Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats

Abstract

Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. Tissue determined LPCs may reside in or near the terminal biliary tree, but it has been difficult to identify authentic LPCs in the normal liver. 2,3 One of the principle lines of evidence for existence of LPCs is appearance of oval cells after different kinds of liver injury, which differentiate into either hepatocytes or cholangiocytes. For example, using carcinogenic regimens where N-2-acetylaminofluorene (AAF) inhibits proliferation of mature hepatocytes, 4,5 oval cells appear in large numbers in duct-like structures. 6,7 Similarly, marked expansions of ductular oval cells occur when centrolobular injury is induced by CCl 4 and bile ductular hyperplasia is stimulated by bile duct ligation 8 or hepatocyte proliferation is inhibited by AAF. 9,10 Although these observations suggest that LPCs exist within biliary ducts, other models implicate nonductal cells in the portal triad. 11 Thus, nondescript periductular oval cells proliferate within 2 days after allyl alcohol (AA)-induced periportal injury, and 2 to 4 days later the periportal necrotic zone is replaced by an expanding population of oval cells and transitional hepatocytes, with little ductular reaction. 12-14 Based on these and earlier observations, 2 we postulated that nontransformed LPC lines, derived from oval cells induced after AA injury, might exhibit different stages of liver-cell differentiation and bipotential capacities of differentiation. We now describe the isolation and characterization of 11 LPC strains and lines from adult livers after periportal injury induced by AA. Materials and Methods Materials.The following materials were obtained commercially: dexamethasone and bFGF (Sigma); Oncostatin M (R&D Systems, Minneapolis, MN); Matrigel (Collaborative Research, Bedford, MA); human epidermoid carcinoma cell line A431 lysates (Santa Cruz Biotechnology, Santa Cruz, CA).Derivation, Culture, and Storage of LPC Strains. Nineweek-old Fisher/344 female rats weighing 140-150 g were obtained from Harlan, India...

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

“…Interestingly, subsequent to isolation, Thy1 mRNA levels appeared to increase in c-kit-positive cell clusters in culture, indicating that Thy1 may be induced in these cells. In agreement with the latter, all functional repopulating HSCs were shown to be contained within a c-kit + /lin − /Sca-1 + BM population [45] and liver progenitor cells cloned from adult rat livers following allyl alcohol injury co-expressed both Thy1 and c-kit [46]. Thus, expression of Thy1 may be induced in c-kit + cells subsequent to injury to promote transdifferentiation of HSCs towards a hepatic progenitor phenotype.…”

“…However, a distinct cell subpopulation retained the ability to propagate in the presence of iMEFs, a prerequisite shared by several hepatic progenitor as well as hematopoietic stem cell lines expressing c-kit and/or Thy1 [46,48,49]. Consistent with the latter, c-kit and Thy1 mRNA were detected in cell clusters, suggesting that these were derived from c-kit-positive HSCs.…”

2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: Confocal and electron microscopic studies

“…25 The hepatocytes have no contact with laminin containing structured basement membrane. Yin et al 26 reported that isolated hepatic stem cells expressed biliary or hepatocytic phenotypes in culture, depending on the presence or absence of basement membrane matrix (Matrigel). Matrigel also was found to play an important role in maintaining the biliary phenotype in tissue culture in other experimental systems.…”

Embryonic Mouse STO Cell–Derived Xenografts Express Hepatocytic Functions in the Livers of Nonimmunosuppressed Adult Rats

“…To investigate differentiation potential of a putative rat liver progenitor cell line, 3 (8)21-EGFP cells [27] were injected into spontaneously mutated DPPIV -rats [21,28]. The DPPIV gene is naturally mutated in DPPIV -rats with lack of cell surface expression and enzyme activity.…”

“…Similarly, stem or progenitor cells lacking DPPIV activity at the outset can be identified in the liver of DPPIV -recipients after cell differentiation along appropriate lineages [21][22][23]. As previously described, 3(8)21-EGFP cells were clonally derived specifically from cocultures of putative 3(8)21 rat liver progenitor cells and STO cells that had been retrovirally transduced with EGFP and neomycin resistance (neo R ) genes [27]. Thus, because 3(8)21-EGFP cells were thought to be derived from the rat liver progenitor cells, they were assumed to be syngeneic with DPPIV -rats and to express properties of adult hepatocytes or biliary cells [27].…”