Name: Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations (Video) | JoVE Journal
Uploaded: 2011-10-26T12:00:00.000+00:00
Duration: 5 min 49 s
Description: Learn to generate functional hepatocytes from human embryonic stem cells with ~90% efficiency. This method enables in vitro liver biology studies and provides an inexhaustible supply for drug toxicity testing.

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5 minutes

Dr Hay was supported by a RCUK Fellowship, Dr West was supported by the Department of Surgery, Dr Medine was supported by a grant from the BHF Core Fund, Mr. Baltasar Lucendo-Villarin was supported by a MRC PhD Studenship. Dr Zhou was supported by a scholarship from the Chinese Government.

<ol><li>Asgari, S., Pournasr, B., Salekdeh, G. H., Ghodsizadeh, A., Ott, M., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cells%3A+a+new+era+for+hepatology%5BTitle%5D)+AND+%22J.+Hepatol%22%5BJournal%5D)">Induced pluripotent stem cells: a new era for hepatology.</a> J. Hepatol. 53, 738-751 (2010).</li>
<li>Hay, D. C., Pernagallo, S., Diaz-Mochon, J. J., Medine, C. N., Greenhough, S., Hannoun, Z., Schrader, J., Black, J. R., Fletcher, J., Dalgetty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unbiased+Screening+of+Polymer+Libraries+to+Define+Novel+Substrates+for+Functional+Hepatocytes+with+Inducible+Drug+Metabolism%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">Unbiased Screening of Polymer Libraries to Define Novel Substrates for Functional Hepatocytes with Inducible Drug Metabolism.</a> Stem Cell Research. 6, 92-101 (2011).</li>
<li>Payne, C. M., Samuel, K., Pryde, A., King, J., Brownstein, D., Schrader, J., Medine, C. N., Forbes, S. J., Iredale, J. P., Newsome, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistence+of+Functional+Hepatocyte+Like+Cells+in+Immune+Compromised+Mice%5BTitle%5D)+AND+%22Liver+International%22%5BJournal%5D)">Persistence of Functional Hepatocyte Like Cells in Immune Compromised Mice.</a> Liver International. 31, 254-262 (2011).</li>
<li>Greenhough, S., Medine, C., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pluripotent+Stem+Cell+Derived+Hepatocyte+Like+Cells+and+their+Potential+in+Toxicity+Screening%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">Pluripotent Stem Cell Derived Hepatocyte Like Cells and their Potential in Toxicity Screening.</a> Toxicology. 278, 250-255 (2010).</li>
<li>Medine, C. N., Greenhough, S., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Role+of+Stem+Cell+Derived+Hepatic+Endoderm+in+Human+Drug+Discovery%5BTitle%5D)+AND+%22Biochemical+Society+Transactions%22%5BJournal%5D)">The Role of Stem Cell Derived Hepatic Endoderm in Human Drug Discovery.</a> Biochemical Society Transactions. 38, 1033-1036 (2010).</li>
<li>Hannoun, Z., Fletcher, J., Greenhough, S., Medine, C. N., Samuel, K., Sharma, R., Pryde, A., Black, J. R., Ross, J. A., Wilmut, I., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Comparison+between+Conditioned+Media+and+Serum+Free+Media+in+Human+Embryonic+Stem+Cell+Culture+and+Differentiation%5BTitle%5D)+AND+%22Cellular+Reprogramming%22%5BJournal%5D)">The Comparison between Conditioned Media and Serum Free Media in Human Embryonic Stem Cell Culture and Differentiation.</a> Cellular Reprogramming. 12, 133-140 (2010).</li>
<li>Dalgetty, D. M., Medine, C., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+and+Future+Challenges+in+Stem+Cell-Derived+Liver+Technologies%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Gastrointestinal+and+Liver+Physiology%22%5BJournal%5D)">Progress and Future Challenges in Stem Cell-Derived Liver Technologies.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 297, 241-248 (2009).</li>
<li>Hay, D. C., Fletcher, J., Payne, C., Terrace, J. D., Gallagher, R. C. J., Snoeys, J., Black, J., Wojtacha, D., Samuel, K., Hannoun, Z., Pryde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+Efficient+Differentiation+of+hESCs+to+Functional+Hepatic+Endoderm+Requires+ActivinA+and+Wnt3a+Signalling%5BTitle%5D)+AND+%22+Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Highly Efficient Differentiation of hESCs to Functional Hepatic Endoderm Requires ActivinA and Wnt3a Signalling.</a> Proceedings of the National Academy of Sciences. 105, 12301-12306 (2008).</li>
<li>Fletcher, J., Cui, W., Samuels, K., Black, J. R., Currie, I. S., Terrace, J. D., Payne, C., Filippi, C., Newsome, P., Forbes, S. J., Ross, J. A., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Inhibitory+Role+of+Stromal+Cell+Mesenchyme+on+Human+Embryonic+Stem+Cell+Hepatocyte+Differentiation+is+Overcome+by+Wnt3a+Treatment%5BTitle%5D)+AND+%22Cloning+and+Stem+Cells%22%5BJournal%5D)">The Inhibitory Role of Stromal Cell Mesenchyme on Human Embryonic Stem Cell Hepatocyte Differentiation is Overcome by Wnt3a Treatment.</a> Cloning and Stem Cells. 10, 331-340 (2008).</li>
</ol>

