Human hepatoblast phenotype maintained by hyaluronan hydrogels
William S. Turner , Eva Schmelzer, Randall McClelland, Eliane Wauthier, Weiliam Chen, Lola M. Reid
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.
5 comments:
how long is the phenotype maintained? In other words how long is "long culture periods"? Is is long enough to be useful in therapies?
How much HA was added? Did the amount of HA that was added affect the cultures or the ability of the cells to be maintained?
What other glycosaminoglycans have been tested in hydrogels and for what other types of cells? What is it about HA that makes it so successful in helping maintain healthy liver tissue?
Clare - according to the findings, the phenotype of intermediate cells (early stage hepatoblasts) between hepatic stem cells and hepatoblasts was stable for greater than four weeks in hyaluronan. In contrast, hepatoblasts cultured on tissue culture plastic lasted only 7-10 days. Unfortunately, the paper does not specify if this is long enough to be useful in therapies. However, it more importantly emphasizes that the use of HA is one step closer to creating scaffolds that enhance the viability and phenotype of early stage hepatoblast cells, which in the future could lead to improvements in therapy.
Adeeti - According to the paper, a 1% aqueous HA solution was crosslinked with 1N HCl to create HA scaffolds. The expansion potential was found to be fairly low with only 2.28% increase/day, which they believe could have been affected by the high resistance to dissolution of the scaffold (in vivo dissolution of HA allows is necessary for the creation of tissue/organs) or the stiffness of the gel. Unfortunately, there were no tests on the effect of various amounts of HA on the phenotypes/viability of the cells. The authors, however, did mention future work on increasing the strength, flexibility, and cross-linking density of HA scaffolds, to increase expansion of cells. Alternate types of HA and ones biosynthesized with ECM components are also being explored.
Zach - The paper did not specify if any other glycosaminoglycans have been tested. The authors used HA because it mimics in vivo conditions in the liver where liver stem cells are stored. Unfortunately, there is no explanation other than it being a naturally occurring ECM for its success in maintaining healthy liver tissue.
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