Rat liver contains progenitor cells located by each of the portal triads and which produce daughter cells that mature through a unidirectional, differentiation process ending at the central vein. Thus, the plates of parenchymal cells within each acinus (in vivo) are lineages of maturing liver cells with age-dependent size, ploidy, growth and differentiative potential. We propose to use these progenitor cells, purified by multiparametric fluorescence activated cell sorting, to establish a bioartificial liver. The cells will be seeded into a hollow fiber bioreactor, of novel design, and under defined ex vivo culture conditions that are entirely or mostly serum-free, contain defined and purified extracellular matrix components as substratum, and defined and purified soluble signals (hormones, growth factors, nutrients). Protocols have been developed, and antigenic profiles defined by which to identify and isolate three subpopulations of hepatic progenitors and two subpopulations of mature parenchymal cells using a combination of panning and multiparametric fluorescence activated cell sorting (FACS): hepatoblasts (pluripotent hepatic progenitors); committed biliary and hepatocytic progenitors; periportal parenchymal cells (presumptive young parenchyma); and 5) pericentral parenchymal cells (presumptive old parenchyma). Also developed are in vivo bioassays for fate studies, ex vivo conditions that permit cell expansion and others that drive differentiation of each of the progenitor subpopulations. The rat bioartificial liver will be established from each of the 5 subpopulations of maturationally staged parenchymal cells by seeding them onto porous, biodegradable mircocarriers coated with matrix components, into a novel form of hollow fiber bioreactor and under appropriate ex vivo expansion conditions. A separate bioreactor for feeder cells will be established and will contain two feeder cell types found to yield paracrine signals that are strict requirements for growth of the progenitors: 1) FACS-purified hemopoietic OCAP cells (myeloid cells that bear an oval cell antigen 3+, OC3+) and the STO embryonic stromal cell line, that has recently been found to replace primary cultures of age- and liver-specific stromal feeder cells (from E14-E16 livers). The bioreactors with the feeder cells will be coupled in tandem with the ones containing the hepatic progenitor cells; if the factors produce by the feeders are too labile to survive the tandemly connected bioreactors, the feeder cells will be treated with mitomycin C and co-seeded into the same bioreactors with the hepatic progenitors. The bioartifical livers and control monolayer cultures will be characterized for fetal and adult liver-specific functions and for hemopoietic markers by means of the Johnson and Johnson dry slide assays, by immunochemistry, biochemical assays, molecular hybridization assays and by flow cytometric analyses. In addition, the bioreactors will be characterized non-invasively using nuclear magnetic resonance and magnetic resonance imaging. The fates of the cells in the bioreactors will be compared with those identified from in vivo bioassays.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052851-03
Application #
6177720
Study Section
Surgery and Bioengineering Study Section (SB)
Program Officer
Serrano, Jose
Project Start
1998-06-24
Project End
2002-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
3
Fiscal Year
2000
Total Cost
$305,528
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Physiology
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Turner, Rachael A; Wauthier, Eliane; Lozoya, Oswaldo et al. (2013) Successful transplantation of human hepatic stem cells with restricted localization to liver using hyaluronan grafts. Hepatology 57:775-84
Jeffries, Rex E; Macdonald, Jeffrey M (2012) New advances in MR-compatible bioartificial liver. NMR Biomed 25:427-42
Lozoya, Oswaldo A; Wauthier, Eliane; Turner, Rachael A et al. (2011) Regulation of hepatic stem/progenitor phenotype by microenvironment stiffness in hydrogel models of the human liver stem cell niche. Biomaterials 32:7389-402
Turner, Rachael; Lozoya, Oswaldo; Wang, Yunfang et al. (2011) Human hepatic stem cell and maturational liver lineage biology. Hepatology 53:1035-45
Wang, Yunfang; Yao, Hsin-Lei; Cui, Cai-Bin et al. (2010) Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates. Hepatology 52:1443-54
Schmelzer, Eva; Reid, Lola M (2009) Human telomerase activity, telomerase and telomeric template expression in hepatic stem cells and in livers from fetal and postnatal donors. Eur J Gastroenterol Hepatol 21:1191-8
McClelland, Randall; Wauthier, Eliane; Zhang, Lili et al. (2008) Ex vivo conditions for self-replication of human hepatic stem cells. Tissue Eng Part C Methods 14:341-51
Wauthier, Eliane; Schmelzer, Eva; Turner, William et al. (2008) Hepatic stem cells and hepatoblasts: identification, isolation, and ex vivo maintenance. Methods Cell Biol 86:137-225
Schmelzer, Eva; Reid, Lola M (2008) EpCAM expression in normal, non-pathological tissues. Front Biosci 13:3096-100
Schmelzer, Eva; Zhang, Lili; Bruce, Andrew et al. (2007) Human hepatic stem cells from fetal and postnatal donors. J Exp Med 204:1973-87

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