Abstract
The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.
Highlights
The liver is capable of regenerating after surgical resection or cytotoxic damage
We previously reported that small hepatocytes (SHs) are a subpopulation of mature hepatocytes (MHs) and possess the potential to be hepatocytic progenitor cells[10,29]
SHs can be obtained from a healthy adult liver, and more than 1% of MHs have the potential to be SHs30
Summary
The liver is capable of regenerating after surgical resection or cytotoxic damage. A few studies have reported the multiplication of primary hepatocytes with differentiated functions These procedures are divided into two major methods: the use of a high concentration of nicotinamide[11,12,13] and a chemically defined nutrient-rich medium supplemented with growth factors and DMSO14,15. Oncostatin M-dependent human hepatocytes introduced through human papilloma genes could expand in medium containing serum[19] These cells possess the capability to proliferate and regain differentiated hepatic functions, they depend on serum and feeder cells or require genetically manipulation. These cells retained their expression of albumin, HNF4α, and CD44, and possessed self-renewal capability for up to 17 weeks after isolation These cells maintained the ability to recover highly differentiated functions and re-establish a network of bile canaliculi (BCs) when overlaid on Matrigel. Long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo
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