Human hepatocyte engraftment in F8KO TK-NOG mice: Creating a novel Hemophilia A mouse model with functional human coagulation cascade Free

Introduction:

Humanized mouse models have been developed to elucidate human-specific responses in various research applications. The TK-NOG mouse model enables human hepatocyte engraftment following ganciclovir-induced hepatic injury, allowing replacement of mouse hepatocytes with human hepatocytes. Since many coagulation and anticoagulation factors are produced in the liver, we expected that the coagulation cascade in humanized TK-NOG mice would more closely resemble that of humans than that of mice. We therefore hypothesized that developing a hemophilia A model using these mice would provide a unique experimental model with functional human coagulation-related factors. In this study, we generated a novel hemophilia A mouse model (TK-NOG F8KO mice) and subsequently humanized these mice by transplanting human hepatocytes. We then characterized the phenotypic and functional properties of these humanized mice.Methods:

Animal experiments were conducted with institutional approval. We first replaced the genetic background of B6;129S-F8^tm1Kaz/J (F8KO) mice with NOD.Cg-Prkdc^scidIl2rg^tm1Sug/ShiJic (NOG) to generate NOG F8KO mice, then crossbred TK-NOG mice with NOG F8KO mice to create TK-NOG F8KO mice. These mice received 0.1 mg/mL valganciclovir hydrochloride in drinking water for 48 hours. Seven days after initiation of water intake, each mouse received 0.76-1.10×10^6 viable normal human hepatocytes via splenic portal injection following recombinant human (rh) FVIII administration for hemostasis. Plasma and liver samples were collected 8 weeks post-transplantation and analyzed for human hepatocyte engraftment, chimerism rate, gene expression of coagulation factors, concentrations of human factor IX (hFIX) and human factor X (hFX), and coagulation activity using histological assessment, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), electrochemiluminescence (ECL), and global coagulation assays.Results:

Human hepatocyte engraftment was confirmed by histological assessment using human mitochondria staining, with chimerism rates ranging from 12.6% to over 95% in 25 humanized TK-NOG F8KO mice as determined by plasma human albumin ELISA. qPCR analysis of liver tissue showed no human gene expression in TK-NOG F8KO control mice, whereas humanized TK-NOG F8KO mice expressed human coagulation and anticoagulation factor genes (F2, F7, F8, F9, F10, F11, protein C (PC), antithrombin (AT)). Both human and mouse FVIII genes were detected, consistent with previous reports of low FVIII gene expression in human hepatocytes (Tatsumi, Cell Transplant., 2012) and residual expression in F8KO mice (Bi, Blood, 1996).

Global coagulation assay revealed that 11 humanized TK-NOG F8KO mice showed FVIII activity below the limit of quantification, while one mouse demonstrated 5.3% FVIII activity. Additionally, 20 mice, including controls, showed less than 4% FVIII activity. PC levels were below the detection limit in 3 humanized TK-NOG F8KO mice, but all other factors showed measurable activity. Although the assay cannot differentiate between human and mouse activities, some factors such as FX, factor XI (FXI), AT, and PC showed tendencies toward 100% activity as chimerism increased, suggesting that human factors were becoming dominant. This phenomenon was supported by ECL measurements, which confirmed hFIX and hFX expression with positive correlation to chimerism rate, indicating that humanized TK-NOG F8KO mice with higher chimerism rates express greater quantities of human coagulation factors.

Finally, activated partial thromboplastin time (APTT) and clot waveform analysis were performed in mice with sufficient plasma volume to evaluate the effects of rhFVIII and emicizumab, that has no cross reactivity to mouse FIX and FX. Both treatments shortened APTT and increased adjusted maximum coagulation velocity. Notably, with emicizumab treatment, APTT tended to shorten as chimerism rate increased, demonstrating the functionality of human coagulation factors in this model.Conclusions:

We successfully developed a novel humanized mouse model expressing human coagulation factors derived from transplanted human hepatocytes. This model demonstrates that human-derived coagulation factors increase proportionally with chimerism rate, enabling effective evaluation of human factor-specific therapeutics. With stable production of high-chimerism mice, applications to various disease research areas can be anticipated.