Hepatic differentiation of human embryonic stem cells is promoted by three-dimensional dynamic perfusion culture conditions.

The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research, and extracorporeal bioartificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture, the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells.

[1]  S. Strom,et al.  To be or not to be: generation of hepatocytes from cells outside the liver. , 2008, Gastroenterology.

[2]  Hanry Yu,et al.  Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices. , 2005, Biomaterials.

[3]  K. Meyer zum Büschenfelde,et al.  High-yield purification and characterization of human asialoglycoprotein receptor. , 1995, Protein expression and purification.

[4]  G. Gerken,et al.  Receptor-mediated entry of hepatitis B virus particles into liver cells , 1997, Archives of Virology.

[5]  Smadar Cohen,et al.  Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds. , 2006, Tissue engineering.

[6]  Aarati R. Ranade,et al.  Mouse fetal liver cells in artificial capillary beds in three-dimensional four-compartment bioreactors. , 2005, The American journal of pathology.

[7]  B J Blaauboer,et al.  The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. , 1990, Biochemical pharmacology.

[8]  T. Nikaido,et al.  Effective hepatocyte transplantation using rat hepatocytes with low asialoglycoprotein receptor expression. , 2004, The American journal of pathology.

[9]  B. Yener,et al.  The natural and engineered 3D microenvironment as a regulatory cue during stem cell fate determination. , 2009, Tissue engineering. Part B, Reviews.

[10]  Robert L. Thompson,et al.  Hepatic maturation of human fetal hepatocytes in four-compartment three-dimensional perfusion culture. , 2010, Tissue engineering. Part C, Methods.

[11]  I M Sauer,et al.  Clinical extracorporeal hybrid liver support – phase I study with primary porcine liver cells , 2003, Xenotransplantation.

[12]  Melissa A. Kinney,et al.  Hydrodynamic modulation of pluripotent stem cells , 2012, Stem Cell Research & Therapy.

[13]  L. Wiebe,et al.  Targeted delivery of macromolecular drugs: asialoglycoprotein receptor (ASGPR) expression by selected hepatoma cell lines used in antiviral drug development. , 2008, Current drug delivery.

[14]  C. Wandrey,et al.  Encapsulation of Huh-7 cells within alginate-poly(ethylene glycol) hybrid microspheres , 2011, Journal of Materials Science: Materials in Medicine.

[15]  벤자민 프라이어,et al.  Differentiation of human embryonic stem cells , 2011 .

[16]  G. Kolios,et al.  Stem Cells in Liver Regeneration and Their Potential Clinical Applications , 2013, Stem Cell Reviews and Reports.

[17]  M. Manns,et al.  Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepatocytes. , 2004, The International journal of developmental biology.

[18]  C. Verfaillie,et al.  Enhanced differentiation of adult bone marrow-derived stem cells to liver lineage in aggregate culture. , 2011, Tissue engineering. Part A.

[19]  Nancy Cheng,et al.  Mature human hepatocytes from ex vivo differentiation of alginate-encapsulated hepatoblasts. , 2008, Tissue engineering. Part A.

[20]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[21]  Shulamit Levenberg,et al.  Transplantation of a tissue-engineered human vascularized cardiac muscle. , 2010, Tissue engineering. Part A.

[22]  Robert Lanza,et al.  Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells , 2008, Stem cells.

[23]  Sason Shaik,et al.  Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. , 2004, Chemical reviews.

[24]  Interwoven Four-Compartment Capillary Membrane Technology for Three-Dimensional Perfusion with Decentralized Mass Exchange to Scale Up Embryonic Stem Cell Culture , 2010, Cells Tissues Organs.

[25]  K. Sasaki,et al.  Embryonic stem cell-derived embryoid bodies in three-dimensional culture system form hepatocyte-like cells in vitro and in vivo. , 2004, Tissue engineering.

[26]  D. Mooney,et al.  Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes. , 2004, Tissue engineering.

[27]  R. Tompkins,et al.  Long‐Term in Vitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration , 1991, Biotechnology progress.

[28]  Hossein Baharvand,et al.  Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture. , 2013, Stem cells and development.

[29]  F. Guengerich.  Uncommon P450-catalyzed reactions. , 2001, Current drug metabolism.

[30]  Lan-juan Li,et al.  Advances in cell sources of hepatocytes for bioartificial liver. , 2012, Hepatobiliary & pancreatic diseases international : HBPD INT.

[31]  T. Ochiya,et al.  Differentiation of embryonic stem cells into hepatocytes: Biological functions and therapeutic application , 2003, Hepatology.

[32]  In vitro differentiation of embryonic stem cells into hepatocytes induced by fibroblast growth factors and bone morphological protein-4. , 2007, The international journal of biochemistry & cell biology.

[33]  Y. Nakajima,et al.  In Vitro Differentiation of Embryonic Stem Cells into Hepatocyte‐Like Cells Identified by Cellular Uptake of Indocyanine Green , 2002, Stem cells.

[34]  A. Boonstra,et al.  Experimental models for hepatitis C viral infection , 2009, Hepatology.

[35]  X. Pei,et al.  Lineage restriction and differentiation of human embryonic stem cells into hepatic progenitors and zone 1 hepatocytes. , 2009, Tissue engineering. Part C, Methods.

[36]  Teruo Okano,et al.  Maturation of the extracellular matrix and cell adhesion molecules in layered co-cultures of HepG2 and endothelial cells. , 2009, Journal of biochemistry.

[37]  Takao Hayakawa,et al.  The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets. , 2012, Biomaterials.

