A Dual Immunologic And Hepatic Reconstitution Mouse From One Donor

A MHC-restricted chimeras mouse will be a most practical tool for studying donor’s immune response against donor’s non-immune cells from organ with pathogen infection, transformation and damage in recipient mice, without putting individuals at risk. It was extensively accepted hematopoietic stem cells (HSC) may engraft and develop into immune system in lethal irradiated or immunodeficient mice, such as NOD-scid112r γ-/-(NSG) recipients or BALB/c-Rag2-/-Il2r γ-/-recipients. Liver chimeric mice were also reproted to be developed by exogenous hepatocytes or embryonic stem cells transplantation, like the uroplasminogen-activator (uPA) transgenic model or fumarylacetoacetate hydrolase (FAH) deficient model. However, neither of the chimeric mice are suitable for further investigation of the interaction between the immune system and pathogen infected organs.Development of humanized mice provides insights into in vivo human biology that would be severely limited by ethical and/or technical constraints. Human immune system (HIS) mice are already established, showing a potential as the available model for the study of human immune response and human lymphotropic pathogens in mice, and human liver chimeric mice were developed for study of human hepatotropic pathogens or preclinical evaluation of anti-hepatitis virus drug candidates. However, further investigation of the pathology, immune correlates, and mechanisms of highly specialized pathogens like HBV, HCV and malaria (at liver stage) needs an excellent mouse model engrafted with MHC-restricted human immune system and pathogen-targeting organs. Recently, AFC8-hu HSC/Hep mice model was developed by meeting this requirement through co-implantation of human CD34+HSCs and hepatocyte progenitor cells from a15-18weeks old fetal liver tissue into BALB/c-Rag2-/-Il2r γ-/-mice. Although this approach successfully provides immune system and liver cells together in recipients, its extensive utilization is limited by obtaining human fetal liver tissues.It was recently reported that HSC may also differentiate into hepatocytes in bone marrow transplanted (BMT) mice. Here, using a strain of mice deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase (fah-/-), we tried to see whether donor’s HSC may concurrently differentiate into immune cells and hepatocytes in recipient, which will greatly benefit exploiting the donor’s MHC-restricted interaction between immune cells and hepatocytes.In this study, we firstly assessed the bodyweight, serum ALT and liver failure in fah-/-mice after NTBC withdrawal. Then, we transplanted mouse or human hepatocytes into fah-/-mice or FRG mice to see whether exogenous hepatocytes could repopulate in the recipient liver. After that, we tried to established chimeric mice model with a dual immunologic and hepatic reconstitution by syngeneic, allogeneic or even xenogeneic bone marrow transplantation. Bodyweight and serum ALT was measured in BMT animals after NTBC withdrawal. To confirm immunologic reconstitution in recipients, PBMC subset were monitored every two weeks in BMT mice, and the mononuclear cells from bone marrow, thymus, spleen, blood, liver and lymph node were detected using flow cytometry after8weeks rebuild time. The histopathology of spleen and lymph node was aslo assessed by H&E staining and immunohistochemistry (IHC). Further more, BMT mice were immunized by HBV vaccine or OVA protain, and antigen-specific antibody was examined by RIA or ELISA. To evaluate the hepatic reconstitution, hepatocytes from long-term survivors were isolated and analyzed using flow cytometry. Liver histology of sections of BMT mice was aslo detected by H&E staining and IHC. The major results of our studies are shown as follows:1. Fah-/-mice suffer from progressive liver failure after NTBC withdrawal.Firstly, we compared the life status of fah-/-mice with or without NTBC. We found that the body weight of fah-/-mice reduced gradually and finally died when the loss of bodyweight exceed30%when NTBC was off. Serun ALT increased week by week indicated progressive liver failure in these mice. Hepatocytes from fah-/-mice without NTBC feeding were more prone to apoptosis. Liver histology of these mice also showed lymphocytes inflitration, and further analysis shown that T, B lymphocytes may play a important role in the progressive liver failure. Together, these results implied that NTBC protect fah-/-mice from death of liver injury, and NTBC cut off could be a switch to initiate hepatocytes necrosis in fah-/-mice.2. Liver repopulation with exogenous hepatocytes in fah-/-mice.Next, we transplanted fresh isolated hepatocytes (EGFP-positive) into fah-/- mice, then we stopped NTBC feeding. In contrast to congtrol fah-/-mice, the body weight and serum ALT from recipient mice with hepatocytes transplantation could soon recovered to normal. Transplanted mice survived far more longer than control mice after NTBC withdrawal. We also detected EGFP-positive hepatocytes in these mice, and such hepatocytes could provide FAH to to restore liver function in recipient. However, when we changed the donor cells to human hepatocytes cell line (LO2), the recipient mice died of hepatoma. The uncontrolled expansion of LO2cells in the liver maybe cause the death, because we found many tumor nodules in the liver from LO2transplanted mice.3. A dual immunologic and hepatic reconstitution in recipient fah-/-mice after syn-BMTFurther, we transplanted syngeneic bone marrow cells into fah-/-/129SvvJ (haplotype H-2b) recipients. We choose two different mice as donor, EGFP-Tg mice and HBs-Tg mice. Most BMT mice survived at least5months after NTBC withdrawal beginning at day28. Surviving recipients also show stable mutilineage hematopoietic reconstitution after syn-BMT. And