Mice Humanized with Hepatocytes and iPS Cells from Patients with Metabolic Diseas

描述（由申请人提供）：特定挑战主题14-DK-101，“诱导多能干细胞-细胞和人源化小鼠”通过诱导多能干细胞（iPSC）技术，来自疾病患者的体细胞（如成纤维细胞）可用于产生细胞系、组织以及可能的器官系统。这种模型可用于阐明疾病的潜在病理学或筛选可用于治疗的药剂。将这种方法与能够接受多种人体组织而不排斥的小鼠品系相结合，可以提供微环境，以支持整个生物体背景下组织的生理功能，从而能够更好地理解疾病发病机制，并为候选药物的临床前测试提供平台。“肝脏执行超过3,000种对维持机体至关重要的功能，从白蛋白，凝血因子和抗蛋白酶等蛋白质的产生和分泌，到药物或毒素等外源性化合物以及激素，胆红素和胆汁酸和胆固醇等内源性化合物的代谢和排泄。肝脏代谢疾病可能由这些关键途径中的基因突变引起。转基因或基因敲除小鼠模型已被创建用于许多这些疾病，但它们并不总是忠实地再现人类疾病。例如，胆盐输出泵（BSEP）中的突变导致需要整个器官移植的人类患者的严重胆汁淤积和纤维化，而敲除小鼠几乎没有表型。虽然许多患有α-1-抗胰蛋白酶（A1 AT）缺乏症的患者多次发生肝纤维化/肝硬化，导致肝移植，但携带突变人类基因的转基因小鼠模型显示出极其温和的表型。鸟氨酸转氨甲酰酶（OTC）活性（氨代谢中的限速步骤）缺乏的小鼠模型是一个合理的模型，然而，动物耐受含有正常量蛋白质的饮食，而严重受影响的人类患者需要严格的蛋白质限制，以防止致命的高氨血症。小鼠（和大鼠）是研究肝纤维化和肝硬化的特别差的模型，这是许多人类肝脏疾病的共同特征。人源化小鼠为肝纤维化和肝硬化的研究和治疗提供了一个平台。这些只是小鼠模型中的几个例子，它们不能忠实地再现人类疾病。我们提出的假设是，人类代谢性肝病的最佳模型是从受影响的人类肝细胞中创建的模型。因此，我们建议通过移植受影响的人肝细胞来“人源化”FRG小鼠的肝脏，以创建真实的人类代谢疾病模型。这些小鼠是免疫缺陷的，并且也缺乏酪氨酸分解代谢酶，富马酰乙酰乙酸水解酶（Fah -/-），如果不治疗，则发展为不可逆的肝衰竭。然而，如果移植Fah-熟练的细胞，它们容易且快速地用供体细胞重新填充天然肝脏，即使供体细胞是人源的（a）。为了创建这些模型，FRG小鼠的肝脏将用来自代谢疾病患者的肝细胞“人源化”。此外，iPSC技术将用于重编程来自代谢疾病患者的肝细胞，并且在肝分化之后，将用iPS衍生的肝细胞对另外的小鼠进行人源化。然后可以将这些人源化小鼠模型与真实的患病肝脏在（患者或动物的）临床化学变化、肝脏组织病理学以及肝脏组织的基因和蛋白质表达谱方面进行比较。以这种方式，我们将能够确定用这些程序开发的人源化模型是否忠实地再现在患者中观察到的表型。除了对代谢性肝病的直接影响之外，这些模型的成功将促进使用人源化小鼠模型来研究其他基于肝脏的疾病，例如威尔逊病和α-1-抗胰蛋白酶缺乏症，并且甚至可能导致用于原发性急性肝功能衰竭和病毒性、酒精性和自身免疫性肝炎以及甚至疟疾的肝脏阶段的更好的人源化模型，特别是如果除了肝脏之外还重建了人类免疫系统。 公共卫生相关性：肝功能遗传缺陷影响大约1/50，000的活产婴儿。对于大多数人来说，没有小动物模型可以忠实地复制人类表型。我们提出了一个假设，即这些肝脏疾病的最合适的模型是用真实的受疾病影响的肝细胞产生的。我们最近成功地通过用人肝细胞重新填充肝脏来使专门小鼠的肝脏人源化。我们建议通过移植代谢性肝病患者的肝细胞来建立人类肝病模型，并表征该模型与人类疾病的相关性。此外，我们将重新编程肝细胞，以产生诱导多能细胞（iPSC），这些细胞也可用于重新填充和人源化小鼠肝脏。我们建议这些人源化小鼠将成为更好地了解人类疾病的技术平台，它将有助于开发基因或细胞疗法来纠正这些毁灭性疾病。

DESCRIPTION (provided by applicant): Specific Challenge Topic 14-DK-101, "Induced Pluripotent stem cells-cellular and humanized mouse" Somatic cells such as fibroblasts from patients with diseases can be used to create cell lines, tissues, and perhaps, organ systems, through induced pluripotent stem cell (iPSC) technology. Such models could be used to elucidate underlying pathology of disease or screen for agents that could be used therapeutically. Combining this approach with mouse strains able to accept multiple human tissues without rejection could provide the microenvironment milieu to support the tissue's physiological function within the context of the whole organism, enabling greater understanding of disease pathogenesis and providing a platform for preclinical testing of drug candidates." The liver performs over 3,000 functions critical to maintaining the organism, ranging from production and secretion of proteins such as albumin, clotting factors and antiproteases, to metabolism and excretion of exogenous compounds such as drugs or toxins as well as endogenous compounds like hormones, bilirubin and bile acids and cholesterol. Liver-based metabolic disease can result from mutations in genes in these critical pathways. Transgenic or knock-out mouse models have been created for many of these diseases, but they do not always faithfully reproduce the human disease. For example mutations in the bile salt export pump (BSEP) results in severe cholestasis and fibrosis in human patients requiring whole organ transplants, yet the knockout mouse is nearly without a phenotype. While many patients with alpha-1-antitrypsin (A1AT) deficiency develop liver fibrosis/cirrhosis many times resulting in liver transplantation, the transgenic mouse model carrying mutant human genes shows an extremely mild phenotype. The mouse model for a deficiency of