[38]  G. Gerken,et al.  The asialoglycoprotein receptor mediates hepatic binding and uptake of natural hepatitis B virus particles derived from viraemic carriers. , 1994, The Journal of general virology.

[39]  T. Cresteil,et al.  Expression of CYP3A in the human liver--evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. , 1997, European journal of biochemistry.

[40]  R. Chamuleau,et al.  Proliferative human cell sources applied as biocomponent in bioartificial livers: a review , 2012, Expert opinion on biological therapy.

[41]  P. Neuhaus,et al.  Hepatocyte culture between woven capillary networks: a microscopy study. , 1994, Artificial organs.

[42]  M. Panhuis,et al.  Gelapin, a degradable genipin cross-linked gelatin hydrogel , 2013 .

[43]  Tao Xu,et al.  Inkjet-mediated gene transfection into living cells combined with targeted delivery. , 2009, Tissue engineering. Part A.

[44]  P. Chakravarty,et al.  Biotransformation of losartan to its active carboxylic acid metabolite in human liver microsomes. Role of cytochrome P4502C and 3A subfamily members. , 1995, Drug metabolism and disposition: the biological fate of chemicals.

[45]  J. Levitsky,et al.  Overview of immunosuppression in liver transplantation. , 2009, World journal of gastroenterology.

[46]  Xulang Zhang,et al.  MODIFIED ALGINATE/CHITOSAN HOLLOW MICROFIBER AS A BIOCOMPATIBLE FRAME FOR BLOOD VESSEL RECONSTRUCTION , 2012 .

[47]  F. Tronche,et al.  Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. , 2009, Gastroenterology.

[48]  A. Lindahl,et al.  Derivation, Characterization, and Differentiation of Human Embryonic Stem Cells , 2004, Stem cells.

[49]  Jane Synnergren,et al.  Hepatic differentiation and maturation of human embryonic stem cells cultured in a perfused three-dimensional bioreactor. , 2013, Stem cells and development.

[50]  E. Kroon,et al.  Efficient differentiation of human embryonic stem cells to definitive endoderm , 2005, Nature Biotechnology.

[51]  E. Schuetz,et al.  Regulation of cytochrome P-450b/e and P-450p gene expression by growth hormone in adult rat hepatocytes cultured on a reconstituted basement membrane. , 1990, The Journal of biological chemistry.

[52]  Karin Noaksson,et al.  Facilitated Expansion of Human Embryonic Stem Cells by Single‐Cell Enzymatic Dissociation , 2007, Stem cells.

[53]  E. Wauthier,et al.  Mature Human Hepatocytes fromEx VivoDifferentiation of Alginate-Encapsulated Hepatoblasts , 2008 .

[54]  Clare Selden,et al.  Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia , 2008, Biotechnology and bioengineering.

[55]  Primary hepatocyte culture: substratum requirements and production of matrix components. , 1981, Federation proceedings.

[56]  J. Yager,et al.  A morphological study of differentiated hepatocytes in vitro , 1995, Hepatology.

[57]  C. Verfaillie,et al.  Stem cells and liver engineering. , 2013, Biotechnology advances.

[58]  J. Itskovitz‐Eldor,et al.  Directed differentiation of human embryonic stem cells into functional hepatic cells , 2007, Hepatology.

[59]  J. Lemasters,et al.  Evidence that activation of Kupffer cells increases oxygen uptake after cold storage. , 1994, Transplantation.

[60]  Shichang Zhang,et al.  Hepatic differentiation of mouse embryonic stem cells in three-dimensional polymer scaffolds. , 2010, Tissue engineering. Part A.

[61]  Wei Yang,et al.  Tight Junction Proteins Claudin-1 and Occludin Control Hepatitis C Virus Entry and Are Downregulated during Infection To Prevent Superinfection , 2008, Journal of Virology.

[62]  J. Gerlach,et al.  Membranes as Substrates for Hepatocyte Adhesion in Liver Support Bioreactors , 1990, The International journal of artificial organs.

[63]  J. Gerlach.  Long-term liver cell cultures in bioreactors and possible application for liver support , 1997, Cell Biology and Toxicology.

[64]  Sean P. Palecek,et al.  Engineering the human pluripotent stem cell microenvironment to direct cell fate. , 2013, Biotechnology advances.

[65]  D. Hay,et al.  Pluripotent stem cell-derived hepatocytes: potential and challenges in pharmacology. , 2013, Annual review of pharmacology and toxicology.

[66]  N. Kobayashi,et al.  Stem cell research and therapy for liver disease. , 2009, Current stem cell research & therapy.

[67]  P. Neuhaus,et al.  Hepatocyte Aggregate Culture Technique for Bioreactors in Hybrid Liver Support Systems , 1993, The International journal of artificial organs.

[68]  D. Moradpour,et al.  Hepatitis C virus infection: in vivo and in vitro models , 2007, Journal of viral hepatitis.

[69]  G. Horgan,et al.  Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR , 2002 .

[70]  Toshio Miki,et al.  Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro. , 2010, Tissue engineering. Part C, Methods.

[71]  Wei-Shou Hu,et al.  Structural polarity and functional bile canaliculi in rat hepatocyte spheroids. , 2002, Experimental cell research.

[72]  J. Vienken,et al.  Gas supply across membranes in bioreactors for hepatocyte culture. , 1990, Artificial organs.

[73]  P. Neuhaus,et al.  Extracorporeal liver support based on primary human liver cells and albumin dialysis--treatment of a patient with primary graft non-function. , 2003, Journal of hepatology.

[74]  J. Encke,et al.  Bioreactor for a larger scale hepatocyte in vitro perfusion. , 1994, Transplantation.