Isolation and differentiation of adult and embryonic stem cells into hepatocytes in vitro

Liver dysfunction is a major health problem in the United States with a variety of causes including acute and chronic liver failure, metabolic disorders, and congenital defects. Over the past few decades, several solutions to support liver function have been proposed and tested. Original work focused on an extracorporeal device much like that used in kidney dialysis. It has become clear that a biological component to the device to replace hepatic function is needed. Despite initial successes with porcine hepatocytes as the biological component in the device, safety worries have hampered implementation of such a bioartificial device. A high quality and steady source of safe, mature and functioning hepatocytes are needed. We investigated whether different stem cell populations can generate functional hepatocyte-like cells in vitro and thus serve as a source of hepatocytes for the "bioartificial liver". We have identified primitive cells in normal human, mouse and rat post-natal bone marrow, termed Multipotent Adult Progenitor Cell or MAPC, that differentiate into most endodermal cells, mesodermal cells, and neuroectodermal cells in vitro, and in vivo into all embryological lineages. We investigated whether MAPC can also differentiate into hepatocytes in vitro. Human, mouse and rat MAPC were cultured on Matrigel(TM) with FGF4 and HGF. Differentiation occurred into epithelioid cells staining positive for endodermal and immature hepatic markers by day-7, and mature hepatic markers by day-28, shown by immunofluorescence, quantitative RT-PCR and Western blot. FGF4 and HGF induced MAPC also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital inducible cytochrome p450, could take up LDL, and stored glycogen. Using a similar methodology, we investigated mouse and human embryonic stem cell differentiation into hepatocytes in vitro. Tests of several different conditions determined that addition of FGF-4 and hepatocyte growth factor in completely serum-free cultures of mES and hES cell-derived embryoid bodies subsequently allowed to attach to type I collagen coated dishes lead to maximal differentiation into cells not only with the morphologic and phenotypic characteristics of hepatocytes but the functional characteristics as well. Thus, MAPC and hES may be an ideal source of cells for use in bioartificial liver devices.