Genome engineering porcine expanded potential stem cells for xenotransplantation

Abstract

Pig-to-human xenotransplantation holds immense promise as a potential solution to the global shortage of organs for transplantation. Traditionally, pig fibroblasts are used for gene editing and subsequent cloning to generate genetically engineered pigs for organ transplantation. However, it is challenging to perform multiple rounds or complex genome editing in fibroblasts. In this thesis, I have attempted to address this challenge by using pig expanded potential stem cells (EPSCs) as a stem cell source for sophisticated gene editing and the subsequent immuno-phenotyping. Porcine EPSCs are derived from pre-implantation embryos. They exhibit some enriched transcriptional features of pre-implantation embryos, possess remarkable proliferation ability and broad developmental potential. These stem cells are genetically stable in long-term cultures, and enable efficient precise editing such as knock-in for making reporter cell lines. In the first part of my thesis project, I optimized the porcine EPSC culture medium. This optimized medium allowed the isolation of pig EPSCs from in vitro fertilization (IVF) pre-implantation embryos, besides from porcine in vivo preimplantation embryos. I subsequently used the optimized porcine EPSCs for xenotransplantation-related gene editing and cell differentiation. I successfully knocked out porcine genes associated with xeno-antigen expression (GGTA1, CMAH, B4GALNT2) and with organ size control (GHR). I further established a pig EPSC master cell line for very efficient Cre-loxP site-specific recombination and introduced human cDNAs such as human CD47, which is associated with macrophage ”don’t eat me” signals. Finally, I differentiated the edited pEPSCs into various cell lineages including endothelial cells for immunophenotyping the functionalconsequences of gene editing. I found that the GGTA1-/- CMAH-/- B4GALNT2-/- GHR-/-(QKO) - human CD47-pEPSC-derived endothelial cells exhibited substantially reduced complement-mediated cytotoxicity, human immunoglobulin binding, and macrophage phagocytosis when compared to the control non-edited EPSC- derived endothelial cells. Therefore, pEPSCs offer a quick and reliable avenue for immunophenotyping the introduced genetic changes without the need to generate a cloned pig for in vivo tissue antigenicity evaluation, highlighting EPSC’s unique advantages in xenotransplantation research. As a supplementary part of my thesis, I studied TP53-knockout pig EPSCs. TP53 mutations in cancer cells can contribute to an immunosuppressive microenvironment and enable immune evasion. Leveraging this immune escape mechanism, I generated TP53-knockout pig EPSCs through both deletion (DNA binding domain knockout, TP53-/-) and point mutation (TP53-R167H/R167H, pig R167H corresponding to human R175H), and investigated their molecular features and developmental potential. These mutant cells exhibited robust culture characteristics and retained the capacity to differentiate into the three germ layers. However, their extraembryonic developmental potential to trophoblast was impaired, in line with another study in our lab where TP53 knockout in human EPSCs caused severe trophoblast defects. I will further test the TP53 mutant cells in immunophenotyping. These TP53-knockout pEPSCs may serve as a proof-of-concept for xenotransplantation research. In conclusion, I have successfully generated multiple gene-edited porcine EPSCs for xenotransplantation and established an in vitro immune phenotyping system for the edited EPSCs. These findings and resources are expected to facilitate pig-to-human xenotransplantation research and promote the development of novel approaches to help address the critical shortage of organs for transplantation.