Human hepatoblast phenotype maintained by hyaluronan hydrogels

William S. Turner 1 *, Eva Schmelzer 2, Randall McClelland 2, Eliane Wauthier 2, Weiliam Chen 3, Lola M. Reid 1 2 4 *

1Department of Biomedical Engineering, UNC School of Medicine, Chapel Hill, North Carolina 27599 2Department of Cell and Molecular Physiology, UNC School of Medicine, Chapel Hill, North Carolina 27599 3Department of Biomedical Engineering, State University of New York, Stony Brook, New York 11794-2580 4Program in Molecular Biology and Biotechnology, Lineberger Cancer Center, UNC School of Medicine, Chapel Hill, North Carolina 27599

J Biomed Mater Res B Appl Biomater. 2006 Dec 20

The matrix of the liver in fetal and embryonic tissues contain significant amounts of hyaluronan (HA), a glycosaminoglycan that aids in matrix stabilization, facilitation of cell migration, transport regulation, acting as a hormonal reservoir, and water/protein homeostasis. In adult liver tissue it is found in the presumed stem cell compartment called the Canals of Hering, often present when adult tissues are undergoing cellular expansion, wound repair, and regeneration. Therefore, because they are in association with hepatic stem cells and their immediate descendants heptoblasts, HAs are hypothesized to be candidates for matrix components in 3-D scaffolds for creating ex-vivo cultures of hepatic progenitors.

In this study, parenchymal cells (hepatoblasts) were isolated from liver tissue from human fetuses. HA matrices were created by crosslinking a pre-made HA solution, which became highly porous HA spongy hydrogels that absorbed water readily. The cultured progenitor cells, rich in hepatoblasts, were then seeded into the HA hydrogels and also in collagen gels and on culture plastic, which were both used as controls. The cultures were then fixed with paraformaldehyde and micro-sectioned for observation; analyzed for albumin and urea production; RNA, DNA, and protein were isolated and quantified; and gene expression was analyzed via quantitative real time RT-PCR. They were also stained with immunofluorescence outside HA hydrogels (using primary and secondary antibody coupling for HA receptors).

Results showed that HA hydrogels were able to maintain early staged hepatic progenitors in viable, proliferative, and phenotypically stable state over long culture periods. Immunostaining showed that human hepatic stem cells and hepatoblasts were positive for the specific HA receptor, CD44, and uptook the conjugates at higher rates than the other cells in the culture, such as stroma and endothelial cells. In the HA hydrogels, cells showed considerable aggregation and expansion and an increase in DNA production over time. Furthermore, hepatoblasts in HA hydrogels also produced less albumin and survived in culture longer than hepatoblasts in plastic culture. Urea production decayed slower in HA hydrogels than in the other two culture conditions. Both results showed that ability of HA hydrogels to maintain hepatoblast phenotype in culture as high levels of albumin and high decay rates of urea are characteristics of mature cells, while the opposite holds true for progenitor cells.

This research was significant as it explored and affirmed the possibility of creating ex-vivo constructs of liver tissue. So far, HAs have been the only culture condition discovered to allow the viability, proliferability, and maintenance of hepatoblasts. Because they will allow for the maintenance of a larger variety of specific cell lineage states (since hepatoblasts are pluripotent), HA hydrogels will be a prime reservoir for replenishing liver tissue/cells in medical research and cell therapy. Furthermore, this article was also significant as it contained many of the assays and imaging techniques that were discussed in lecture and performed in lab.