Enhancement of pancreatic beta cell generation in vitro and in vivo by post-translational regulation of Neurogenin3

许多中老年疾病是由组织功能的逐渐丧失引起的，包括阿尔茨海默病、帕金森病、心力衰竭，尤其是糖尿病。这些慢性疾病通常需要长期药物治疗，但最近主要基于基础生物学的惊人进展提出了直接替换有缺陷细胞的可能性。胰岛细胞是糖尿病中缺失或功能失调的一种细胞，增加胰岛细胞的质量被认为可以改善1型糖尿病，也可能是许多2型糖尿病患者的有效治疗方法。事实上，成功的胰岛移植已被证明可以减轻对胰岛素治疗的需求和减轻糖尿病相关症状。所谓的干细胞，既可以无限增殖，又可以在培养皿中诱导形成更专门的细胞类型，可以作为β细胞替代的来源。然而，一个主要的障碍仍然存在，即干细胞必须被诱导成β细胞的命运，并在移植回患者体内时无限期地保持这种命运，以保留功能并避免癌性肿瘤形成的风险。人们普遍认为，要实现这一目标，干细胞必须遵循胚胎发育过程中β细胞通常走过的发育途径。基于这些发育途径，已经发表了一些方案，其中人类胚胎干细胞可以被诱导形成相对不成熟的β细胞，而它们在体内植入动物体内会导致进一步成熟和β细胞功能。然而，对这种方法的一个警告是，偶尔也会观察到癌症的形成，这表明需要移植分化到更成熟状态的β细胞，以尽量减少这种风险。这是目前在体外产生更成熟的β细胞的一个障碍，我们可能已经发现了一种方法。在发育过程中，所有β细胞的形成都需要蛋白质Neurogenin 3 (Ngn3)，许多实验表明，操纵其活性可以促进干细胞的β细胞形成。然而，正常的Ngn3只会产生相当不成熟的β细胞，这些细胞不能像正常的β细胞那样对血糖水平做出反应。我们发现，通过阻止磷酸盐的加入来改变神经原素蛋白的化学修饰可以显著增强神经原素驱动细胞成熟的能力，我们假设这可能使修饰形式的Ngn3比正常蛋白更好地产生成熟的β细胞。该项目旨在验证这一假设，如果是正确的，就能理解这种增强细胞成熟的机制。为了帮助我们将这个项目推向临床应用，我们正在与哈佛干细胞研究所的道格拉斯·梅尔顿教授合作，他专注于为人体试验产生足够质量的β细胞。

Many illnesses of middle and old age result from a progressive loss of tissue function including Alzheimers, Parkinsons Disease, heart failure and notably diabetes. These chronic conditions generally require long-term medication, but recent spectacular advances mostly grounded in basic biology have raised the possibility that defective cells could be directly replaced. An increase in the mass of beta islets cells, the type of cells that are missing or malfunctioning in diabetes, has been proposed to ameliorate type 1 diabetes, and is also likely to be an effective treatment for many sufferers of Type 2 diabetes. Indeed, successful islet transplantation has been shown to result in relief from a requirement for insulin therapy and relief from diabetes-related symptoms. So called stem cells, cells that can both proliferate indefinitely and can be induced to form more specialised cell types in a culture dish, could act as a source for beta cell replacement. However, a major obstacle remains in that stem cells must be induced to adopt beta cell fate and maintain that fate indefinitely when transplanted back into the patient, retaining functionality and avoiding the risk of cancerous tumour formation. It is widely acknowledged that for this to occur, stem cells must be taken down the developmental pathway that beta cells usually take when undergoing formation in the developing embryo. Based on these developmental pathways, protocols have been published where human embryonic stem cells can be induced to form relatively immature beta cells, while their in vivo implantation back into animals results in further maturation and beta cell functionality. However, a caveat to this approach is that cancer formation was also occasionally observed, indicating the need for tranplantation of beta cells that are differentiated to a more mature state to minimise this risk. This is currently a road-block to generating more mature beta cells in vitro that we may have uncovered a way of moving through.The protein Neurogenin 3 (Ngn3) is needed for formation of all beta cells during development and many experiments indicate that manipulating its activity could enhance beta cell formation from stem cells. However, normal Ngn3 only causes rather immature beta cells to be generated and these cannot respond to blood glucose levels in the same way as normal beta cells. We see that changing the chemical modifications of Neurogenin protein by preventing the addition of phosphates can significantly enhance the ability of Neurogenins to drive cell maturation and we hypothesise that this may make a modified form of Ngn3 much better at generating mature beta cells than the normal protein. This project is designed to test this hypothesis and, if correct, to understand the mechanism of this enhanced cellular maturation. To help us move this project towards clinical application, we are working with Prof Douglas Melton at the Harvard Stem Cell institute who is focussed on generating beta cells of sufficient quality for human trails.