderm-derived hepatic organoids (eHEPOs), iPSCs were directly first differentiated into endodermal cells, and then FACS-enriched EpCAM-positive (EpCAM+) cells were used to establish hepatic organoids using the expansion medium. We provide a fast and efficient method to generate hepatic organoids within 2 weeks. The generated organoids mimic the essential properties and functions of hepatocytes, such as albumin secretion, glycogen storage, and cytochrome P450 enzyme activity. Besides the liver-specific gene expression similarities, eHEPOs comprise polarized epithelial cells with bile canaliculi in between. In addition, eHEPOs can be expanded and serial passages long term (1 year) without losing their capacity to differentiate into mature hepatocytes. Thus, eHEPOs provide an alternative source to produce functional hepatocytes.

This protocol describes the generation of fast and reproducible endodermal hepatic organoids (eHEPOs). With this protocol, eHEPOs can be produced within 2 weeks and expand long-term (more than 1 year) without losing their differentiation and functionality.

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The ...

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

Samenvatting

Meer video's verkennen