We thank Bing Lim&#160;for discussions and the Stanford Institute for Stem Cell Biology &#38; Regenerative Medicine for infrastructure support. This work was supported by the California Institute for Regenerative Medicine (DISC2-10679) and the Stanford-UC Berkeley Siebel Stem Cell Institute (to L.T.A. and K.M.L.) and the Stanford Beckman Center for Molecular and Genetic Medicine as well as the Anonymous, Baxter and DiGenova families (to K.M.L.).

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	<li>Bernal, W., Wendon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Liver+Failure.">Acute Liver Failure.</a> New England Journal of Medicine. 369, 2525-2534 (2013).</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>Fisher, R. A., Strom, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Hepatocyte+Transplantation:+Worldwide+Results.">Human Hepatocyte Transplantation: Worldwide Results.</a> Transplantation. 82, 441-449 (2006).</li><li>Dhawan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+human+hepatocyte+transplantation:+Current+status+and+challenges.">Clinical human hepatocyte transplantation: Current status and challenges.</a> Liver transplantation. 21, S39-S44 (2015).</li><li>Salama, 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=Autologous+Hematopoietic+Stem+Cell+Transplantation+in+48+Patients+with+End-Stage+Chronic+Liver+Diseases.">Autologous Hematopoietic Stem Cell Transplantation in 48 Patients with End-Stage Chronic Liver Diseases.</a> Cell Transplantation. 19, 1475-1486 (2017).</li><li>Tan, A. K. Y., Loh, K. M., Ang, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+regenerative+potential+and+functionality+of+human+liver+cells+in+mice.">Evaluating the regenerative potential and functionality of human liver cells in mice.</a> Differentiation. 98, 25-34 (2017).</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>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=Mapping+the+Pairwise+Choices+Leading+from+Pluripotency+to+Human+Bone,+Heart,+and+Other+Mesoderm+Cell+Types.">Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.</a> Cell. , 451-467 (2016).</li><li>Zhao, D., 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=Promotion+of+the+efficient+metabolic+maturation+of+human+pluripotent+stem+cell-derived+hepatocytes+by+correcting+specification+defects.">Promotion of the efficient metabolic maturation of human pluripotent stem cell-derived hepatocytes by correcting specification defects.</a> Cell Research. 23, 157-161 (2012).</li><li>Si Tayeb, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+generation+of+human+hepatocyte-like+cells+from+induced+pluripotent+stem+cells.">Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.</a> Hepatology. 51, 297-305 (2010).</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=Hepatic+differentiation+of+human+pluripotent+stem+cells+in+miniaturized+format+suitable+for+high-throughput+screen.">Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen.</a> Stem Cell Research. 16, 640-650 (2016).</li><li>Avior, 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=Microbial-derived+lithocholic+acid+and+vitamin+K2+drive+the+metabolic+maturation+of+pluripotent+stem+cells-derived+and+fetal+hepatocytes.">Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells-derived and fetal hepatocytes.</a> Hepatology. 62, 265-278 (2015).</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>Rostovskaya, M., Bredenkamp, N., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+consistent+generation+of+pancreatic+lineage+progenitors+from+human+pluripotent+stem+cells.">Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 370, 20140365 (2015).</li><li>Haridass, D., 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=Repopulation+Efficiencies+of+Adult+Hepatocytes,+Fetal+Liver+Progenitor+Cells,+and+Embryonic+Stem+Cell-Derived+Hepatic+Cells+in+Albumin-Promoter-Enhancer+Urokinase-Type+Plasminogen+Activator+Mice.">Repopulation Efficiencies of Adult Hepatocytes, Fetal Liver Progenitor Cells, and Embryonic Stem Cell-Derived Hepatic Cells in Albumin-Promoter-Enhancer Urokinase-Type Plasminogen Activator Mice.</a> The American Journal of Pathology. 175, 1483-1492 (2009).</li></ol>

The authors have nothing to disclose.

This method enables the generation of enriched populations of liver bud progenitors, and subsequently hepatocyte-like cells, from hPSCs. The ability to generate enriched populations of human liver cells is important for the practical utilization of such cells. Previous methods to generate hepatocytes from hPSCs yielded impure cell populations containing both liver and non-liver cells that, upon transplantation into rodents, yielded bone and cartilage in addition to liver tissue15. Hence the explic...

