Reprogramming adult human hepatocytes into liver progenitors with unlimited self-renewal, efficient differentiation, and transplantation capacities

将成人肝细胞重编程为具有无限自我更新、高效分化和移植能力的肝祖细胞

• 批准号: MR/V005537/1
• 负责人: Keisuke Kaji
• 金额: $ 78.71万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV005537%2F1
• 关键词: Reprogramming; adult; human; hepatocytes; into

The liver is the largest internal organ in the body and performs ~500 tasks, including detoxification of certain substances and balancing energy metabolism. While it has an excellent regeneration capacity, chronic damage due to years of alcohol abuse or high fat diets can result in severe liver function failure. Liver transplantation is currently the only available treatment for terminal liver diseases, but the liver transplant waiting list is getting longer in recent days due to the limited number of donors and the increasing number of patients. Hepatocytes, which make up 55-68% of the liver mass, are the major players of metabolism and drug biotransformation in our body, and the use of human hepatocyte for the toxicology tests in the laboratory can reduce the numbers of animals commonly used. It will also allow more precise prediction of the drug toxicity, because different species have different metabolic activities. However, mature hepatocytes cannot be propagated in the laboratory and their supply for the toxicology tests also depends on the limited number of donors. Excitingly, recent research has enabled the conversion of the normally non-proliferative state of mature hepatocytes from rat liver to a proliferative state by using a specific culture condition in the laboratory. These 'reprogrammed' cells are in a similar state to 'hepatocyte progenitors', which transiently proliferate and then become fully functional mature hepatocytes in the developing or regenerating liver in the body. Importantly, the artificially created rat hepatocyte progenitors showed an unlimited proliferation capacity and a capacity to go back to non-proliferative, fully functional mature hepatocytes when placed in another culture condition. If the same cell state conversion, called cellular reprogramming, is successfully achieved with human hepatocytes, we can generate an unlimited number of fully functional hepatocytes from one donor. However, human hepatocyte reprogramming with the same or similar modified conditions so far have resulted in only a limited success. The hepatocyte progenitors derived from the adult human liver could proliferate only <1 month. Those from the infant liver could gain an unlimited proliferation capacity, reflecting the highly proliferative nature of neonatal hepatocytes. However, they lost the capacity to make fully functional hepatocytes, called a differentiation capacity, after 2 weeks in the dish. In order to achieve successful reprogramming of human hepatocytes into the right progenitor state, we will use techniques called genetic engineering. With this technique, we will supply multiple candidate genes, which are missing in the partially reprogrammed non-functional human progenitors when compared to the fully functional rat progenitors, to the human progenitors. This would result in fully reprogrammed human hepatocyte progenitors with an unlimited proliferation and efficient differentiation capacities in a genetic modification-dependent manner. In parallel, we will look for environmental cues that can help to maintain the reprogrammed state in the absence of the genetic manipulation. This will be performed by eliminating each one of all ~20,000 genes in the genome simultaneously in millions of cells using the state-of-the-art technology called CRISPR/Cas9 genome editing. This strategy will inform us which environmental cues are essential to maintain the reprogrammed human hepatocyte progenitors, allowing us to supplement these essential components (protein/chemicals) in the culture condition, instead of the genetic modifications. In summary, this project aims to develop a strategy to generate fully functional human liver progenitors that will be an unlimited source of fully functional mature hepatocytes, used for drug screening, toxicology tests, as well as cell therapies, in the absence of genetic modifications.

肝脏是人体最大的内脏器官，执行约500项任务，包括某些物质的解毒和平衡能量代谢。虽然它具有极好的再生能力，但由于多年酗酒或高脂肪饮食造成的慢性损害可能会导致严重的肝功能衰竭。肝移植是目前治疗终末期肝病的唯一方法，但最近几天，由于捐赠者数量有限，患者数量不断增加，等待肝移植的名单越来越长。肝细胞占肝脏质量的55-68%，是人体代谢和药物生物转化的主要参与者，利用人肝细胞进行实验室毒理试验可以减少常用动物的数量。它还将允许更准确地预测药物毒性，因为不同的物种具有不同的代谢活动。然而，成熟的肝细胞不能在实验室中繁殖，其毒理学测试的供应也取决于捐赠者的有限数量。令人兴奋的是，最近的研究通过在实验室中使用特定的培养条件，使成熟肝细胞的正常非增殖状态从大鼠肝脏转变为增殖状态。这些“重新编程”的细胞处于类似于“肝细胞前体细胞”的状态，后者在体内发育或再生的肝脏中短暂增殖，然后成为功能齐全的成熟肝细胞。重要的是，人工培养的大鼠肝细胞前体细胞显示出无限的增殖能力，并且当放置在另一种培养条件下时，能够返回到非增殖的、功能完全的成熟肝细胞。如果在人类肝细胞中成功地实现了同样的细胞状态转换，称为细胞重编程，我们可以从一个捐赠者那里产生无限数量的全功能肝细胞。然而，到目前为止，使用相同或相似修改条件的人类肝细胞重新编程只取得了有限的成功。成人肝脏来源的肝细胞前体细胞只能增殖1个月。来自婴儿肝脏的细胞可以获得无限的增殖能力，反映了新生儿肝细胞的高度增殖特性。然而，在培养2周后，他们失去了产生完全功能的肝细胞的能力，这种能力被称为分化能力。为了成功地将人类肝细胞重新编程为正确的祖细胞状态，我们将使用称为基因工程的技术。通过这项技术，我们将向人类祖细胞提供多个候选基因，这些基因与完全功能的大鼠祖细胞相比，在部分重新编程的非功能性人类祖细胞中缺失。这将导致完全重新编程的人肝细胞前体细胞具有无限的增殖和高效的分化能力，并以基因修饰依赖的方式。同时，我们将寻找环境线索，在没有基因操纵的情况下帮助维持重新编程的状态。这将通过使用称为CRISPR/Cas9基因组编辑的最先进技术在数百万个细胞中同时消除基因组中所有约20,000个基因中的每一个来实现。这一策略将告诉我们哪些环境线索是维持重新编程的人类肝细胞前体细胞所必需的，使我们能够在培养条件下补充这些必要的成分(蛋白质/化学物质)，而不是遗传修饰。综上所述，该项目旨在开发一种策略，以产生全功能的人类肝脏前体细胞，在没有遗传修饰的情况下，将成为全功能成熟肝细胞的无限来源，用于药物筛选、毒理学测试以及细胞治疗。