Optimising human stem cell models to decipher signals and responses during organogenesis

哺乳动物胎儿是在胚胎植入子宫后，通过组织规范的渐进过程形成的。每个物种的器官都是通过3个不同的层逐渐分化而发育的：外胚层、内胚层和中胚层，在称为“原肠胚形成”的过程中，它们在物理上和功能上分开。由于这种情况发生在母亲体内，大多数研究都需要移除胚胎，主要使用小鼠作为模型系统。原肠胚可以在体外培养数天，但这需要大量的小鼠（大约4只雌性小鼠培养20个胚胎）和从4-5只大鼠的血液中纯化的血清。胚胎干细胞（ESCs）来源于植入前胚胎，在培养中可以无限扩增，同时保持分化成身体任何组织的能力。通常在基因缺失或过度表达后将ESCs注射到植入前胚胎中，并将新形成的“嵌合体”移植到养母体内。然后选择转基因的后代进行进一步繁殖。为了绕过对动物解决某些科学问题的要求，ESCs可以在3D悬浮培养中生长，使用一种简单的方案，允许它们经历类似于原肠胚形成的过程。这些3D“类胃原体”可以被引导分化成可识别的组织和基本器官，与胚胎具有很强的物理和分子相似性。类胃原体是一种有价值的、易于操作的工具，使研究人员能够减少发育研究所需的胚胎数量。然而，由于小鼠对人类的发育有局限性，我们和其他人已经从人类ESCs中产生了类胃原质。由于伦理方面的考虑，人类胚胎不能用于研究原肠胚形成，人类原肠样细胞为研究人类发育打开了一扇窗，否则是无法进入的。值得注意的是，在类原肠中启动对称断裂的过程是自发的，但这使得很难解开正常原肠形成和器官形成所需的信号。因此，我们面临着一个重大挑战，即确定正常人类胚胎中的过程是如何启动的，这阻碍了我们发现胚胎异常原因的能力。我们的项目将设计一个系统来控制信号提示的位置、集水区和持续时间，以加强对人类胃样蛋白特定身体部位的理解和控制。我们打算专注于一个内部器官，肠道，和一个外部结构，肢体芽。在类胃中可以诱导出初步的肠管，显示出一些类似于胎儿消化道的明显区域。我们将研究专门的“神经嵴细胞”（NCCs）的作用，这些细胞被招募到各种发育中的组织中。在正常发育中，NCCs从发育中的神经管中出现，并迁移产生各种类型的细胞，包括那些在发育中的肠道中形成神经细胞的细胞。我们还将注射NCCs，以确定这些特殊神经元是如何被招募到肠道的，以及它们是否能促进肠道的结构和功能发育。这对于理解先天性巨结肠病等缺陷尤为重要。此外，我们将在定制的凝胶中封装正在发育的类胃原液，以便局部应用已知的可诱导肢体芽形成和图案的物质。由小鼠ESCs产生的类胃原体在侧翼有不同的区域，表达参与肢体形成的基因。我们将利用人类胃原体促进肢体芽的发育，并将精确定位的信号因子与在肢体发育中发挥作用的局部提供的ncc衍生物结合起来。该项目将展示人类类胃原体如何提供一种可行的替代动物模型，可以优化研究肠道和肢体发育，并为未来的项目奠定基础。

The mammalian foetus is formed by a gradual process of tissue specification after the embryo has implanted in the uterus. Organs develop in an arrangement characteristic of each species through progressive differentiation from 3 distinct layers: ectoderm, endoderm and mesoderm, that separate physically and functionally during a process known as 'gastrulation'. Since this occurs within the mother, most studies have required removal of embryos, largely using the mouse as a model system. Gastrulating embryos can be cultured for several days outside the body, but this requires large numbers of mice (around 4 females for 20 embryos) and serum purified from blood of 4-5 rats. Embryonic stem cells (ESCs) are derived from preimplantation embryos and can be expanded indefinitely in culture whilst retaining capacity to differentiate into any tissue of the body. This is illustrated by injecting ESCs, usually after gene deletion or over-expression into preimplantation stage embryos and transferring the newly formed 'chimaeras' into foster mothers. Genetically-modified offspring are then selected for further breeding. To bypass the requirement for animals to address certain scientific questions, ESCs can be grown in 3D suspension culture using a simple protocol that allows them to undergo a process similar to gastrulation. These 3D 'gastruloids' can be guided to differentiate into recognisable tissues and rudimentary organs bearing strong physical and molecular resemblance to those of the embryo. Gastruloids are valuable, tractable tools, allowing researchers to reduce the number of embryos required for developmental studies. However, as mouse has limitations for human development, we and others have generated gastruloids from human ESCs. As human embryos cannot be used to study gastrulation due to ethical considerations, human gastruloids open a window to study human development that is otherwise inaccessible. Remarkably, the process that initiates symmetry breaking in gastruloids is spontaneous, but this makes it difficult to disentangle signals required for normal gastrulation and organ formation. Thus, we are faced with a major challenge to determine exactly how processes are initiated in a normal human embryo, which impedes our ability to uncover causes of embryonic abnormalities. Our project will tackle this problem by devising a system to control position, catchment area and duration of signalling cues to enhance understanding and enable controlled development of specific body parts in human gastruloids. We intend to focus on one internal organ, the gut, and an external structure, the limb bud. Rudimentary gut tubes can be induced in gastruloids showing some distinct regions approximating the foetal digestive tract. We will investigate the role of specialised 'neural crest cells' (NCCs), which are recruited to various developing tissues. In normal development NCCs emerge from the developing neural tube and migrate to produce various cell types, including those that form nerve cells in the developing gut. We will also inject NCCs to determine how these special neurons are recruited to the gut and whether they can contribute to its structural and functional development. This is particularly relevant for understanding defects such as Hirschsprung's disease. In addition, we will encapsulate developing gastruloids in customised gels to enable local application of substances known to induce formation and patterning of limb buds. Gastruloids generated from mouse ESCs have distinct regions in the flanks that express genes involved in limb formation. We will enhance limb bud development using human gastruloids and combine precisely positioned signalling factors with localised provision of NCC-derivatives that play a role in limb development. This project will demonstrate how human gastruloids provide a viable alternative to animal models that can be optimised to study gut and limb development and set the scene for future projects.