p27^Kip1 Inactivation Provides a Proliferative Advantage to Transplanted Hepatocytes in DPPIV/Rag2 Double Knockout Mice after Repeated Host Liver Injury

Abstract

Studies were conducted to develop a new DPPIV–/–/Rag2–/– mouse model for hepatocyte transplantation by allogeneic and xenogeneic cells and to compare the proliferative capacity of p27 null hepatocytes versus normal hepatocytes in this system. Dipeptidyl peptidase IV (DPPIV) gene knockout mice, in which wild-type (wt) DPPIV+ donor hepatocytes can be readily identified by enzyme histochemistry, were bred with Rag2 null mice to prepare immunotolerant DPPIV–/–/Rag2–/– double knockout mice. DPPIV–/–/Rag–/– mice were transplanted with wt hepatocytes or p27 null mouse hepatocytes, which show enhanced cell cycle activity due to disruption of the Kip1 cyclin kinase inhibitor gene, and liver repopulation was assessed under nonproliferative versus proliferative experimental conditions. After their initial engraftment, transplanted wt hepatocytes did not proliferate in untreated livers or increase significantly in response to an acute liver regenerative stimulus. p27 null hepatocytes engrafted with the same efficiency as wt hepatocytes, but showed a noticeable, although not statistically significant, increase in proliferation in response to partial hepatectomy or acute CCl4 administration. Repeated treatments with CCl4 substantially increased proliferation and liver repopulation by p27 null hepatocytes but not by wt hepatocytes. These results suggest that p27 gene inactivation does not overcome proliferative restrictions imposed on hepatocytes by the normal liver, but that after repeated episodes of toxic liver injury, the augmented proliferative capacity of p27 null hepatocytes leads to significant liver repopulation compared with wt hepatocytes. These properties of p27-deficient hepatocytes could prove useful as a target for liver repopulation in patients with intermittent or a low level of chronic liver injury.

Deletion of Smad2 in Mouse Liver Reveals Novel Functions in Hepatocyte Growth and Differentiation

“…The average number of cells per cluster was also increased significantly in S2HeKO compared to both controls (Fig. 7H) toxic liver injury and liver regeneration by using a model of toxic liver injury induced by a single injection of CCL4 (52). CCL4 exposure induced localized centrilobular necrosis/apoptosis/steatosis peaking at 48 h that was not significantly different between Ctrl, S2HeKO, and S3KO mice (Fig.…”

“…Primary hepatocytes were transplanted into dipeptidyl peptidase IV Ϫ/Ϫ (DPPIV Ϫ/Ϫ )Rag2 Ϫ/Ϫ mice by intrasplenic injection, as previously reported (52). The control transplantation group includes transplantations with donor hepatocytes isolated from WT (C57BL/6J) and Smad2 f/f mice, respectively.…”

“…Multiple sections from both the middle and left liver lobes were taken from each recipient at various times after cell transplantation. DPPIV staining was used to quantify donor cell engraftment and proliferation, as described previously (52). The slides were examined independently by two observers and scored in a blinded fashion (by A.O.…”

“…To validate whether S2HeKO hepatocytes have a constitutive sustained growth advantage in vivo, we used a liver cell transplantation model as previously described (52). To control for both genetic background of ES cell lineages and the absence or presence of Smad2, we used donor hepatocytes generated from Smad2 and C57BL/6J donor hepatocytes at 1, 2, and 3 months posttransplantation, as indicated (Fig.…”

Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing

“…To address the issue of whether liver injury increases the frequency of hematopoietic contributions to hepatocytes, two adult vav ancestry mice were injected with carbon tetrachloride (CCl 4 ) at a concentration that induces necrosis in ~60% of hepatocytes (Yuan et al, 2003). Thirty hours after injection, we measured the blood plasma levels of alanine amino transferase (ALT), an indicator for injury to the liver parenchyma.…”