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

项目摘要/摘要 目前有3万多名患者等待接受肝脏移植。这一数字正在增加。 由于美国人口老龄化伴随着慢性肝病发病率的增加 患有酒精性肝病、肝炎、非酒精性脂肪肝和非酒精性脂肪肝等疾病。尽管努力说服 作为器官捐赠者，器官移植越来越供不应求。一 这个问题的解决方案是在动物体内为肝脏和肝细胞产生人的肝脏的能力 移植。尽管有许多分化人类胚胎干细胞(HESCs)的方案， 而可诱导的多能干细胞(IPSCs)在体外分化为多种细胞类型，它们遇到了显著的 翻译到诊所的挑战。然而，现在可以再生器官/细胞的复制品。 第二种动物体内的一种动物。这涉及到特定的击倒(KO) 物种二囊胚中的发育基因和囊胚内注射多能干细胞 来自一个物种的细胞，以产生携带来自该捐赠者的器官/细胞类型的后代。这个 这种方法的翻译需要一种高效的基因编辑技术。事实上，新的TALEN/CRISPR/CAS9 技术提供了一种快速、经济高效的方法来产生转基因动物。 因此，我们建议使用TALEN/CRISPR技术来敲除与 猪囊胚中肝脏的发育。我们假设非人灵长类动物的肝脏可以在 将绒猴胚胎干细胞(ESCs)注射到TALEN和/或CRISPR-KO猪体内 胚泡。绒猴经常被用于研究人类的衰老和疾病，因为它们的身体 与人类非常接近。我们设计了三个具体目标来检验我们的中心假设。 具体地说，我们将(1)通过胚泡互补作为原理证明来产生猪-猪肝嵌合体；(2) 在非人灵长类动物-猪嵌合体模型中开发绒猴肝脏，除了表征它们的 功能；以及(3)询问产生人-猪嵌合肝脏所涉及的遗传程序。 主要由非人灵长类肝细胞组成的完整肝脏的生成 移植的绒猴ESCs将代表着范式的转变，并提供必要的临床前证据 最终在动物身上创造出人类的肝脏。如果成功，这项拟议的研究将改变游戏规则 可以想象，这将为在大型动物(如猪)身上生产人类肝脏铺平道路。 器官和/或肝细胞移植，专门为每个患有慢性病的患者量身定做 肝病。此外，这种新颖的方法，尽管有点高风险，但绕过了许多问题 与数十年的异种移植研究有关。对改善医疗保健的潜在影响 美国和世界各地的肝病都很好。

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.