(170f) Directed Differentiation of Human Embryonic Stem Cells and Multipotent Adult Progenitor Cells Toward a Hepatic Cell Fate

Human embryonic stem cells (hESCs), derived from the inner cell mass of the blastocyst stage of the human embryo, exhibit virtually unlimited self-renewal and pluripotent differentiation potential with the ability to differentiate into every cell type of the adult human. Multipotent adult progenitor cells (MAPCs), derived from the bone marrow of adult rodents, swine, or humans, demonstrate a higher level of self-renewal and potency than traditional adult stem cell populations, exhibiting self-renewal in vitro for extended periods of culture time and multipotent lineage differentiation to cell types of all three germ layers. The ability of these stem cell populations to differentiate into hepatocytes holds the potential for applications in liver cellular therapies, drug toxicity studies, bioartificial liver devices, and for use as model systems for studying development. We have recently demonstrated the ability to direct the differentiation of both hESCs and rat MAPCs (rMAPCs) toward a hepatic fate via a four-step, 20 day differentiation protocol designed to mimic the sequential signaling events experienced by cells in the developing embryo. The progression and efficiency of differentiation have been evaluated by quantification of gene expression of definitive endoderm markers, including Goosecoid and CXCR4, hepatic precursor markers AFP and Transthyretin, and mature hepatocyte markers such as albumin, alpha-1-antitrypsin, and others, each of which demonstrates a time-dependent expression pattern during differentiation that is consistent with developmental events. The maturity of hepatocytes was determined by functional assays such as albumin and urea secretion, glycogen storage, and glutathione S-transferase activity in addition to the presence of hepatocyte-specific protein products, such as albumin and PEPCK, as determined by immunohistochemistry and fluorescence-activated cells sorting (FACS). We have further demonstrated the ability of rMAPCs to self-assemble into three-dimensional aggregates and the ability to both maintain and expand these aggregates without loss of potency, as demonstrated by expression of transcription factors, such as Oct4, and the ability to differentiate into cell types of all three germ layers. Previous studies have shown that primary hepatocytes maintain function and viability longer in culture when allowed to assemble into three-dimensional aggregates, or spheroids. We are therefore currently evaluating the potential of directed differentiation of these three-dimensional aggregates to improve the efficiency of differentiation toward the hepatic cell fate during the 20 day differentiation. In addition to the 3D culture system, we are also investigating the ability to further enhance differentiation, both in efficiency and in functional maturity of the hepatocytes, by selection of hepatic precursor populations and by studying their temporal and spatial interactions with other cell types by promoter-reporter transgenic techniques. Episomal vectors are transgenic vector systems that are maintained episomally, without integration in the host genome, and replicate once per cell cycle along with the host. These vectors are therefore ideal for stem cell systems because they escape silencing, undesired phenotypic changes, and other limitations inherent to other transgenic systems. Two episomal vectors, one expressing GFP from a constitutive promoter (pEP-EG) and one expressing GFP from the alpha feto protein (AFP) promoter (pEP-AFP-GFP), were used to investigate the spatial and temporal distribution of hepatic precursors in vitro during differentiation. Confocal laser scanning microscopy was used to visualize the location of hepatic precursors during differentiation and to observe their interactions with other cell types. Confocal microscopy was also used to visualize the localization of mature hepatocytes by functional assays such as cytochrome P450 activity. The episomal vectors were further used to select for a population of hepatic precursors by sorting of AFP-GFP+ cells at different times during differentiation and to investigate the potential to increase functional maturity of stem cell-derived hepatocytes by enrichment of these precursors. These studies provide insight into the mechanisms of differentiation and into the roles played by other cell types, such as endothelial cells, during the differentiation of stem cells toward a hepatic fate.