Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture

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.

Self‐Renewal Capability of Hepatocytic Parental Progenitor Cells Derived From Adult Rat Liver Is Maintained Long Term When Cultured on Laminin 111 in Serum‐Free Medium

“…The adhesion rates of cells on day 1 were 15% to 20% and 40% to 50% on LN111 and LN511/521, respectively (data not shown), which was consistent with our preliminary results. (10) The sizes of the attached cells on day 1 were measured, and those on LN511/521 were significantly larger than those on LN111 (Supporting Fig. S2A).…”

“…We first confirmed our preliminary results. (10) First-passage cells were plated on dishes coated with ECMs, including LN-α1 (LN111 and Matrigel) or α5 (LN511 and LN521). The adhesion rates of cells on day 1 were 15% to 20% and 40% to 50% on LN111 and LN511/521, respectively (data not shown), which was consistent with our preliminary results.…”

“…(7) The details of the methods for cell culture and passage have been reported. (10) In the experiments using LNs, second-passage cells were plated on 12-well plates coated with LN111, LN511, LN521, and Matrigel (see Supporting Information for details). The medium was replaced every other day, and the cells were passaged every 4 weeks.…”

“…(9) We recently reported that hepatocytic parental progenitor cells (HPPCs) exist among CD44 + SHs. (10) HPPCs continue to proliferate and generate daughter cells after passages, which indicates that they possess the ability to self-renew. In fact, rat HPPCs can potentially divide more than 50 times in a period of 17 weeks and over four passages.…”

“…(12,13) Preliminary experiments have shown that only 15% to 20% of CD44 + SHs attach to LN111-coated or Matrigel-coated dishes, although more than 40% attach to LN511-coated ones. (10) In the present study, we investigated how HPPCs' self-renewal capability can be maintained and how LN isoforms play a role aRtiCle inFoRmation:…”