Ex vivo conditions for self-replication of human hepatic stem cells

Human hepatic stem cells (hHpSCs), identifiable by a unique antigenic profile, have been isolated from human livers and established ex vivo under expansion conditions permissive for self-replication. The conditions consist of a substratum of type III collagen, ideally on Transwell inserts, and Kubota's medium, a serum-free medium developed for hepatic progenitors. Under these conditions the cells demonstrated a doubling time of approximately 24 h, generating at least a 16-fold increase in cell number within 7-10 days; were stable at confluence for up to 2 weeks; could be passaged, if on type III collagen, to initiate colonies that went through log-phase growth and saturation density kinetics; and expressed telomerase, indicative of regenerative capacity. The hHpSC colonies remained morphologically and phenotypically stable throughout expressing epithelial cell adhesion molecule, neural cell adhesion molecule, albumin, cytokeratins 8, 18, and 19, but not alpha-fetoprotein, or intercellular adhesion molecule-1 (ICAM-1). Those maintained under self-replication conditions for more than a month were transplanted and found to engraft in the livers of SCID/nod mice yielding human liver tissue expressing adult liver-specific proteins. The conditions for self-replication should offer ideal culture conditions for generating large numbers of hHpSCs for use in commercial and clinical programs.