The effect of donor age on the function and therapeutic efficacy of human hepatocyte-like cells

项目总结： 全世界有8.44亿人患有肝病，每年的死亡率接近200万人。 肝移植是某些病例的首选治疗方法，但受高质量的可获得性限制。 来自年轻捐赠者(&lt；55岁)的器官，以及与手术相关的发病率。基于细胞的治疗 使用原代人肝细胞(PHHs)是治疗精选肝脏的一种微创选择 保留结构但存在代谢紊乱的病理，如急性肝功能衰竭 和代谢性肝病。细胞代谢功能的优化是细胞移植成功的关键 治疗。然而，PHH治疗受到捐赠者稀缺的严重限制，而且 老年供者PHHs的代谢功能。人肝细胞样细胞(h-iHLC) 诱导多能干细胞(h-IPSCs)作为PHHs的替代细胞出现在部分肝脏的治疗中 条件。H-iHLC有三个好处：(1)H-iHLC来自一种无限的、可再生的来源：H-IPSCs。 (2)它们绕过了与使用胚胎干细胞有关的伦理问题。(3)他们有潜力 通过利用患者自身的细胞来防止移植后的同种异体免疫反应。虽然， 年龄对新陈代谢功能的有害影响已被描述为PHHs，供体年龄的影响 对h-iHLCs代谢的研究尚未见报道。在这里，我们的目标是确定捐赠者年龄相关的变化 通过研究青年h-iHLCs的转录组和蛋白质组研究h-iHLCs的整体代谢 并将结果与来自相同捐赠者的PHH进行比较。我们将详细研究这一表达 细胞色素P450超家族在h-iHLCs和PHHs中的功能与供者年龄的关系。 与年龄相关的DNA甲基化改变下调了PHHs的代谢功能，包括CYP450活性。 因此，我们将研究和尝试在h-iHLC中调节这种调节机制，以期达到优化的目的 H-iHLCs的整体代谢功能包括细胞色素P450活性。我们将检查其治疗效果。 H-iHLCs代谢性肝移植在急性肝功能衰竭小鼠模型中的生成 失败，I型暴食症(Fah‘’/Rag2‘’/IL2RG‘on nod-品系Back(FRGN))小鼠。由此产生的结果是 研究将提供有关供者年龄对h-iHLCs代谢及其调控的影响的关键信息。 并将(1)帮助选择适合同种异体h-iHLCs移植的新陈代谢合适的供体，(2)允许未来 通过重新编程、分化和调节机制的改变调节功能和调节机制 基因编辑，以生产高质量的h-iHLC，具有优化的代谢功能，用于异体和自体基因 移植。

Project Summary: Worldwide, 844 million people are afflicted with liver disease, with mortality nearing 2 million deaths per year. Liver transplantation is the preferred treatment for selected cases but is limited by the availability of high-quality organs from young donors (<55 years old), and the morbidity associated with the operation. Cell-based therapy using primary human hepatocytes (PHHs) is a minimally invasive alternative for treatment of select liver pathologies where architecture is preserved but there are metabolic derangements, such as in acute liver failure and metabolic liver disease. Optimal metabolic function of the cells is critical for the success of cell-based therapies. However, PHH therapy is severely limited by the scarcity of donors, and the dramatic decrease in metabolic function of PHHs from older donors. Human hepatocyte-like cells (h-iHLCs) derived from human induced pluripotent stem cells (h-iPSCs) emerged as an alternative to PHHs for treatment of select liver conditions. H-iHLC have three benefits: (1) H-iHLCs are produced from an unlimited, renewable source: h-iPSCs. (2) They bypass ethical concerns associated with the use of embryonic stem cells. (3) They have the potential to prevent an allogeneic immune response following transplantation by utilizing the patients’ own cells. Although, the deleterious impact of age on the metabolic function has been described for PHHs, the impact of donor age on the metabolism in h-iHLCs has not been studied. Here, we aim to identify the donor age-associated changes in the overall metabolic profile of h-iHLCs by studying the transcriptome and proteome of h-iHLCs from young and old donors and compare the results to PHHs from the same donors. We will study in detail the expression and function of the cytochrome P450 (CYP450) superfamily in h-iHLCs and PHHs as a function of donor age. Age-related changes in DNA-methylation down-regulate metabolic function including CYP450 activity in PHHs. Therefore, we will study and attempt to modulate this regulatory mechanism in h-iHLCs with the goal to optimize the overall metabolic function including CYP450 activity in h-iHLCs. We will examine the therapeutic efficacy of the generated h-iHLCs in a murine model of acute liver failure by transplanting h-iHLCs into metabolic liver failure, tyrosemia type I (Fah¯′¯/Rag2¯′¯/Il2rg¯′¯ on NOD-strain background (FRGN)) mice. The results from this study will provide critical information about the impact of donor age on metabolism and its regulation in h-iHLCs and will (1) assist in selecting metabolically fit donors for allogeneic h-iHLCs transplantation, (2) allow future modulation of functional and regulatory mechanisms through alterations in reprogramming, differentiation and gene editing, to produce high-quality h-iHLCs with optimized metabolic function for allo- and autogeneic transplantation.