Replication of mature hepatocytes can ensure hepatic tissue renewal in normal liver. However, in certain physiopathologic conditions, cells with stem-like properties can proliferate and/or be recruited from extra-hepatic compartments. For a better understanding of the mechanisms related to liver regeneration, more data on the morphological and differentiative profiles of liver stem cells are needed. This study was aimed at the immunophenotypic characterization of circulating and intrahepatic stem cells in organ donors and in patients with chronic liver disease.
We performed a four-colors flow cytometric evaluation of liver mononuclear cell suspensions (L-MNC) obtained after enzymatic digestion of liver biopsy specimens from 8 patients and 6 multi-organ donors. Paired peripheral blood mononuclear cells (PB-MNC) from the same subjects were also examined. L-MNC and PB-MNC were incubated with antibodies to the following markers: CD34, CD133, CD45, CD117 (c-kit) and CXCR4 (Stromal derived factor-1 receptor). Cell viability was performed using 7-aminoActinomycin D to exclude dead cells.
In patients, the stem cell marker CD34 was detected on the surface of 0.6±0.9% of the L-MNC, a proportion similar to that observed in donors (0.6±0.5%). In both patients and donors, the majority of CD34+ cells co-expressed CD45 (86±9% and 60±31% respectively). The subset of CD34+/CD45- cells expressing c-kit was increased in L-MNC from patients as compared to donors (13.8±12% vs.1.5±1% of the total CD34+ cells; p<0.05 by Student t test), while it was scarcely represented or not detectable in PB-MNC. The proportion of CD34+ cells with a definite hematopoietic profile (CD45+/c-kit+) did not differ between patients and donors, both in L-MNC (50±32% vs. 57±13%) and in PB-MNC (66±20% vs. 45±23%). However, in the latter compartment we identified a subset of cells expressing both CD133 and CXCR4. This subset of CD34+ cells was more frequently found in diseased (74±25%) and normal liver (50±18%) as compared to peripheral blood from both patients and donors (24±22% and 30±30% respectively), (p<0.01; paired-t test).
Flow cytometry analysis of intrahepatic CD34+ stem cells allows the identification of at least two phenotipically distinct populations. The first population co-expresses c-kit, is increased in the presence of chronic hepatocellular damage and is likely resident in the liver, being rarely detectable in peripheral blood. The second population resembles the classical hematopoietic stem cell and harbors CD133 and the chemokine receptor CXCR4 for trafficking to the liver in response to liver injury.