No conflicts of interest declared.

We have developed a simple, homogeneous and highly reproducible in vitro model to generate scalable levels of human HLCs. Our model has been validated by a number of external collaborating laboratories. We routinely characterise stem cell derived HLCs using our in house tool box of developmental markers and liver specific functional assays (most of which are commercially available). The critical stages in our process are: the maintenance of stem cell pluripotency; the ability to direct stem cell differentiation ...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Matrigel coating plates and flasks &lt;/p&gt;&lt;ol&gt;&lt;li&gt; Matrigel (10 mL, BD Biosciences, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Tissue culture plates (6 well, 12 well, Corning, UK) &lt;/li&gt;&lt;li&gt;Tissue culture flask (25 cm2 vented, Corning, UK)&lt;/li&gt;&lt;/ol&gt;&lt;p class=&quot;jove_step&quot;&gt;hESC Maintenance &lt;/p&gt;&lt;ol&gt;&lt;li&gt;Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R &amp;amp; D Systems, USA); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;BSA solution (50 mL, Sigma Aldrich, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Human basic fibroblast growth factor (100 &amp;#x03BC;g, Peprotech, USA); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;/ol&gt;&lt;p class=&quot;jove_step&quot;&gt;Passaging hESCs with collagenase&lt;/p&gt;&lt;ol&gt;&lt;li&gt;Confluent well or flask of hESCs.&lt;/li&gt;&lt;li&gt;Matrigel coated wells or flasks as appropriate.&lt;/li&gt;&lt;li&gt;Phospate buffered saline (-MgCl&lt;sub&gt;2&lt;/sub&gt;, -CaCl&lt;sub&gt;2&lt;/sub&gt;) (500 mL, Gibco, Invitrogen, UK); store at room temperature.&lt;/li&gt;&lt;li&gt;Collagenase IV (1 g, Gibco, Invitrogen, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R &amp;amp; D Systems, USA).&lt;/li&gt;&lt;li&gt;Human basic fibroblast growth factor (100 &amp;#x03BC;g, Peprotech, USA).&lt;/li&gt;&lt;/ol&gt;&lt;p class=&quot;jove_step&quot;&gt;Differentiation of hESCs to hepatic endoderm&lt;/p&gt;&lt;ol&gt;&lt;li&gt;RPMI 1640 (500 mL, Gibco, Invitrogen, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;B27 Supplement (10 mL, Gibco, Invitrogen, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Activin A (2 &amp;#x03BC;g, Peprotech, USA); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Recombinant mouse Wnt3a (2 &amp;#x03BC;g, R &amp;amp; D Systems, USA); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;KO-SR (500 mL, Gibco, Invitrogen, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Non-essential amino acids (100 mL, Gibco, Invitrogen, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;&amp;#946;-Mercapt&amp;#8211;thanol (10 mL, Gibco, Invitrogen, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;DMSO (Sigma Aldrich, UK); store at room temperature&lt;/li&gt;&lt;li&gt;Leibovitz L-15 culture medium (500 mL, Sigma Aldrich, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Tryptose phosphate broth (100 mL, Sigma Aldrich, UK); store at 4&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;F&amp;#8211;tal bovine serum, heat inactivated (500 mL, Gibco, Invitrogen, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Hydrocortisone 21-hemisuccinate (100 mg, Sigma Aldrich, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Insulin (bovine pancreas) (100 mg, Sigma Aldrich, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;L-Glutamine (100 mL, Gibco, Invitrogen, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Ascorbic acid (25 g, Sigma Aldrich, UK); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Human HGF (10 &amp;#x03BC;g, Peprotech, USA); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Recombinant Human Oncostatin M (OSM) (50 &amp;#x03BC;g, R &amp;amp; D Systems, USA); store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Syringe driven filter unit 0.22 &amp;#x03BC;m (Millipore, UK)&lt;/li&gt;&lt;/ol&gt;&lt;p class=&quot;jove_step&quot;&gt;Characterisation of hESC derived Hepatic Endoderm&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Immunostaining &lt;/p&gt;&lt;ol&gt;&lt;li&gt;Phosphate buffer saline (-MgCl&lt;sub&gt;2&lt;/sub&gt;, -CaCl&lt;sub&gt;2&lt;/sub&gt;) (500 mL, Gibco, Invitrogen, UK); store at room temperature.&lt;/li&gt;&lt;li&gt;PBST, PBS made up with 0.1% TWEEN 20 (Sigma-Aldrich, UK).&lt;/li&gt;&lt;li&gt;Paraformaldehyde (PFA) (Sigma-Aldrich, UK) is made up in PBS, store -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Glycerol (Sigma-Aldrich, UK), store at room temperature.&lt;/li&gt;&lt;li&gt;Tris Base (Sigma-Aldrich, UK), store at room temperature.&lt;/li&gt;&lt;li&gt;Ethanol&lt;/li&gt;&lt;li&gt;Serum (AbD Serotech, UK), store at -20&amp;#x00B0;C.&lt;/li&gt;&lt;li&gt;Secondary Antibody, Alexa Fluorophores (Molecular Probes, Invitrogen, UK).&lt;/li&gt;&lt;li&gt;MOWIOL 4-88 (Polysciences Inc, USA) is made up in Tris HCL and glycerol as per manufacturers instructions. DAPI (Pierce, Thermo Fisher Scientific, UK) is added to the MOWIOL solution at a 1:1000 dilution.&lt;/li&gt;&lt;/ol&gt;&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Primary antibodies&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Antigen*&lt;/td&gt; &lt;td&gt;Type&lt;/td&gt; &lt;td&gt;Supplier&lt;/td&gt; &lt;td&gt;Dilution&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;ALB&lt;/td&gt; &lt;td&gt;Mouse Monoclonal&lt;/td&gt; &lt;td&gt;Sigma Aldrich&lt;/td&gt; &lt;td&gt;1/500&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;E-Cadherin&lt;/td&gt; &lt;td&gt;Mouse Monoclonal&lt;/td&gt; &lt;td&gt;Millipore&lt;/td&gt; &lt;td&gt;1/100&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&amp;alpha;-fetoprotein&lt;/td&gt; &lt;td&gt;Mouse Monoclonal&lt;/td&gt; &lt;td&gt;Sigma&lt;/td&gt; &lt;td&gt;1/500&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;SSEA-4 FITC&lt;/td&gt; &lt;td&gt;Mouse Monoclonal&lt;/td&gt; &lt;td&gt;Biolegend&lt;/td&gt; &lt;td&gt;1/100&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;IgG&lt;/td&gt; &lt;td&gt;Mouse Monoclonal&lt;/td&gt; &lt;td&gt;DAKO&lt;/td&gt; &lt;td&gt;1/500&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Secondary antibodies&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Anti-mouse FITC conjugate&lt;/td&gt; &lt;td&gt;Goat Monoclonal&lt;/td&gt; &lt;td&gt;Invitrogen&lt;/td&gt; &lt;td&gt;1/400&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table 2.&lt;/strong&gt; The antibodies used for hESC derived hepatic endoderm immunostaining, the concentrations used, the species developed in and the companies they are purchased from.&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Functional Analysis of Hepatic Endoderm and Normalisation (per mg protein)&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Cytochrome P450 Assays&lt;/p&gt;&lt;ol&gt;&lt;li&gt;p4-GLO CYP3A4, CYP1A2, Kits and luminometer (Promega, USA).&lt;/li&gt;&lt;li value=&quot;1&quot;&gt;White flat bottom 96 well assay plate (BD Biosciences, UK).&lt;/li&gt;&lt;li&gt;BCA Assay Kit (Pierce, Thermo Fisher Scientific, UK).&lt;/li&gt;&lt;li&gt;Transparent 96 well assay plate (IWAKI, UK)&lt;/li&gt;&lt;/ol&gt;</td></tr></tbody></table>

robust generation of hepatocyte-like cells from human embryonic stem cell populations

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 ...

