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

哺乳动物的胎儿是在胚胎植入子宫后，通过逐渐的组织特化过程形成的。器官通过从3个不同层（外胚层、内胚层和中胚层）的逐步分化以每个物种特有的排列方式发育，所述外胚层、内胚层和中胚层在被称为“原肠胚形成”的过程中物理上和功能上分离。由于这发生在母亲体内，大多数研究都需要取出胚胎，主要使用小鼠作为模型系统。可以在体外培养几天，但这需要大量的小鼠（大约4只雌性小鼠，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（ESC）来源于植入前胚胎，可以在培养中无限扩增，同时保留分化成身体任何组织的能力。这是通过注射胚胎干细胞，通常在基因缺失或过度表达到植入前阶段的胚胎和转移新形成的“嵌合体”到寄养母亲。然后选择转基因后代进行进一步育种。为了绕过动物解决某些科学问题的要求，ESC可以使用简单的方案在3D悬浮培养中生长，使它们经历类似于原肠胚形成的过程。这些3D“类胃体”可以被引导分化成可识别的组织和基本器官，这些组织和器官与胚胎的组织和器官具有很强的物理和分子相似性。Gastruloids是有价值的，易于处理的工具，使研究人员能够减少发育研究所需的胚胎数量。然而，由于小鼠对人类发育的限制，我们和其他人已经从人类ESC中产生了类胃。由于人类胚胎不能用于研究原肠胚由于伦理方面的考虑，人类gastruloid打开了一扇窗户，以研究人类的发展，否则无法进入。值得注意的是，在类胃体中启动对称性破缺的过程是自发的，但这使得很难解开正常原肠胚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。我们的项目将通过设计一个系统来解决这个问题，以控制位置，集水区和信号提示的持续时间，以提高对人类类胃体的理解，并使特定身体部位的发育受到控制。我们打算集中在一个内部器官，肠道，和一个外部结构，肢芽。在胃状体中可以诱导出未发育的肠管，显示出接近胎儿消化道的一些不同区域。我们将研究专门的“神经嵴细胞”（NCC）的作用，这些细胞被招募到各种发育组织中。在正常发育中，NCC从发育中的神经管中出现，并迁移以产生各种细胞类型，包括在发育中的肠道中形成神经细胞的细胞。我们还将注射NCC，以确定这些特殊的神经元是如何被招募到肠道的，以及它们是否有助于肠道的结构和功能发育。这对于理解先天性巨结肠等缺陷尤其重要。此外，我们将在定制的凝胶中封装发育中的类胃体，以使已知诱导肢芽形成和图案化的物质能够局部应用。由小鼠胚胎干细胞产生的类胃窦在侧翼有不同的区域，表达参与肢体形成的基因。我们将使用人类类胃体来促进肢芽发育，并将联合收割机精确定位的信号传导因子与在肢体发育中发挥作用的NCC衍生物的局部提供相结合。该项目将展示人类类胃体是如何为动物模型提供一种可行的替代方案，可以优化动物模型以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.