Recipients were sacrificed, and liver samples were harvested selleckchem for detection of human hepatocyte engraftment. Fah?/? Rag2?/?Ilr2g?/? recipients on NTBC were as controls. FAH antibody immunostaining indicated that liver samples from 5 of 8 Fah?/?Rag2?/?Ilr2g?/? recipients had FAH-positive hepatocytes at levels from 3.1% to 71.3% of total hepatocytes (Figure 1A, Supplemental Table 1 at http://ajp.amjpathol.org). However, the Fah?/?Rag2?/? Il2rg?/? mouse breeders have a very small litter size in our colony breeding process (Supplemental Figure 1 at http://ajp.amjpathol.org). For this reason, Fah?/?Rag2+/-Il2rg+/? mice could be used as breeders to generate Fah?/?Rag2?/?Il2rg?/? mice. Number of Fah?/?Rag2?/? Il2rg?/? mice was much lower than expected, and only between 1 in 30 and 1 in 20 offspring had the triple homozygous mutant genotype.
The pups with genotype of Fah?/?Rag2?/?Il2rg?/? were also less fit than Fah?/? and Fah?/?Rag2?/? littermates. They had about 60% more death during colony breeding and after surgery for cell transplantation (Supplemental Figure 2 at http://ajp.amjpathol.org). Because of the significantly low numbers of Fah?/?Rag2?/?Il2rg?/? mice during colony breeding and the significant numbers of Fah?/?Rag2?/?Il2rg?/? recipients lost after cell transplantation, Fah?/?Rag2?/? Il2rg?/? mice could not be used for efficient generation of humanized mice. Thus, we chose the Fah?/?Rag2?/? mouse to study the capacity for human hepatocyte repopulation after pharmacological treatment to deplete NK cells.
Figure 1 Depletion of NK cells by anti-asialo GM1 enabled liver xeno-repopulation from human hepatocytes in Fah?/?Rag2?/? mice. Comparison of FAH-positive hepatocytes found in Fah?/?Rag2?/?Il2rg?/? … Human Hepatocyte Engraftment in Fah?/?Rag2?/? Mice after NK Cell Depletion Using the same experimental protocol and under same conditions, we determined the capacities of repopulation from human hepatocytes in Fah?/?Rag2?/? mice. Fah?/? Rag2?/? recipients on NTBC were used as controls that were free of induced liver injury. Three of 17 Fah?/? Rag2?/? recipients were found with detectable FAH-positive hepatocytes, existing in single cells and small nodules (Figure 1B; Supplemental Table 1 at http://ajp.amjpathol.org). Xeno-engraftment of human hepatocytes in Fah?/?Rag2?/? recipients was not successful in a previous report.
7 Without treatment of gradual withdrawal of NTBC before cell transplantation, but instead with abrupt removal of NTBC after cells were transplanted, none of eight Entinostat Fah?/?Rag2?/?Ilr2g?/? and eight Fah?/?Rag2?/? recipients had any detectable FAH positive hepatocytes (Supplemental Table 1 at http://ajp.amjpathol.org). In addition, eight Fah?/?Rag2?/?Ilr2g?/? and eight Fah?/?Rag2?/? recipients that were always kept on NTBC had no detectable FAH positive hepatocytes (Supplemental Table 1 at http://ajp.amjpathol.org).