The overall goal of the following experiment is to produce homogeneous levels of human hepatocytes to study human liver biology in vitro. This is initiated by driving human embryonic stem cells or human ESCs to definitive endoderm using physiologic stimuli active in A and WINT three A.As a second step definitive endoderm is highly efficiently driven to hepatic endoderm. Next human ESC derived hepatic endoderm is matured in vitro to produce functional human hepatocytes.Results from PCR Western blotting, immunofluorescence, Eliza's, and drug metabolism indicate that this method derives hepatocytes from human ESCs at an efficiency of around 90%With this technique, we can, in theory, generate inexhaustible supplies of primary human hepatocytes, which exhibit appreciable levels of human liver function. In the future, this will benefit accurate prediction of drug toxicity and provide human liver support. The trick is to drive solar differentiation and the core density of human cells.Visual demonstration of this method is critical as the core cell density is essential to generate homogeneous cell population. We have trained over 15 individuals over four sites internationally to perform this technique successfully. Begin with a culture of human embryonic stem cells and propagate on matrigel coated plates with mouse embryonic fibroblasts conditioned media ORM cm supplemented with basic FGF.It is important to carefully characterize the human ESCs maintained prior to hepatic differentiation. For example, these cells exhibit human ESC morphology. They also express the pluripotent stem cell markers, OCT four and nano in levels comparable to the positive control H seven human ESC line.Additionally, the human ESCs are positive for SS EEA four. When human E ESCs reach 30 to 60%co fluency replace the ME CM with freshly prepared differentiation medium. To initiate hepatic differentiation, the medium should be replaced every 24 hours for three days.After three days, cells take on a triangular appearance as they begin to differentiate towards an endodermal phenotype. Switch the medium to SR DMSO differentiation. Medium culture for four to five days.Replacing the medium every 48 hours. Following this five day period, cells would begin to take on a polygonal appearance characteristic of hepatic cells. Differentiation of human ESC to hepatic endoderm is reflected in a series of profound morphological changes by day nine.Moreover, expression of Octa four over the nine day time course decreases progressively. In contrast, liver transcripts of a FP and albumin increase from day seven onwards. Continue by maturing the cells and maintaining hepatic phenotype in final medium for nine days with fresh medium every 48 hours.Finally, monitor the cultured cells by phase contrast Microscopy for gradual morphological changes from a spiky triangular shape to a characteristic liver morphology displaying a polygonal appearance to quantitatively evaluate hepatic endoderm function assay. For cytochrome P four 50 enzymatic activity, specifically SIP three A four or SIP one A two. First incubate day 17.Human ESC derived hepatic endoderm cells with the specific substrate performing reactions in triplicates. Remember to use tissue culture media as a negative control incubate at 37 degrees Celsius for five hours. Collect the supernatants and measure the relative levels of basal enzymatic activity as detailed in manufacturer's instructions.Now, for each sample, calculate enzymatic activity per milligram of protein as determined by A-B-C-A-S. A immuno staining of typical cultures derived by this method of in vitro maturation of hepatic endoderm. Demonstrate homogeneous expression of three human liver markers, albumin a FP and ein.After watching this video, you should have a good understanding of how to efficiently differentiate human and learning stem cells to hepatocytes. Once mastered, this technique can be done on a daily basis with a commitment of around two and a half minutes per million cells. It's very important to check morphological changes on a daily basis during the differentiation process.

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Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

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.

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

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.

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

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.

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

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.

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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.

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

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.

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

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).

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

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

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.

Generation of Functional Endodermal Hepatic Organoids

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Learn to generate functional hepatocytes from human embryonic stem cells with ~90% efficiency. This method enables in vitro liver biology studies and provides an inexhaustible supply for drug toxicity testing.

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

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