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

优化人类干细胞模型以破译器官发生过程中的信号和反应

• 批准号: NC/X001938/1
• 负责人: Jennifer Nichols
• 金额: $ 25.37万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FX001938%2F1
• 关键词: Optimising; human; stem; cell; models

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.

哺乳动物胎儿是胚胎植入子宫后通过组织规范的逐渐过程形成的。器官按照每个物种特有的排列方式发育，通过从 3 个不同的层逐步分化而来：外胚层、内胚层和中胚层，这些层在称为“原肠胚形成”的过程中在物理上和功能上分离。由于这种情况发生在母亲体内，因此大多数研究都需要去除胚胎，主要使用小鼠作为模型系统。原肠胚可以在体外培养数天，但这需要大量小鼠（大约 4 只雌性小鼠培养 20 个胚胎）以及从 4-5 只大鼠血液中纯化的血清。胚胎干细胞（ESC）源自植入前胚胎，可以在培养物中无限增殖，同时保留分化成身体任何组织的能力。这可以通过注射ESC来说明，通常是在基因删除或过度表达到植入前阶段胚胎中之后，并将新形成的“嵌合体”转移到养母体内。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，可以使用简单的方案在 3D 悬浮培养物中培养 ESC，使其经历类似于原肠胚形成的过程。这些 3D“类原肠胚”可以被引导分化为可识别的组织和基本器官，与胚胎的物理和分子具有很强的相似性。原肠胚是有价值且易于处理的工具，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有限制，我们和其他人已经从人类胚胎干细胞中产生了类原肠胚。由于出于伦理考虑，人类胚胎不能用于研究原肠胚形成，而人类类原肠胚为研究人类发育打开了一扇否则无法进入的窗口。值得注意的是，类原肠胚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，我们面临着一项重大挑战，即确定正常人类胚胎中的过程是如何启动的，这阻碍了我们揭示胚胎异常原因的能力。我们的项目将通过设计一个系统来控制信号线索的位置、集水区域和持续时间来解决这个问题，以增强理解并实现人类原肠胚中特定身体部位的受控发育。我们打算重点关注一个内部器官（肠道）和一种外部结构（肢芽）。可以在类原肠胚中诱导出初级肠管，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。在正常发育过程中，NCC 从发育中的神经管中出现并迁移产生各种细胞类型，包括那些在发育中的肠道中形成神经细胞的细胞类型。我们还将注射 NCC，以确定这些特殊神经元如何被招募到肠道，以及它们是否有助于其结构和功能发育。这对于了解先天性巨结肠等缺陷尤其重要。此外，我们将把发育中的类原肠胚封装在定制的凝胶中，以便能够局部应用已知诱导肢芽形成和图案化的物质。由小鼠胚胎干细胞产生的类原肠胚在侧翼有不同的区域，表达参与肢体形成的基因。我们将利用人类原肠胚促进肢芽发育，并将精确定位的信号因子与在肢体发育中发挥作用的 NCC 衍生物的局部供应相结合。该项目将展示人类原肠胚如何为动物模型提供可行的替代方案，可以优化该动物模型来研究肠道和肢体发育，并为未来的项目奠定基础。