Utilization of Immune Compromised Rats for an Allogeneic Rat Hepatocyte Transplant Model
Authors: Rasmussen Shauna A , Rudemiller Nathan P , Geurts Aron M 
Published: November 2023

Using immune compromised mice as models for human tissue xenotransplantation has led to major advances in both the understanding and treatment of various human diseases. However, the rat has potential to be a favorable species for the study of physiological processes due to its larger size and ease of surgical manipulation and sampling. Therefore, development of immune compromised rats is vital for the generation of rat xenotransplantation models. The first aim was to phenotype rats with mutations in interleukin 2 receptor gamma (Il2rg) and recombination activating gene 2 (Rag2) or the combination of the two. The second aim was to explore the ability for allogeneic hepatocytes to engraft in these immune compromised models following various liver environment manipulations to enhance engraftment.

We used Wildtype (WT), Il2rg−/−, Rag2−/−, and Rag2−/− Il2rg−/− (R2G) rats to assess levels of circulating lymphocytes by means of fluorescence‐activated cell sorting (FACS) and IgG serum concentration via enzyme‐linked immunosorbent assay (ELISA). We also performed a complete blood count with differential to assess overall immune cell status. For the second study, we used all three models for allogeneic hepatocyte transplantation using green fluorescent protein (GFP) expressing hepatocytes as the donor hepatocytes at 8 weeks of age. We used three different liver regeneration stimuli: 2/3 partial hepatectomy (2/3 PHx), retrorsine administration, and the combination of 2/3 PHx and retrorsine. We also performed no manipulation prior to transplant as our control. Upon completion of experimental period, 30 and 90 days post‐transplant, livers were imaged grossly under green fluorescent light to assess for engraftment and repopulation.

FACS analysis on peripheral blood mononuclear cells (PBMCs) from WT, Rag2−/−, Il2rg−/−, and R2G rats showed an alteration of T, B, and Natural Killer cell populations. An ELISA for IgG levels revealed that all three mutant strains had decreased levels. We observed in all three mutants an overall decrease in total white blood cells, along with a decline in lymphocyte and increase in neutrophil populations in comparison to WT. When assessed for the ability to engraft donor hepatocytes, Rag2−/− animals were unable to engraft donor hepatocytes following all liver regeneration stimuli, Il2rg−/− animals experienced minimal repopulation of donor hepatocytes following all liver regeneration stimuli, and R2G mutant animals displayed varying levels of repopulation with donor hepatocytes following all liver regeneration stimuli.

Successful generation of immune compromised rats opens the door for further exploration into human disease pathophysiology through the xenotransplantation of human tissue. Due to the need for an improved liver xenotransplantation model, we tested whether our immune compromised rat models could engraft healthy hepatocytes from a donor rat. We conclude that the Rag2−/− Il2rg−/− rats were the most successful in engrafting and becoming repopulated with donor hepatocytes, and would be applicable for further humanization experiments.

Support or Funding Information

This project is supported by the NIH grants T32 HL007852 and the DP2 OD008396