Development of dynamic 3D culture systems for maintenance and expansion of pluripotent embryonic stem cells

干细胞在我们的生命中一直存在于我们的体内，并且非常重要，因为它们确保当受损和磨损的细胞死亡时，有新细胞的供应来取代它们。一些器官和组织有巨大的能力来取代细胞，例如血液系统，肝脏和肠道内壁。然而，其他组织，例如大脑和心脏，几乎没有再生能力。干细胞已经从发育非常早期的胚胎中分离出来（称为胚胎干细胞），也可以从各种成人来源中分离出来，包括骨髓，肠道和肌肉。研究表明，这些干细胞有两个显著的特性。它们可以分裂并形成两个相同的干细胞，即自我更新，或者它们可以通过称为分化的过程形成许多其他类型的细胞。目前，人们对利用干细胞的这些独特特性非常感兴趣，因为通过使用干细胞，可以在体外即实验室中产生细胞，然后可以用于替换体内受损的组织。许多慢性疾病目前无法有效治疗，因为细胞损失是根本原因。一些脑部疾病的发生是因为某些神经细胞死亡，例如帕金森氏病。在儿童糖尿病中，通常控制血糖水平的胰腺细胞被破坏，这意味着患者依赖于定期注射胰岛素来调节血糖水平。基于干细胞的疗法为这些疾病的患者提供了潜在的令人兴奋的替代疗法。然而，在将这些进步带入现代医学实践之前，需要对干细胞及其行为进行更多的研究。通常支持干细胞在体内生长和存活的环境具有三维结构，越来越多的证据表明这种3D微环境对于维持干细胞表型至关重要。然而，目前在实验室中用于扩增然后研究干细胞的方法主要依赖于二维培养。在这个提议中，我们希望开发出在三维动态培养系统中维持和扩增胚胎干细胞的方法，因为这将更接近地模拟它们在宿主发育中的囊胚内的自然环境。这种三维培养可以提供比二维培养显著的优势。我们将研究由不同材料制成的具有不同表面修饰的支架维持ES细胞生长和自我更新的能力。我们将进一步研究不同生物反应器形式和不同生长因子对ES细胞维持和生长的影响。我们的目标是开始研究小鼠胚胎干细胞的行为，但同时启动人类胚胎干细胞的实验。我们希望这些研究将使我们能够优化最合适的动态3D培养系统，以允许hES细胞的扩增和维持。如果基础干细胞研究要成功地转化为临床，这是一个需要解决的重要目标。这种扩增对于需要大量未分化的人ES细胞的情况下的未来应用是至关重要的，例如，这些开发的应用可以使大量未分化的干细胞生长成为可能，这些干细胞随后可以分化成例如神经细胞、肝细胞等，并用于替代患有慢性疾病的患者中受损的细胞和组织。这种基于细胞的策略为许多此类疾病的患者提供了真实的希望，这里提出的研究可能在中长期内具有真实的益处。

Stem cells are present within our bodies throughout our lives and are very important because they make sure that as damaged and worn out cells die, there is a supply of new cells to replace them. Some organs and tissues have a tremendous capacity to replace cells, for example the blood system, the liver and the inner lining of the gut. However, other tissues, for example the brain and heart, have almost no capacity for regeneration. Stem cells have been isolated from embryos at very early stages of development (termed embryonic stem cells) and also from a variety of adult sources including bone marrow, gut and muscle. Research has shown that these stem cells have two remarkable properties. They can divide and form two identical stem cells, i.e. self-renew themselves, or they can form many other types of cells by a process called differentiation. There is currently great interest in harnessing these unique properties of stem cells because by using stem cells it may be possible to generate cells outside the body i.e. in the laboratory, that can then be used to replace damaged tissues inside the body. Many chronic diseases cannot currently be effectively treated because loss of cells is the underlying cause. Several brain disorders occur because certain nerve cells die, e.g. in Parkinson's disease. In childhood diabetes, the cells of the pancreas that normally act to control the level of sugar in the blood are destroyed meaning patients are reliant on regular injections of the hormone insulin in an attempt to regulate their blood sugar levels. Stem cell-based therapies offer potentially exciting alternative treatments for the sufferers of such diseases. However, much more research needs to be carried out on stem cells and their behaviour before such advances will be brought into modern day medical practice. The environment that normally supports the growth and survival of stem cells within the body has 3-dimensional architecture and there is increasing evidence that this 3D microenvironment is critical for maintenance of the stem cell phenotype. However, the methods currently used in laboratories to expand and then study stem cells rely largely on 2-dimensional cultures. In this proposal we want to develop ways in which embryonic stem cells can be maintained and expanded in 3-dimensional dynamic culture systems, since this would more closely mimic their natural environment within the developing blastocyst of the host. Such 3-dimensional culture could afford significant advantages over culture in 2-dimensions. We will investigate the ability of scaffolds made of different materials, with different surface modifications, to maintain ES cell growth and self-renewal. We will further investigate the effects of different bioreactor formats and the influence of different growth factors on ES cell maintenance and growth. Our aim is to begin by studying the behaviour of ES cells derived from mice, but in parallel to initiate experiments with human ES cells. We hope that these studies will enable us to optimise the most appropriate dynamic 3D culture system that allows for expansion and maintenance of hES cells. This is an important goal that needs to be addressed if basic stem cell research is to be successfully translated into the clinic. Such expansion is critical for future applications in situations where large numbers of undifferentiated human ES cells are required, for example application of these developments could make it possible to grow large numbers of undifferentiated stem cells that can subsequently be differentiated into e.g. nerve cells, liver cells etc and used to replace damaged cells and tissues in patients suffering from chronic diseases. Such cell-based strategies offer real hope for the sufferers of many such diseases and the research proposed here could be of real benefit in the medium to longer-term.