Generating liver using blastocyst complementation: Opportunities and challenges

Abstract

Orthotopic liver transplantation (OLT) is the only definitive treatment option for many patients with end‐stage liver disease. Current supply of donor livers for OLT is not keeping up with the growing demand. To overcome this problem, a number of experimental strategies have been developed either to provide a bridge to transplant for patients on the waiting list or to bioengineer whole livers for OLT by replenishing them with fresh supplies of hepatic cells. In recent years, blastocyst complementation has emerged as the most promising approach for generating whole organs and, in combination with gene editing technology, it has revolutionized regenerative medicine. This methodology was successful in producing xenogeneic organs in animal hosts. Blastocyst complementation has the potential to produce whole livers in large animals that could be xenotransplanted in humans, thereby reducing the shortage of livers for OLT. However, significant experimental and ethical barriers remain for the production of human livers in domestic animals, such as the pig. This review summarizes the current knowledge and provides future perspectives for liver xenotransplantation in humans.

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production

“…Hepatic stellate cells (HSCs) possess features of all three primitive germ layers ( Friedman, 2008 ). Complex interaction between the foregut anterior endoderm, cardiac mesoderm, and septum transversum mesenchyme (STM) leads to the development and maturation of the liver bud ( Aravalli, 2021 ; Arterbery and Bogue, 2014 ; Saito et al., 2012 ; Si-Tayeb et al., 2010 ) The major pathways in this interaction are regulated by GATA4, inductive signals from fibroblast growth factors (FGFs), and transforming growth factor beta (TGFβ), working in parallel to regulate the early gene induction that leads to the formation of the liver and pancreas ( Wandzoich and Zaret, 2009 ).…”

“…Although Hhex knockouts have successfully hosted donor-generated livers in porcine intraspecies blastocyst complementation ( Matsunari et al., 2020 ), no interspecies Hhex studies exist. A main concern with Hhex is that its effects on organogenesis are not limited to the liver and could result in off-target donor cell growth ( Aravalli, 2021 ; Bort et al., 2006 ; Cong et al., 2006 ; Jackson et al., 2015 ; Paz et al., 2010 ; Rankin et al., 2011 ). Tbx3 , OC1/2 , Mab21l2 , and UbC have never been tested as targets for blastocyst complementation.…”

“…Generation of a liver via blastocyst complementation has been explored. Hhex , a homeobox gene that regulates liver bud and hepatocyte development, has been disabled in recipient embryos in intraspecies blastocyst complementation, resulting in WT-predominant livers in these chimeras ( Aravalli, 2021 ; Keng et al., 2000 ; Matsunari et al., 2020 ). However, Hhex mutations affect many organs and developmental processes, posing ethical issues for interspecies experiments.…”

Limited Expansion of Human Hepatocytes in FAH/RAG2-Deficient Swine

“…The FAH defect leads to apoptosis of native hepatocytes due to abnormal tyrosine metabolism while wild type (WT) donor hepatocytes engraft and expand robustly after transplantation. While other xenogeneic chimerism models have been proposed, FAH-deficiency is uniquely devoid of significant off-target systemic effects and can be manipulated via treatment with nitisinone (NTBC) to exert or remove selective pressure favoring WT donor hepatocyte expansion 6,7 . The most successful immunodeficiency for expansion of human hepatocytes in mice results from the combined inactivation of recombinase activating gene 2 (RAG2) and interleukin 2 receptor-gamma (IL2rg) with the FAH-deficiency to produce "FRG" mice 8 .…”

“…While other xenogeneic chimerism models have been proposed, FAH deficiency is uniquely devoid of significant off-target systemic effects and can be manipulated through treatment with nitisinone (NTBC) to exert or remove selective pressure favoring WT donor hepatocyte expansion. 6 , 7 The most successful immunodeficiency for expansion of human hepatocytes in mice results from combined inactivation of the recombinase-activating gene 2 ( RAG2 ) and interleukin-2 receptor gamma ( IL2rg ) with the FAH deficiency to produce FRG mice. 8 Although FRG mice support the expansion of human hepatocytes, the use of mice is suboptimal due to their diminutive size and potential transmission of murine retroviruses.…”