Authors R. MANCO (1), L. CLERBAUX (1), I. LECLERCQ (1) / [1] Université Catholique de Louvain, Brussels, Belgium, Laboratory of Hepato-Gastroenterology Introduction Self-renewal of mature hepatocytes supports homeostasis and regeneration of adult liver. Recent studies indicate that liver progenitor cells (LPC) are recruited upon injury as a facultative reservoir for generation of hepatocytes, although only a small number of mature hepatocytes were shown to derive from LPC in vivo. Models used for these studies do not recapitulate long lasting chronic hepatocellular damage and fibrosis seen in human chronic liver disease and cirrhosis. Aim Our aim is therefore to follow the dynamics of ductular reaction (DR) and the LPC’s fate during chronic liver injury in mice. Methods We used tamoxifen-inducible Osteopontin-Cre (OPN-CreERT2) mice crossed with yellow fluorescent protein (YFP) reporter mice to follow the fate of LPC and biliary cells with an efficiency >85%. Long-term chronic injury was induced by repeated carbon tetrachloride (CCl4) injections 3x/week for 4, 6, 8, 16 and 24 weeks, resulting in chronic fibrosis and eventually cirrhosis. Livers from 8 and 16 weeks were also analysed after 4 weeks and 2 and 4 weeks of CCl4-free recovery period, respectively. Results After 4 weeks CCl4, DR is minimal with few ck19+/YFP+ positive cells in periportal area and LPC-derived hepatocytes (traced as YFP+ hepatocytes) are inconspicuous. After 6 weeks, DR is similar in intensity but small foci of YFP+ hepatocytes adjacent to portal area are readily seen; these have a median size of 3010µm². As fibrotic disease increases in severity, the DR is negligible while patches of YFP+ hepatocytes become larger (median size of 3850µm² and 7040µm² at 8 and 16weeks, respectively) and extend to into the parenchyma. In the cirrhotic liver (24 weeks CCl4) some regenerative nodules are entirely composed of YFP+ hepatocytes. The number of YFP+ hepatocytes does no rise accordingly to the size of the patches as they represent 4.2 ± 2.4% of the lobule area in 6 weeks’ samples, increases up to 11.5 ± 3.8 % in 8 weeks’ samples and stabilizes around 5% thereafter, suggesting that not all YFP+ hepatocytes expand into growing patches. At 6 weeks, YFP+ hepatocytes are significantly smaller cells than YFP- native hepatocytes (750 vs 981 µm²) but in 16 weeks’ samples YFP+ and YFP- hepatocytes have the same size (996 and 1001 µm²). The dynamic of the YFP+ hepatocytes was also evaluated upon recovery: in the 4 weeks following 8 weeks of CCl4, the area occupied by YFP+ hepatocytes has a tendency to decrease from 11.5 ± 3.8% to 5.03 ± 3.8% (p=NS), while in the 2 and 4 weeks of recovery after 16 weeks of CCl4 the area significantly increases from 4.58 ± 1.7% to 7.7 ± 3.3% (p=NS), up to 13.8 ± 0.7 % (p<0.001), respectively. Whereas, upon recovery the size of the YFP+ hepatocytes, in all the different time points, is the same of the native hepatocytes. Conclusions Our data demonstrate a significant contribution of LPC to the hepatocytes regeneration in a model of chronic liver injury leading to cirrhosis. The kinetic study supports that when DR is present, LPC differentiate into small hepatocytes, some of these subsequently increase in number, to form growing patches, and in size, becoming undistinguishable from the native hepatocytes. Upon recovery the growth of the patches of the LPC-derived hepatocytes depends on the severity of the underlying injury. Clonality studies are ongoing to test this hypothesis.
