Cell Cycle Factor FoxM1Promotes Liver Repopulation

As a substitute strategy of orthotopic liver transplantation in treating end-stage liver diseases, hepatocyte transplantation has been studied for decades. However, therapeutic liver repopulation is hampered by the inability to efficiently expand transplanted cells in recipient liver, which is the largest obstacle in the way to establish efficient hepatocyte transplantation therapy procedure.The phenomenon, that hepatocytes and their descendants take part in liver plate reconstruction along with normal liver cell functions, is named liver repopulation. In order to achieve therapeutic liver repopulation, transplanted cells must possess selective advantage over host liver cells. Previously reports indicate that there are two ploys to offer donor cells the selective advantages:1) Suppress the proliferation of host liver cells, by blocking the regenerative capacity of host cells using chemicals such as retrorsine; or acquiring the micro-environment that support proliferation of transplant hepatocytes by inducing death of host hepatocytes in genetic modified mice models including uPA mice and Fah knock-out mice. This strategy, although can be implemented in animal models, is hardly clinical usable.2) Enhance the survival and growth ability of transplanted cells, such as by manipulating the expression of cell cycle factors in donor hepatocytes in order to promote the proliferation capacity therefore improve liver repopulation. Then, how to select the efficacious factor is the crucial issue of the research area.Transcription factor FoxM1, regulating the expression of cell cycle proteins, which are essential for Gl/S and G2/M progression, is required for initiating mitosis. As reported, FoxMl regulated the expression of proteins required for G1/S transition as Skp2, Cksl, p21, p27and Cdc25A, as well as mitosis-related proteins including cyclin B, Cdc25B, surviving, Aurora B kinase, Polo-like kinase1and CENP-A/B/F. Which have been proved that, growth hormone and adenovirus mediating increase in FoxM1level could restore the liver regeneration capacity in old-aged mice. Increasing FoxMl expression caused diminished cyclin-dependent kinase inhibitor p27protein level, elevated Cyclin B1, Cyclin B2, Cdc25B, Cdk1, and p55CDC mRNA level and stimulating Cdc25B nuclear localization promoted liver regeneration. Hepatic over-expression of FoxM1resulted an8-hour acceleration in the onset of hepatocyte DNA replication and mitosis in a transgenic mice model, which involved stimulating DNA replication and premature activation of mitosis required genes. Another study showed that suppressing of FoxM1expression in hepatocytes resulted in significant a reduction in hepatocyte DNA replication and mitosis inhibition after partial hepatectomy. It proved from another aspect that FoxM1regulates expression of cell cycle proteins are essential for hepatocyte entry into DNA replication and mitosis. Transthyretin (TTR) promoting hepatocyte-specific overexpression of FoxM1did not cause primary hepatocellular carcinoma (HCC). Treated with a diethylnitrosamine/Phenobarbital (DEN/PB) tumor induction protocol, mice with sustained FoxM1expression showed no detectable elevated level on the cell proliferation rate of HCCs, nor on the formation or growth of these advanced tumors. All together, FoxM1can promote cell proliferation without bring the risk of tumor-genesis. Based on those reports, we choose FoxMl the target gene of this study. By up-regulating FoxM1in hepatocytes, we can investigate the effect on enhancing liver repopulation capacity of the hepatocytes.To analysis the repopulation capacity of hepatocytes with sustained FoxM1expression, TTR-FoxM1mice were used as donors for liver repopulation experiments. Equal number of hepatocytes from TTR-FoxM1mice and wild-type mice were separately, as well as equally mixed transplanted into FAH knockout mice (fah-/-mice) respectively. At four time points, livers from recipients were harvested for analyzing repopulation levels. The repopulation levels of those received hepatocytes with sustained expression of FoxM1were higher than those received WT hepatocytes, and it took about2weeks less than WT hepatocytes complete liver repopulation, the repopulation capacity of hepatocytes with sustained FoxM1expression is1.5fold of the capacity of wild type cells. Equal number of hepatocytes from TTR-FoxM1mice and wild-type mice were transplanted into2/3-part hepatectomy (PHx) mice model. TTR-FoxM1hepatocytes can repopulate5%～9%host liver, while control group can only reach less than1%, which also proved that hepatocyte with sustained FoxM1expression have enhanced repopulation capacity.For future clinical application consideration, using DNA delivery system to genetic manipulate normal liver cells to obtain FoxM1+hepatocytes is the essential procedure. In order to generate hepatocytes with sustained expression of FoxM1, we introduced plasmid vectors using the sleeping beauty DNA transposon system, which were capable of transgene integration and long-term expression. Plasmids were injected into fah-/-mice via tail vein. The survival rates of those received plasmid carries FoxM1gene tail vein injection were clearly higher than those received control plasmids. Analyzing repopulation capacity of hepatocytes transfected in vivo, the repopulation rates were higher in mice received FoxMl+cell transplantation than those received control cells. With in vivo imaging system (IVIS), we found that SB-FoxMl hepatocytes can complete liver repopulation2weeks earlier than wild type cells. Similarly, SB-FoxMl hepatocytes repopulated4%～9%(with15%maximum) host liver in2/3PHx model. The result of evaluation of cell proliferation in vitro with BrdU immunofluorescence shows that FoxMl+hepatocytes possess higher proliferation activity than wild-type hepatocytes. All the results above robustly proved that, FoxMl gene can be delivered by SB vector, and hepatocytes genetic modified possess enhanced proliferation and liver repopulation capacity. Then we obtained FoxMl expressing hepatocytes by ex vivo transfection. Analyzed by transplantation experiments, these cells possess enhanced capacity of liver repopulation. This procedure could be a possible strategy for putting FoxMl in clinical hepatocyte transplantation therapy. In this way, endogenous virus recombination, gene-insert activation, strong immune response and other risks brought by viral vectors can be avoided. Also, a new technology for hepatic genetic modification with broad clinical prospects was introduced.For over-expression cell cycle factor may bring the risk of tumor-genesis, we eliminated the possibility of cancer formation by hepatocytes from TTR-FoxMl and modified in vivo. Those FAH positive areas in recipient mice formed by transplanted hepatocytes showed no clue of tumor genesis or any abnormality morphologically nor genetically. There is neither tumor genesis found in all of the recipient mice in serial transplantation experiments, nor in recipient mice that were kept more than1year.In this study, we demonstrated that up-regulating FoxM1would enhance the proliferation as well as repopulation capacity of mice hepatocytes, which provides a theoretical solution for improve transplanted hepatocyte proliferation efficiency in host liver. For the first time, we obtained hepatocytes with sustained expression of FoxMl by non-viral vector, which offers a possible technological strategy to achieve therapeutic repopulation after hepatocyte transplantation.