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

许多中老年人的疾病是由于组织功能的逐渐丧失，包括老年痴呆症、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已经提出了直接替换缺陷细胞的可能性。增加β胰岛细胞的质量，这种细胞在糖尿病中缺失或发生故障，已被提出改善1型糖尿病，并且也可能是许多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缓解对胰岛素治疗的需求和缓解糖尿病相关症状。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的细胞，可以作为β细胞替代的来源。然而，一个主要的障碍仍然是，干细胞必须被诱导采用β细胞命运，并在移植回患者体内时无限期地保持这种命运，保持功能并避免癌性肿瘤形成的风险。人们普遍认为，要做到这一点，干细胞必须沿着β细胞在发育胚胎中形成时通常采取的发育途径进行。基于这些发育途径，已经公布了可以诱导人胚胎干细胞形成相对不成熟的β细胞的方案，而将其体内植入动物体内会导致进一步成熟和β细胞功能。然而，这种方法的一个警告是，偶尔也会观察到癌症形成，这表明需要移植分化为更成熟状态的β细胞，以最大限度地降低这种风险。这是目前在体外产生更多成熟β细胞的一个障碍，我们可能已经发现了一种方法。神经生成素3（Ngn 3）蛋白是发育过程中形成所有β细胞所必需的，许多实验表明，操纵其活性可以增强干细胞的β细胞形成。然而，正常的Ngn 3只会导致相当不成熟的β细胞产生，这些细胞不能以与正常β细胞相同的方式对血糖水平做出反应。我们看到，通过阻止添加磷酸盐来改变神经生成素蛋白的化学修饰可以显著增强神经生成素驱动细胞成熟的能力，我们假设这可能使Ngn 3的修饰形式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，如果正确，则了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，他专注于为人类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.