ornithine transcarbamylase (OTC) activity, the rate limiting step in ammonia metabolism, is a fair model, however the animals tolerate a diet containing normal amounts of protein, while severely affected human patients require severe protein restriction to prevent lethal hyperammonemia. Mice (and rats) are particularly poor models for studying liver fibrosis and cirrhosis, common features of many human liver diseases. Humanized mice may offer a platform to both the study and treatment of hepatic fibrosis and cirrhosis. These are only a few examples of mouse models that do not faithfully recreate the human disease. We propose, the hypothesis, that the best models for human metabolic liver disease are those created from the affected human hepatocytes. Thus, we propose to" humanize" the liver of FRG mice by transplantation of affected human hepatocytes to create authentic models of human metabolic disease. These mice are immunodeficient and also deficient in the tyrosine catabolic enzyme, fumarylacetoacetate hydrolase (Fah -/-) and develop irreversible liver failure if left untreated. However, if Fah-proficient cells are transplanted, they readily and rapidly repopulate the native liver with donor cells, even if the donor cells are of human origin (a). To create these models, the liver of FRG mice will be "humanized" with hepatocytes derived from patients with metabolic disease. In addition, iPSC technology will be utilized to reprogram the liver cells from metabolic disease patients and following hepatic differentiation, additional mice will be humanized with iPS-derived hepatocytes. These humanized mouse models can then be compared to the authentic diseased liver with respect to changes in clinical chemistry (of the patient or animal), the histopathology of the liver and gene and protein expression profiling of liver tissue. In this manner, we will be able to determine if the humanized models developed with these procedures faithfully reproduce the phenotype observed in the patient. In addition to the direct effects on metabolic liver disease, success with these models will facilitate the use of humanized mouse models to investigate other liver based diseases such as Wilson's and Alpha-1-antitrypsin deficiency and may even lead to better-humanized models for primary, acute liver failure and viral, alcoholic and autoimmune hepatitis and even the hepatic stage of malaria, especially if a human immune system were reconstituted in addition to the liver. PUBLIC HEALTH RELEVANCE: Genetic defects in liver functions affect approximately 1/50,000 live births. For most, there are no small animal models that faithfully reproduce the human phenotype. We propose the hypothesis that the most appropriate model for these liver based diseases is one that is produced with authentic disease-affected hepatocytes. We have recently succeeded in humanizing the liver of specialized mice by repopulating the liver with human hepatocytes. We propose to develop models of human liver disease by transplantation of liver cells from patients with metabolic liver diseases and characterize the model for relevance to the human disease. In addition we will reprogram the liver cells to produce induced pluripotent cells (iPSC) that can also be used to repopulate and humanize the mouse liver. We propose these humanized mice will be a technology platform to better understand the human disease and it will aid in the development of gene or cellular therapy to correct these devastating diseases.