Hyaluronan hydrogels, 3-dimensional scaffolds for ex vivo maintenance of human hepatic stem cells and hepatoblasts Public Deposited

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  • March 21, 2019
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  • Turner, William S.
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
Abstract
  • Sourcing of human liver cells has remained a challenge obviating ex vivo studies of human cells and clinical therapies such as cell transplantation and assist devices using bioartificial livers. The expansion potential of human hepatic progenitors under appropriate conditions can overcome this problem, and the potential medical applications include the treatment of liver disease by cell therapies such as cell transplantation and/or bioartificial livers. Human hepatic stem cells (hHpSCs) in vivo are located in ductal plates in fetal livers and neonatal livers and in canals of Herring in pediatric and adult livers, are ~9 [mu]m in diameter, can be isolated by immunoselection for epithelial cell adhesion molecule (EpCAM) or neural cell adhesion molecule (NCAM), and constitute ~ 0.5-1.5% of parenchymal cells in non-ischemic livers of all donor ages. They express cytokeratins 8, 18 and 19, CD133/1, CD44H, telomerase, NCAM, weakly express albumin, are negative for ICAM-1, for markers for adult liver cells (e.g. P450s), hemopoietic cells (e.g. CD45, CD34), endothelia (e.g. VEGFr, Van Willebrand Factor), and other mesenchymal cells ([alpha]-smooth muscle actin, desmin, CD146) and for [alpha]-fetoprotein (AFP). Self-replication (also called self-renewal) ex vivo occurs in hHpSCs cultured in a serum-free medium tailored for hepatic progenitors, "Kubota's Medium" (KM), yielding doubling times of ~35 hrs (plastic) or <24 hrs (type III collagen) and a stable phenotype for more than 150 population doublings. Colonies of hHpSCs are in close association with angioblasts and hepatic stellate cell precursors; elimination of them results in slowed growth and reduced viability. Ex vivo expansion conditions comprise serum-free KM with varying soluble signal supplements in combination with monolayer substrata versus 3-dimensional (3-D) scaffolds of purified extracellular matrix components (used individually or in combinations). The best of the 3-D scaffolds for the hHpSCs and their progeny, hepatoblasts, and committed progenitors have thus far proven to be complexes of chemically modified hyaluronans combined with other matrix components. The precise mix of matrix molecules with the hyaluronans enables the cells to be in a 3-dimensional format and yet kept in an undifferentiated state (certain combinations) versus driven to differentiate (other combinations). The hydrogels offer stable support of the cells with ex vivo maintenance for longer than four weeks in culture. Human hepatic progenitors cultured in the hydrogels and in KM have been characterized by classic cell biological techniques including growth analyses, immunohistochemistry and RT-PCR. In parallel, nuclear magnetic resonance spectroscopy (NMR) has been used to monitor the cells without the use of techniques that destroy them. Metabolomic profiling of the cells within their 3-D microenvironment has allowed tracking of metabolites such as glucose, lactate, glutamine, alanine and others which allows for the elucidation of the cellular regulation of glycolysis, the Krebs cycle and other unique metabolic pathways.
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  • Reid, Lola
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