Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.

Hedgehog signaling through its receptor, Patched, activates transcription of genes, including Patched, that regulate the fate of various progenitors. Although Hedgehog signaling is required for endodermal commitment and hepatogenesis, the possibility that it regulates liver turnover in adults had not been considered because mature liver epithelial cells lack Hedgehog signaling. Herein, we show that this pathway is essential throughout life for maintaining hepatic progenitors. Patched-expressing cells have been identified among endodermally lineage-restricted, murine embryonic stem cells as well as in livers of fetal and adult Ptc-lacZ mice. An adult-derived, murine hepatic progenitor cell line expresses Patched, and Hedgehog-responsive cells exist in stem cell compartments of fetal and adult human livers. In both species, manipulation of Hedgehog activity influences hepatic progenitor cell survival. Therefore, Hedgehog signaling is conserved in hepatic progenitors from fetal development through adulthood and may be a new therapeutic target in patients with liver damage.

Human hepatoblasts and hepatic stem cells, pluripotent hepatic progenitors that give rise to hepatocytes and biliary cells, were isolated from fetal livers and found to express hyaluronan receptors (CD44) in both the freshly isolated cells and after culture. This implicates an in vivo connection to hyaluronan (HA), an embryonic matrix component, as a candidate 3-dimensional (3-D) scaffold for hepatic progenitor cell expansion and/or differentiation. To assess HAs as scaffolds, hepatoblasts and hepatic stem cells were seeded into HA hydrogels with a serum-free, hormonally defined medium tailored for expansion of hepatic progenitors. Cell aggregates formed within the HA hydrogels and remained viable, proliferative, and demonstrated a stable phenotype intermediate between that of hepatic stem cells and hepatoblasts throughout more than 4 weeks of culturing, with little evidence of lineage restriction towards either hepatocytic or biliary pathways. The phenotype consisted of stable co-expression of both hepatocytic and biliary markers such as biliary-specific cytokeratin, CK19, low levels of expression of albumin, and urea synthesis. HA hydrogels are ideal as 3-D scaffolds for pluripotent hepatic progenitors and should be useful for generating cells to be used in bioartificial livers or tissue engineered liver grafts.

Cell therapies are potential alternatives to organ transplantation for liver failure or dysfunction but are compromised by inefficient engraftment, cell dispersal to ectopic sites, and emboli formation. Grafting strategies have been devised for transplantation of human hepatic stem cells (hHpSCs) embedded into a mix of soluble signals and extracellular matrix biomaterials (hyaluronans, type III collagen, laminin) found in stem cell niches. The hHpSCs maintain a stable stem cell phenotype under the graft conditions. The grafts were transplanted into the livers of immuno-compromised murine hosts with and without carbon tetrachloride treatment to assess the effects of quiescent versus injured liver conditions. Grafted cells remained localized to the livers resulting in a larger bolus of engrafted cells in the host livers under quiescent conditions and with potential for more rapid expansion under injured liver conditions. By contrast, transplantation by direct injection or via a vascular route resulted in inefficient engraftment and cell dispersal to ectopic sites. Transplantation by grafting is proposed as a preferred strategy for cell therapies for solid organs such as liver.

Establishing suitable oxygen transport pathways within bioartificial liver replacement devices continues to be an important engineering challenge. Oxygen delivery is critical since this is one of the nutrients necessary to maintain hepatocyte viability and function. In the current study, the microporosity of a collagen extracellular matrix has been modified to permit both diffusion and convection mass transport. Using fluorescent visualization, the enhancement technique was found to extend the oxygen transport distance from 170 microns to 360 microns. Furthermore, in hepatocyte culture studies, the enhancement technique was observed to yield a sixfold increase in the amount of viable hepatocytes able to be sustained by a single O2 source. Normalized function studies confirm that hepatocyte function was also improved in the enhanced collagen configurations.