The present invention relates to a population of cells comprising hepatic stem-like cells and therapeutic use thereof, for the treatment and the prevention of fulminant liver disorders. The hepatic stem-like cells according to the invention may be safely and reproducibly generated from pluripotent stem cells. In addition, although the hepatic stem-like cells according to the invention do not display the phenotype of physiologically mature hepatic cells, as they are lacking the albumin expression marker (ALB), they may still be transplanted in a diseased liver with acute failure, rescue the diseased liver and promote liver regeneration. Moreover, various protocols of preparation of hepatic stem-like cells according to the invention may be implemented, all resulting in high quality and high yield of production. Finally, the hepatic stem-like cells according to the invention may be cryopreserved and may also be prepared as spheroid particles.

The present invention relates to directed differentiation and maturation of mammalian hepatocytes, such as human hepatocytes. The hepatocyte obtained in accordance with the present invention show a phenotype which is more similar to that of primary hepatocytes than previously shown. In particular, the present invention relates to exposure of mammalian hepatocytes, such as human hepatocytes, to at least one maturation factor selected from the group consisting of Src kinase inhibitors, vitamin D including precursors, metabolites and analogs thereof, hypoxia inducing compounds, sphingosine and sphingosine derivatives, activators of peroxisome proliferator-activated receptors (PPARs), platelet-activating factor (PAF), PKC inhibitors, and combinations thereof.

Directed differentiation and maturation of mammalian hepatocytes, such as human hepatocytes. The hepatocyte obtained show a phenotype which is more similar to that of primary hepatocytes than previously shown. In particular, exposure of mammalian hepatocytes, such as human hepatocytes, to at least one maturation factor selected from the group consisting of Src kinase inhibitors, vitamin D including precursors, metabolites and analogs thereof, hypoxia inducing compounds, sphingosine and sphingosine derivatives, activators of peroxisome proliferator-activated receptors (PPARs), platelet-activating factor (PAF), PKC inhibitors, and combinations thereof.

The present invention provides, in order to prepare matured hepatocytes analogous in various points to primary hepatocytes, a method for preparing hepatocytes or cells that can be differentiated into hepatocytes from pluripotent stem cells, comprising the steps of: (1) culturing the pluripotent stem cells in a medium containing an activator of an activin receptor-like kinase-4,7; (2) culturing the cells obtained in the step (1) in a medium containing a bone morphogenetic factor and a fibroblast growth factor; (3) culturing the cells obtained in the step (2) in a medium containing an activator of a hepatocyte growth factor receptor and an activator of an oncostatin M receptor; and (4) culturing the cells obtained in the step (3) to obtain hepatocytes or cells that can be differentiated into hepatocytes, wherein in at least one of the steps (2), (3) and (4), cells are cultured on a high-density collagen gel membrane.

Methods and materials are described for producing immortalized hybrid cells composed of a fusion of immortalized multi-lineage progenitor cells (MLPC) and primary hepatocytes. The hybrid cells express the biological activity of the primary hepatocytes and the immortality and expansion capacities of the immortalized MLPC. The methods of culture and expansion of the resultant hybrid cells and the methods to confirm the characteristics of the hybrid cells are described.

Culture medium formulations are described that are capable of large-scale expansion of stem cell-derived hepatocyte-like cells while sustaining the activity of those cells to maintain the phenotype and biological characteristics of normal hepatocytes.

Provided is a selective method for inducing differentiation from pluripotent stem cells to enterocyte-like cells. Also provided is an excellent enterocyte-like cell expressing drug-metabolizing enzymes and drug transporters. More specifically, provided is an enterocyte-like cell having properties closer to those of primary enterocytes, which are difficult to acquire. The foregoing is achieved by adding an ALK5 inhibitor (SB431542), Wnt3a, and EGF to a culture system of definitive endoderm cells obtained by differentiation induction from pluripotent stem cells and extending a culture time. The foregoing is also achieved by introducing CDX2 gene and/or FOXA2 gene into the pluripotent stem cells or the definitive endoderm cells. The foregoing is also achieved by overlaying a basement membrane matrix on the enterocyte-like cells.

Disclosed herein are human hepatocytes for example isolated human hepatic progenitor cells, artificial tissue or organ thereof, and kits or compositions thereof. Also disclosed herein are various methods of using a human hepatocyte disclosed herein.

Methods for producing hepatocytes from pluripotent human stem cells are disclosed herein. The stem cells are plated on a cell culture substrate comprising two laminins. The stem cells are then exposed to different cell culture mediums to induce differentiation. The resulting hepatocytes have higher metabolic capacity compared to hepatocytes cultured on different substrates.

Provided is an isolated population of human pluripotent stem cells comprising at least 50% human pluripotent stem cells characterized by an OCT4+/TRA1-60/TRA1-81/SSEA1+/SSEA4 expression signature, and novel methods of generating and maintaining same in a pluripotent, undifferentiated state a suspension culture devoid of cell clumps. Also provided are novel culture media, cell cultures and methods for culturing pluripotent stem cells in a suspension culture or a two-dimensional culture system while maintaining the cells in a proliferative, pluripotent and undifferentiated state. The novel culture media comprise interleukin 11 (IL11) and Ciliay Neurotrophic Factor (CNTF); bFGF at a concentration of at least 50 ng/ml and an IL6RIL6 chimera; or an animal contaminant-free serum replacement and an IL6RIL6 chimera. Also provided are methods for generating lineage-specific cells from the pluripotent stem cells.