Isolation of adult liver stem cells is difficult because the cells share many morphologic
and phenotypic features with other hepatic cells. Previous studies in our laboratory
indicated that adult liver stem cells might be more resistant to ischemic stress than
the differentiated liver cell populations. Therefore, it could be possible to enrich
for liver stem cells through damaging the other liver cells by ischaemic treatment
of the tissue. The aim of this study was to examine the effect of an ischemic treatment
of human liver tissue on the subsequent isolation of adult liver stem cells. Human
liver tissue pieces utilized for this study were obtained from partial hepatectomies.
The resected liver piece was placed in culture medium at 4°C and kept under hypoxic
conditions for varying periods up to 120 hours until cell isolation. After the cold
ischemic period the tissue was perfused with 40ml of a phosphate based buffer solution
that contained EDTA followed by perfusion with 40ml of a buffer solution containing
0.1% Pronase E, 0.1% Collagenase IV and 0.025% DNase I. After perfusion, the liver
tissue was mechanically minced and incubated for 30 min at 37°C in the enzyme solution.
Then filtered through sterile gauze and cells were pelleted by centrifugation. Remaining
erythrocytes were removed by lysis. Finally, cells were seeded into collagen coated
culture vessels. Cultured cells were analysed by light microscopy and immunocytochemistry.
Stem cell like cells and fibroblast like cells could be observed in cultures after
all lengths of ischemia but hepatocytes were only found in cultures after short therm
ischemia. The stem like cells formed colonies consisting of small cells with a low
cytoplasm to nucleus ratio that coexpressed the hepatocyte marker CK18, the cholangiocyte
markers CK19, CK7, and HEA–125. In addition, albumin expression could be detected
in some colonies, suggesting differentiation towards the hepatocytic and cholangiocytic
lineages. The examination of the effect of ischemic treatment on the isolation result
confirmed that stem cell like cells, but also fibroblast like cells are more resistant
to hypoxia than the differentiated parenchymal cells. Thus, it was possible to enrich
for the stem cell like cells, while complete purification was not achieved. Further
studies are necessary to optimize the developed isolation method.
Schlüsselwörter
adult stem cells - ischemia - isolation
