title: Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.
abstract: Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.
authors.affiliation: 1 Department of Chemical Engineering and Materials Science, University of Minnesota , Minneapolis, Minnesota.
authors.forename: Kartik
authors.initials: K
authors.lastname: Subramanian
authors.forename: Derek Jason
authors.initials: DJ
authors.lastname: Owens
authors.forename: Ravali
authors.initials: R
authors.lastname: Raju
authors.forename: Meri
authors.initials: M
authors.lastname: Firpo
authors.forename: Timothy D
authors.initials: TD
authors.lastname: O'Brien
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Verfaillie
authors.forename: Wei-Shou
authors.initials: WS
authors.lastname: Hu
mesh_terms: D000418:Albumins / Q000096:biosynthesis; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D037263:Asialoglycoprotein Receptor / Q000096:biosynthesis; D002454:Cell Differentiation; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D053595:Embryonic Stem Cells / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology*; D010078:Oxazines / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000096:biosynthesis; D018874:Spheroids, Cellular / Q000378:metabolism*
chemical_list: C546927:ASGR1 protein, human; D000418:Albumins; D037263:Asialoglycoprotein Receptor; D010078:Oxazines; C014180:resorufin; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C569463:CYP1A1 protein, human; C504573:CYP1A2 protein, human; C585599:CYP2B6 protein, human; C107082:CYP2B7P protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; D019764:Phosphoenolpyruvate Carboxykinase (ATP)
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