Hepatocyte transplantation may provide a therapy for a variety of liver diseases. The applicant hypothesizes that the inability of current hepatocyte transplant systems to provide long-term replacement of lost or deficient liver function may be caused by a loss of liver-specific gene expression in the transplanted cells. The applicant proposes to quantitate and experimentally optimize the liver-specific gene expression of transplanted hepatocytes. A large number of previous studies have indicated that the phenotype of cultured hepatocytes is regulated both by the presence of soluble hormones or growth factors and by cell adhesion to specific extracellular matrix (ECM) adhesion ligands. The applicant hypothesizes that the liver-specific phenotype of transplanted hepatocytes can be maintained by presenting them with specific and defined combinations of ECM molecules and growth factors. Controlled drug delivery technology will be utilized to incorporate hepatocyte growth factors and cofactors (epidermal growth factor, insulin and dexamethasone) into polymer microspheres which will release the factors over extended periods of time. The polymer microspheres will be mixed with hepatocyte suspensions and seeded into hollow fibers with a defined ECM gel matrix (laminin, type I collagen, alginate, and no ECM). Hollow fiber devices fabricated with semipermeable membranes will be used to prevent cellular migration into or out of hollow fibers, and thus prevent alteration of the ECM by cells other than the seeded hepatocytes. The ability of specific combinations of growth factors and ECM matrices to promote long-term maintenance of liver-specific gene expression in vitro and in vivo will be tested. The alterations in the ECM surrounding hepatocytes will be analyzed to determine whether cells present in different microenvironments selectively secrete and organize a new matrix over time that supplements the original ECM. These studies may aid in bringing hepatocyte transplantation closer to a clinically relevant therapy. Additionally, the issues addressed in this proposal are common to any attempt to engineer a functional new tissue, and novel systems developed in this proposal may find application in a variety of engineered tissues.
