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.

哺乳动物胎儿是由胚胎植入子宫后的组织规范过程形成的。器官通过与3种不同层的渐进分化的渐进分化以每个物种的布置特征发展：外胚层，内胚层和中胚层，它们在称为“胃肠道”的过程中在物理和功能上分离。由于这发生在母亲内部，因此大多数研究都需要去除胚胎，这在很大程度上将小鼠用作模型系统。可以在体内培养几天的胃胚胎，但这需要大量小鼠（约4个女性，用于20个胚胎），并从4-5只大鼠的血液中纯化血清。胚胎干细胞（ESC）源自植入前胚胎，可以在培养物中无限期地扩展，同时保留能力以区分体内任何组织。通常在基因缺失或过表达过表达后将ESC注射到植入前胚胎并将新形成的“ Chimaeras”转移到寄养母亲之后来说明这一点。然后选择遗传改性的后代进行进一步的繁殖。为了绕过对动物解决某些科学问题的要求，可以使用简单的方案在3D悬架文化中种植ESC，该方案使他们能够经历类似于胃口的过程。这些3D“胃底子”可以被引导分化成具有强大的物理和分子相似性的可识别组织和基本的器官。胃底子是有价值的，可拖动的工具，使研究人员能够减少发育研究所需的胚胎数量。但是，由于小鼠对人类的发展有局限性，我们和其他人从人类ESC产生了胃突。由于人类胚胎不能因道德上的考虑而用于研究胃肠道，因此人类胃肠道为研究人类发育的窗口打开了否则无法访问的窗口。值得注意的是，引发对称性破裂的过程是自发的，但这使得很难脱离正常胃肠道和器官形成所需的信号。因此，我们面临着一个重大挑战，即确定在正常的人类胚胎中如何启动过程，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控制位置，集水区和信号提示的持续时间来解决这个问题，以增强理解并能够控制人类胃lu脚的特定身体部位的发展。我们打算专注于一个内部器官，肠道和外部结构，即肢体芽。可以在胃类似的胃诱导的一些不同区域中诱导肠道消化道。我们将研究专业的“神经rest细胞”（NCC）的作用，这些作用被募集到各种发育中的组织中。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，以确定这些特殊的神经元如何招募到肠道，以及它们是否可以为其结构和功能发展做出贡献。这与理解诸如Hirschsprung氏病这样的缺陷特别重要。此外，我们将封装在自定义凝胶中的开发胃底子，以实现已知诱导肢体芽形成和模式的局部应用。由小鼠ESC产生的胃类动物在表达与肢体形成的基因的侧面中具有不同的区域。我们将使用人类胃lu虫增强肢体发育，并将精确定位的信号传导因子与NCC衍生物的局部提供，这些信号传导因素在肢体发育中发挥作用。该项目将展示人类胃lu虫如何为动物模型提供可行的替代品，可以优化以研究肠道和肢体开发并为未来的项目奠定场景。