Survival‐Assured Liver Injury Preconditioning (SALIC) Enables Robust Expansion of Human Hepatocytes in Fah^–/–Rag2^–/–IL2rg^–/– Rats

Abstract

Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah -/-Rag2 -/-IL2rg -/-(FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species.

Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model

“…The success of the FRG humanized liver mouse model has led to demand for an equivalent recipient rat model. However, despite the successful generation of Fah null and Rag2 null, and Il2rg null rats by different groups more than 5 years ago 10,13,16 , bringing all 3 alleles together in rats through crossing is time consuming and logistically challenging, and the first FRG rat model was just published recently 9 . In this report, we sequentially introduced www.nature.com/scientificreports/ additional mutations through gene targeting in one-cell-stage embryos of animals carrying the homozygous alleles already modified.…”

“…A recent study 9 described a similar model of humanized rat livers, with several potentially relevant technical differences. First, although NTBC on-and-off cycling (albeit with slightly different schedule) was applied after implantation in both the current study and the other FRG rat humanized liver model described by Zhang et al 9 , we pre-conditioned recipient rats through dosing with adenovirus encoding urokinase-plasminogen activator (uPA) 24 hours before hepatocyte implantation, rather than treating with retrosine 2 weeks before cell transplantation followed by gradual NTBC withdrawal. In addition, the approaches to genetically alter the rat ES cells were somewhat different.…”

“…In particular, both Rag1 and Rag2 genes were inactivated in our approach, rather than just Rag2 gene. Further, in addition to using donor liver cells freshly thawed from cryo-preservation as described in the other report 9 , we also successfully engrafted cultured human hepatocytes.…”

“…Recently, efforts to generate similar chimeric rats were published [6][7][8][9] , which might overcome shortcomings of humanized liver mouse models, including small body and liver size, small total blood volume, and greater physiological differences to human. One study described an FRG rat model 9 , in which animals were genetically modified to induce hepatotoxicity and crossed into severe immuno-compromised background. This model overcame many of the limitations of earlier humanized liver rat models [6][7][8] and greatly enhanced its generalization and application potential.…”

IL-6–GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models

“…Humanized liver mouse and rat models, in which donor human hepatocytes repopulate recipient rodent livers, have been widely used to study human liver biology, diseases, and therapeutics (1)(2)(3)(4)(5)(6). However, it has been widely observed that engrafted human hepatocytes in both humanized liver mice and rats show defects, including increased lipid droplet accumulation (5)(6)(7).…”