A Novel Stem Cell-based Approach for Generating Non-Human Primate Livers in Pigs

项目总结/摘要 目前有3万多名患者等待接受肝脏移植。这个数字正在增加 由于美国人口老龄化，伴随着慢性肝病的发病率增加， 酒精性肝病、肝炎、NAFLD和NASH等疾病。尽管努力劝说 随着人们成为器官捐献者，器官移植的需求越来越超过供应。一 这个问题的解决方案是能够在动物体内产生人类肝脏，用于肝脏和肝细胞 移植虽然有许多方案来分化人类胚胎干细胞（hESC）， 和诱导性多能干细胞（iPSC）离体转化为多种细胞类型，他们遇到了显著的困难。 翻译到诊所的挑战。然而，现在有可能再生器官/细胞的复制品， 一种动物在另一种动物体内。这涉及特异性的敲除（KO）。 发育基因在第二物种的囊胚;和囊胚内注射多能干细胞 从物种1中提取细胞，以产生携带来自该供体的器官/细胞类型的后代。的 这种方法的翻译需要高效的基因编辑技术。事实上，新型TALEN/CRISPR/Cas9 技术为生产转基因动物提供了一种快速和具有成本效益的手段。 因此，我们建议采用TALEN/CRISPR技术敲除与以下相关的特定基因： 肝脏在猪胚泡中的发育。我们假设非人灵长类动物的肝脏可以在 通过将绒猴胚胎干细胞（ESC）注射到TALEN和/或CRISPR-KO猪中， 囊胚绒猴经常被用于人类衰老和疾病的研究，因为它们的身体 非常接近人类。我们设计了三个具体目标来检验我们的中心假设。 具体来说，我们将（1）通过囊胚互补产生猪肝嵌合体作为原理证明;（2） 在非人灵长类-猪嵌合体模型中开发绒猴肝脏， 功能性;和（3）询问参与产生人-猪嵌合肝脏的遗传程序。 产生主要由来源于非人灵长类动物肝细胞组成的全肝， 植入的绒猴胚胎干细胞将代表一种范式转变，并提供必要的临床前证据， 最终在动物身上制造出人类肝脏。如果成功的话，这项拟议中的研究将改变游戏规则 可以想象，这可能为在大型动物（如猪）中生产人类肝脏铺平道路， 器官和/或肝细胞移植，特别是为患有慢性胰腺炎的每个患者量身定制。 肝脏疾病此外，这种新颖的，尽管有点高风险的方法规避了许多问题 与几十年的异种移植研究有关。对改善卫生保健的潜在影响， 美国和世界范围内的肝病是伟大的。

Project Summary/Abstract At present there are more than 30,000 patients waiting to receive liver transplants. The number is increasing due to an aging US population accompanied by an increasing incidence of chronic liver diseases associated with such disorders as alcoholic liver disease, hepatitis, NAFLD and NASH. In spite of efforts to persuade people to serve as organ donors, the demand increasingly outstrips the supply for organ transplantation. One solution to this problem is the ability to generate human livers in animals for liver as well as hepatocyte transplantation. Although there are numerous protocols to differentiate human embryonic stem cells (hESCs), and inducible pluripotent stem cells (iPSCs) ex vivo to a variety of cell types, they have encountered significant challenges in translation to the clinic. However, it is now possible to regenerate the replica of organs/cells from one species of animal within the body of a second species. This involves the knockout (KO) of specific developmental genes in the blastocyst of species two; and the intra-blastocyst injection of pluripotent stem cells from species one to generate offspring that carry organs/cell types derived from that donor. The translation of this approach requires an efficient gene-editing technology. In fact, novel TALEN/CRISPR/Cas9 technologies provide such a rapid, and cost-effective means to generate genetically modified animals. Accordingly, we propose to employ the TALEN/CRISPR technology to knockout specific genes associated with liver development in the pig blastocyst. We hypothesize that non-human primate liver can be generated in the pig by the injection of marmoset embryonic stem cells (ESCs) into TALEN and/or CRISPR-KO porcine blastocysts. Marmosets are often used for research on human aging and disease because their bodies are very close to those of humans. We have designed three Specific Aims to test our central hypothesis. Specifically, we will (1) generate pig-pig liver chimeras by blastocyst complementation as proof-of-principle; (2) develop marmoset livers in a model of non-human primate-pig chimeras in addition to characterizing their functionality; and (3) interrogate the genetic programs involved in generating human-porcine chimeric livers. The generation of whole livers that are comprised primarily of non-human primate hepatic cells derived from implanted marmoset ESCs would represent a paradigm shift and provide the necessary preclinical evidence for ultimately creating human livers in animals. If successful, the proposed research would be a game-changer that could conceivably pave the way for the production of human livers in large animals, such as the pig, for organ and/or hepatocyte transplantation that is specifically tailored for each patient suffering from a chronic liver disease. In addition, this novel, albeit somewhat high-risk approach circumvents many of the problems associated with decades of research on xenotransplantation. The potential impact on improved health care in the U.S. and world wide for liver diseases is great.