Supporting data for "Genome Engineering Porcine Expanded Potential Stem Cells For Xenotransplantation".

Pig-to-human xenotransplantation holds immense promise as a potential so- lution to the global shortage of organs for transplantation. Traditionally, pig fi- broblasts 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 ge- netically 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 intro- duced 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 substan- tially reduced complement-mediated cytotoxicity, human immunoglobulin bind- ing, 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 microen- vironment and enable immune evasion. Leveraging this immune escape mecha- nism, I generated TP53-knockout pig EPSCs through both deletion (DNA binding domain knockout, TP53-/-) and point mutation (TP53-R167H/R167H, pig R167H cor- responding to human R175H), and investigated their molecular features and de- velopmental 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 tro- phoblast defects. I will further test the TP53 mutant cells in immunophenotyping. These TP53-knockout pEPSCs may serve as a proof-of-concept for xenotransplan- tation research.In conclusion, I have successfully generated multiple gene-edited porcine EP- SCs for xenotransplantation and established an in vitro immune phenotyping sys- tem for the edited EPSCs. These findings and resources are expected to facili- tate pig-to-human xenotransplantation research and promote the development of novel approaches to help address the critical shortage of organs for transplantation.

猪-人异种移植作为一种解决全球器官移植短缺问题的潜在方案，具有巨大的发展前景。传统上，利用猪成纤维细胞进行基因编辑和后续克隆，以生成用于器官移植的基因工程猪。然而，在成纤维细胞中执行多轮或复杂的基因组编辑是一项挑战。在本论文中，我试图通过利用猪扩展潜能干细胞（EPSCs）作为基因编辑和随后免疫表型分析的干细胞来源来应对这一挑战。猪EPSCs来源于早期胚胎。它们展现出一些早期胚胎丰富的转录特征，具备显著的增殖能力和广泛的发展潜力。这些干细胞在长期培养中保持基因稳定性，并能够实现高效的精确编辑，如敲入以构建报告细胞系。在我论文项目的第一部分，我优化了猪EPSC培养介质。这种优化的介质不仅允许从体外受精（IVF）早期胚胎中分离猪EPSCs，而且还可以从体内早期胚胎中分离。随后，我使用优化的猪EPSCs进行异种移植相关的基因编辑和细胞分化。我成功敲除了与异种抗原表达（GGTA1、CMAH、B4GALNT2）和器官大小控制（GHR）相关的猪基因。我进一步建立了一个猪EPSC主细胞系，以实现高效的Cre-loxP位点特异性重组，并引入了如人类CD47等人类cDNAs，CD47与巨噬细胞“不要吃我”信号相关。最后，我将编辑后的pEPSCs分化成各种细胞谱系，包括内皮细胞，以免疫表型分析基因编辑的功能性后果。我发现，与对照非编辑EPSCs来源的内皮细胞相比，GGTA1-/- CMAH-/- B4GALNT2-/- GHR-/-（QKO）-人类CD47-pEPSC衍生的内皮细胞显示出显著降低的补体介导的细胞毒性、人类免疫球蛋白结合和巨噬细胞吞噬。因此，pEPSCs为快速且可靠的免疫表型分析引入的遗传变化提供了途径，无需生成克隆猪进行体内组织抗原性评估，凸显了EPSC在异种移植研究中的独特优势。作为论文的补充部分，我研究了TP53敲除猪EPSCs。肿瘤细胞中的TP53突变可能导致免疫抑制性微环境和免疫逃逸。利用这种免疫逃逸机制，我通过删除（DNA结合域敲除，TP53-/-）和点突变（TP53-R167H/R167H，猪R167H对应人类R175H）两种方式生成了TP53敲除猪EPSCs，并研究了它们的分子特征和发育潜力。这些突变细胞表现出稳健的培养特征，并保留了分化为三个胚层的潜力。然而，它们在滋养层发育中的潜能受损，这与我们实验室的另一项研究一致，其中人类EPSCs中的TP53敲除导致严重的滋养层缺陷。我将进一步测试TP53突变细胞在免疫表型分析中的应用。这些TP53敲除pEPSCs可能作为异种移植研究的概念验证。总之，我成功生成了多个用于异种移植的基因编辑猪EPSCs，并建立了一个用于编辑EPSCs的体外免疫表型分析系统。这些发现和资源有望促进猪-人异种移植研究，并推动新型方法的发展，以帮助解决器官移植的严重短缺问题。