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

项目摘要： 全世界有8.44亿人患有肝病，每年死亡率接近200万人。 肝移植是选定病例的首选治疗方法，但受到高质量药物供应的限制 年轻供体（<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（PHH）是治疗选定肝脏的微创替代方法 结构保留但存在代谢紊乱的病理，如急性肝衰竭 和代谢性肝病。细胞的最佳代谢功能对于基于细胞的免疫治疗的成功至关重要。 治疗然而，PHH治疗受到供体稀缺的严重限制， 来自老年供体的PHH的代谢功能。来源于人的人肝细胞样细胞（h-iHLC） 诱导多能干细胞（h-iPSC）作为PHH的替代品出现，用于治疗选择的肝脏 条件H-iHLC具有三个益处：（1）H-iHLC由无限的可再生来源产生：h-iPSC。 (2)他们绕过了与使用胚胎干细胞相关的伦理问题。(3)它们有可能 通过利用患者自身的细胞来防止移植后的同种异体免疫应答。不过， 年龄对代谢功能的有害影响已被描述为PHH，供体年龄的影响 对h-iHLCs中代谢的影响尚未研究。在这里，我们的目标是确定供体年龄相关的变化， 通过研究年轻h-iHLC的转录组和蛋白质组， 并将结果与来自相同供体的PHH进行比较。我们将详细研究 以及细胞色素P450（CYP 450）超家族在h-iHLCs和PHH中的功能作为供体年龄的函数。 DNA甲基化中的CYP 450相关变化下调代谢功能，包括PHH中的CYP 450活性。 因此，我们将研究并尝试调节h-iHLCs中的这种调节机制，以优化 总体代谢功能，包括h-iHLCs中的CYP 450活性。我们将研究 通过将h-iHLCs移植到代谢性肝脏中在急性肝衰竭的鼠模型中产生的h-iHLCs 失败，I型酪氨酸血症（FGN）小鼠（NOD品系背景上的Fah/Rag 2/Il 2 rg）。结果是 这项研究将提供关于供体年龄对h-iHLCs代谢及其调节的影响的关键信息 并且将（1）帮助选择代谢适合的供体用于同种异体h-iHLCs移植，（2）允许将来的移植。 通过改变重编程、分化和分化来调节功能和调节机制 基因编辑，以产生具有优化的代谢功能的高质量h-iHLC，用于同种异体和自体移植。 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.