there was little difference in myeloid and lymphoid development between chimeras and donor mice. NK, B, CD4and CD8T cells could reconstitute normally in chimeras’ peripheral blood, and there was a similar ratio of T cells in all immune organs of recipient mice to that of donor mice. Spleen and inguinal lymph node histology of serial sections further confirmed successful immunologic reconstitution in BMT mice. To directly test the immune response after reconstitution, mice were immunized2times with HBV vaccine. All recipient mice produced specific anti-HBsAg antibodies in serum, similar to the donor mice. To evaluate the hepatic reconstitution, long-term survivors were sacrificed and analyzed. Liver histology showed that donor-derived hepatocytes (FAH-positive) were organized in a cell cluster. Further study demonstrated that myelomonocytic cells (CD45+F4/80+Gr-1+CD11b+CD11c-) were the progenitors for the bone marrow-derived hepatocytes. Together, these data imply that HSC from a syngeneic mouse may concurrently reconstitute immunologic and hepatic system in recipient fah-/-mice.4. A dual immunologic and hepatic reconstitution in recipient fah-/-mice after allo-BMT. After successful liver and immune reconstitution after syn-BMT in fah-/-mice, we carried out allo-BMT. We also choose two allogeneic mice as donor, C3H/HeJ mice (haplotype H-2k) and HBV-Tg mice (haplotype H-2d). About60%of BMT mice survived at least5months after NTBC withdrawal. We monitored PBMC subset from BMT mice, and found contents of NK, B, CD4and CD8T cells became normal in chimeras. Meanwhile, spleen and inguinal lymph node histology of serial sections further confirmed successful immunologic reconstitution in BMT mice. BMT mice produced specific anti-OVA antibodies in serum, similar to donor mice after immunization with OVA protein. Meanwhile, hepatocytes from BMT mice partially expressed donor MHC class Ⅰ antigen, and such hepatocytes were partially positive for recipient MHC class Ⅰ antigen and CD45antigen, implying cellular fusion between donor BMC and resident hepatocytes.5. A dual immunologic and hepatic reconstitution in recipient fah-/-mice after xeno-BMT.We next attempted to define whether xenogeneic HSC could successfully reconstitute immunologic and hepatic system in fah-/-mice. We transplanted S.D. rat (RT1A,fah+/+) bone marrow into fah-/-/129SvJ (haplotype H-2b) recipients. About50%of BMT mice survived at least5months after NTBC withdrawal, and serum ALT almost kept in a normal level. Surviving recipients also show stable mutilineage hematopoietic reconstitution after Xeno-BMT. Nearly100%of the PBMCs from the chimeras were RT1A+similar to donor cells, and there was little difference in contents of myeloid and lymphoid cells between chimeras and recipient mice. NK, CD4and CD8T cells could reconstitute normally in chimeras’peripheral blood, and there was a similar ratio of T cells in all immune organs of recipient mice to that of donor rat. Meanwhile, hepatocytes from Rat-BMT mice were partially positive for donor MHC class I antigen (RT1A+), implying the generation of bone marrow derived hepotocytes. Liver histology also showed that FAH-positive cells were organized in a cell cluster. Altogether, HSC from xenogeneic donor could indeed reconstitute immunologic and hepatic system in fah-/-mice.6. Immunologic and hepatic reconstitution in recipient fah-/-rag2-/-mice after human HSC transplantation.Based on the inspiring results from syn/allo/xeno-BMT, we then transplanted purified human hematopoietic stem cells (CD34+cord blood cells) into immunodeficient fah-/-/129SvJ.B6recipients (fah-/-rag2-/-). After NTBC withdrawal, the body weight of most chimeras was about10%loss at the beginning because of sub lethal irradiation, but soon they recovered and kept getting on body weight gradually. Serum ALT from these mice also kept in a normal level, implying that human HSC transplantation partially restored liver function in chimeras. Meanwhile, most chimeras survived longer than no cell transferred mice. Additionally, the liver function restoration was relative to the engraftment of human HSC. We compared different recipient mice, and found that chimeras in fah-/-rag2-/-112r γ-/-mice survived better than that in fah-/-mice, and we indeed detected the most human cells in these mice. H&E staining of the liver section clearly distinguished human hepatocyte clusters, which were larger and less eosinophilic and hence appear paler than mouse cells. Since the poor engraftment of human HSC in B6recipients (fah-/-rag2-/-), we could only detected few FAH-positive hepatocytes in liver tissue. Thus, after HSC transplantation, recipient mice could be partially repopulated with human origin hepatocytes, which provided FAH to alleviate live injury.Conclusion:The major finding of the current study was that HSCs from one individual were successfully differentiated into both immune cells and hepatocytes by using an irradiated or imunodeficient fah-/-mouse. We, for the first time, developed a mouse model with immune and liver reconstitution from one donor’s HSCs, which was carried out by systematically performing syngeneic, allogeneic or even xenogeneic BMT. We further showed that the purified human hematopoietic stem cells transplantation could partially restore liver function in fah-rag2double knockout mice. These finding indicated that a better humanized mouse model, with a HLA-identity between human immune cells and human hepatocytes, would be developed by using a more imunodeficient fah-/-mouse, such as fah-/-NSG in future. Although further improvements of our model need to be done, we expect that such model provide a new opportunity to perform pre-clinical testing and to investigate many human biological processes which are happened between donor immune cells and organs with MHC-identity.