Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Abstract

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca(2+)-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for alpha-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as alpha-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.

In vitro generation of myofibroblasts-like cells from liver epithelial progenitor cells of rhesus monkey (Macaca mulatta)

“…Having considered all these facts, we hypothesized that TGF-β would induce hepatic epithelial progenitors to convert into myofibroblasts-like cells through the process of EMT. Previously, we reported the isolation of liver epithelial progenitor cells from both adult and newborn monkeys (Jin et al 2009;Ji et al 2010). Here, we provide in vitro evidence for the first time that adult rhesus monkey liver epithelial progenitor cells, mLEPCs, acquire myofibroblastslike phenotype via EMT.…”

“…Adult mLEPCs were isolated and characterized as previously described (Jin et al 2009;Ji et al 2010). FACS analysis of cell surface molecules revealed that adult mLEPCs were homogenously positive for hepatic progenitor markers such as E-cad (Nitou et al 2002;Nierhoff et al 2005), CD44 (Kon et al 2006) and CD73 (Kakinuma et al 2009; Fig.…”

“…Adult mLEPCs were isolated as previously described (Jin et al 2009;Ji et al 2010). Fluorescence-activated cell sorting (FACS) (FACSVantage SE, BD Biosciences, Franklin Lakes, NJ) was then employed to yield E-cad+CD34-CD45-CD90-cells.…”

Demonstration of expression of hepatic stellate cell markers in cells from liver epithelial progenitor cells of rhesus monkey

“…However, further research is needed to evaluate the HSC potential of hepatic progenitors. Previously, we reported the isolation of rhesus monkey liver epithelial progenitor cells (mLEPCs) from both adult and newborn liver (2, 3). Here, we have reported that fluorescence‐activated cell sorting (FACS)‐purified E‐cadherin‐positive adult mLEPCs expressed HSCs‐specific protein when co‐cultured with monkey ear skin fibroblasts (MESFs).…”

“…Adult mLEPCs were isolated and prepared as described previously (2, 3). Briefly, cell aggregates were prepared in Ca 2+ ‐containing medium from fresh digested hepatic cells.…”