The purpose of this protocol is to efficiently differentiate human pluripotent stem cells (hPSCs) into enriched populations of liver bud progenitors and hepatocyte-like cells2. Access to a ready supply of human liver progenitors and hepatocyte-like cells will accelerate efforts to investigate liver function and disease and could enable new cellular transplantation therapies for liver failure3,4,5. This has proven challenging in the past since hPSCs (which include embryonic and induced pluripotent stem cells) can differentiate into all the cell-types of the human body; consequently, it has been difficult to exclusively differentiate them into a pure population of a single cell-type, such as liver cells6.
To precisely differentiate hPSCs into liver cells, first it is critical to understand not only how liver cells are specified but also how non-liver cell-types develop. Knowledge of how non-liver cells develop is important to logically suppress the formation of non-liver lineages during differentiation, thereby exclusively guiding hPSCs towards a liver fate2. Second, it is essential to delineate the multiple developmental steps through which hPSCs differentiate towards a liver fate. It is known that hPSCs sequentially differentiate into multiple cell-types known as the primitive streak (APS), definitive endoderm (DE), posterior foregut (PFG) and liver bud progenitors (LB) before forming hepatocyte-like cells (HEP). Earlier work revealed the signals specifying liver fate and the signals that suppressed the formation of alternate non-liver cell-types (including stomach, pancreatic, and intestinal progenitors) at each developmental lineage choice2,7,8.
Collectively, these insights have given rise to a serum-free, monolayer method to differentiate hPSCs towards primitive streak, definitive endoderm, posterior foregut, liver bud progenitors and finally, hepatocyte-like cells2. Overall the method involves the seeding of hPSCs in a monolayer at an appropriate density, preparing six cocktails of differentiation media (containing growth factors and small molecules that regulate various developmental signaling pathways), and sequentially adding these media to induce differentiation over the course of 18 days. During the process, no passaging of cells is needed. Of note, because this method explicitly includes signals that suppress the formation of non-liver cell-types, this differentiation approach1 more efficiently generates liver progenitors and hepatocyte-like cells by comparison to extant differentiation methods2,9,10,11,12. Furthermore, the protocol described in this text enables the faster generation of hepatocytes that ultimately express higher levels of hepatic transcription factors and enzymes than those produced by other protocols9,10,11,12.
The protocol described here has certain advantages over current differentiation protocols. First, it entails monolayer differentiation of hPSCs, which is technically simpler compared to three-dimensional differentiation methods, such as those that rely on embryoid bodies13. Second,&#160;this method exploits a recent advance whereby definitive endoderm cells (an early precursor to liver cells) can be efficiently and rapidly generated within 2 days of hPSC differentiation2,7, thus enabling the subsequent production of hepatocytes with increased purity. Third, in side-by-side comparisons, the hepatocyte-like cells produced by this method2 produce more ALBUMIN&#160;and express higher levels of hepatic transcription factors and enzymes compared to hepatocytes produced in other methods10,11,12.

<table><tbody><tr><td>&amp;nbsp;Geltrex</td><td>Thermofisher Scientific</td><td>A1569601</td><td></td></tr><tr><td>1:1 DMEM/F12</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>0.2&amp;nbsp;&amp;mu;m pore membrane filter</td><td>Millipore</td><td>GTTP02500</td><td></td></tr><tr><td>mTeSR1</td><td>Stem Cell Technologies</td><td>5850</td><td></td></tr><tr><td>Thiazovivin</td><td>Tocris Bioscience</td><td>3845</td><td></td></tr><tr><td>&amp;nbsp;Accutase</td><td>Gibco or Millipore&amp;nbsp;</td><td>Gibco A11105, Millipore&amp;nbsp; SCR005</td><td></td></tr><tr><td>IMDM, GlutaMAX&amp;trade; Supplement</td><td>Thermofisher Scientific</td><td>31980030</td><td></td></tr><tr><td>Ham&#039;s F-12 Nutrient Mix, GlutaMAX&amp;trade; Supplement</td><td>Thermofisher Scientific</td><td>31765035</td><td></td></tr><tr><td>KOSR, Knockout serum replacement</td><td>Thermofisher Scientific</td><td>10828028</td><td></td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>P8136</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>10652202001</td><td></td></tr><tr><td>Chemically Defined Lipid Concentrate</td><td>Thermofisher Scientific</td><td>11905031</td><td></td></tr><tr><td>Human Activin</td><td>R&amp;amp;D</td><td>338-AC</td><td></td></tr><tr><td>CHIR99201</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>PI103</td><td>Tocris</td><td>2930/1</td><td></td></tr><tr><td>Human FGF2</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>DM3189</td><td>Tocris</td><td>6053/10</td><td></td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939/10</td><td></td></tr><tr><td>Human BMP4</td><td>R&amp;amp;D</td><td>314-BP</td><td></td></tr><tr><td>C59</td><td>Tocris</td><td>5148</td><td></td></tr><tr><td>TTNPB</td><td>Tocris</td><td>0761/10</td><td></td></tr><tr><td>Forskolin</td><td>Tocris</td><td>1099/10</td><td></td></tr><tr><td>Oncostatin M</td><td>R&amp;amp;D</td><td>295-OM</td><td></td></tr><tr><td>Dexamethasone</td><td>Tocris</td><td>1126</td><td></td></tr><tr><td>Ro4929097</td><td>Selleck Chem</td><td>S1575</td><td></td></tr><tr><td>AA2P</td><td>Cayman chemicals</td><td>&lt;em&gt;16457&lt;/em&gt;</td><td></td></tr><tr><td>Human recombinant Insulin</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>11061-68-0</td><td></td></tr></tbody></table>

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.

After 24 h of APS differentiation, colonies will generally adopt a different morphology than undifferentiated colonies concomitant with a loss of the bright border that typically circumscribes hPSC colonies. Morphologically, primitive streak cells generally have ragged borders and are more spread and less compact than hPSCs-this is evocative of an epithelial-to-mesenchymal transition as pluripotent epiblast cells differentiate and ingress into the primitive streak in vivo. If the colony s...

Watch this Scientific Journal Video about Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells at JoVE.com

Efficient Differentiation of Human Pluripotent Stem Cells into Liver 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.

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

efficient-differentiation-human-pluripotent-stem-cells-into-liver

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.5K Views.  Stanford University School of Medicine. 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.

Video: Efficient Differentiation of Human Pluripotent Stem Cells into Liver 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 ...

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 ...

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

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

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

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

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 ...

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

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

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

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

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.

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 ...

Immunology and Infection

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 ...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...