Cell fate regulation during gastrulation in humans and pigs

人和猪原肠胚形成过程中的细胞命运调控

• 批准号: BB/S000178/1
• 负责人: Ramiro Alberio
• 金额: $ 71.65万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS000178%2F1
• 关键词: Cell; fate; regulation; during; gastrulation

To the renowned embryologist Lewis Wolpert is attributed the quote: "It is not birth, marriage or death, but gastrulation which is truly the most important time in your life". Gastrulation is the period of embryo development (around the second week of pregnancy in humans) when the major cell precursors that will form the organs in our body are determined. It is a critical time because if such development is disrupted, it can lead to irreversible abnormalities or have long term detrimental consequences for health in later life. How the gastrulation process occurs in humans is very poorly understood because human embryos cannot be directly studied during this period for ethical reasons. Mouse embryos have served as good models for understanding general principles of mammalian development, but we also know that mice and humans are very different in their development. Indeed, some of the key developmental differences correlate with distinct embryo morphology in mice and humans. Mouse embryos develop as a conical structure in which interactions with tissues contributing to the placenta are very different from other large mammals. Humans develop flat embryos, which establish different relationships with placental tissues. We have studied the significance of these differences in detail in pig embryos, which also develop as flat discs like humans. Our research showed that the genes that regulate the formation of sperm and egg precursor cells are different between pigs and mice. Importantly, we also showed that the mechanism used by pig is also used in human and other primate embryos. This landmark study showed for the first time that the molecular identity of a cell is different between mice and other larger mammals. Furthermore, it indicated that pig embryos can be used as a model for non-rodents mammals and be used to help understand human development. Further research is needed to find out whether differences also occur during the formation of other cells and organs, such as the muscle, liver, gut and pancreas. Such new understanding will ultimately help inform methods for driving human stem cells towards specific cell types with therapeutic potential. The first objective of the research is to determine the genetic program of different cell precursors during gastrulation in pig embryos. Using single cell molecular analysis we will build a map of how cells become restricted towards different cellular identities. This data will be then compared with available data from mouse and human stem cell differentiation studies that will allow us to establish similarities in developmental mechanisms between species. Next, we will validate these findings using new genome editing technologies to disrupt protein function in embryos and determine the morphology of mutant embryos. We will determine cell identity in mutant embryos and establish the roles played by these key proteins during this critical period of development. The final objective of the research is to determine the developmental pathway of human cells in pig/human chimeric embryos. Embryos generated from two species, known as chimeras, are excellent tools for assessing cell developmental potential in vivo. Here we will test the capacity of newly establish hESC and other progenitors combined with chemical methods to prevent cell death to enhance chimera integration. We will use labelling technologies to track cell development and determine cell fate in pig embryos. Understanding how pig/human chimeras develop will be an important step towards generating human organs in large animals for xenotransplantation. A better understanding of the biological barriers and technical limitations of these approaches will improve the safety and efficacy of these new transplantation technologies.

著名的胚胎学家刘易斯·沃伯特（Lewis Wolpert）曾说过：“生命中最重要的时刻不是出生、结婚或死亡，而是原肠胚形成。”原肠胚形成是胚胎发育的时期（人类怀孕的第二周左右），在此期间，将形成我们身体器官的主要细胞前体被确定。这是一个关键的时刻，因为如果这种发展被破坏，它可能导致不可逆转的异常或对晚年的健康产生长期的有害后果。人类的原肠胚形成过程是如何发生的，人们对此知之甚少，因为出于伦理原因，人类胚胎在这一时期无法直接研究。小鼠胚胎是理解哺乳动物发育一般原理的良好模型，但我们也知道小鼠和人类在发育过程中非常不同。事实上，一些关键的发育差异与小鼠和人类不同的胚胎形态有关。小鼠胚胎发育为圆锥形结构，其中与胎盘组织的相互作用与其他大型哺乳动物非常不同。人类发育扁平胚胎，与胎盘组织建立不同的关系。我们已经在猪胚胎中详细研究了这些差异的意义，猪胚胎也像人类一样发育为扁平圆盘。我们的研究表明，调控精子和卵子前体细胞形成的基因在猪和小鼠之间是不同的。重要的是，我们还表明，猪使用的机制也适用于人类和其他灵长类动物的胚胎。这项具有里程碑意义的研究首次表明，小鼠和其他大型哺乳动物之间的细胞分子身份不同。此外，这表明猪胚胎可以用作非啮齿类哺乳动物的模型，并用于帮助了解人类发育。还需要进一步的研究来确定在其他细胞和器官的形成过程中是否也存在差异，如肌肉，肝脏，肠道和胰腺。这种新的理解将最终有助于为驱动人类干细胞向具有治疗潜力的特定细胞类型发展的方法提供信息。本研究的第一个目的是确定猪胚胎原肠胚形成过程中不同细胞前体的遗传程序。使用单细胞分子分析，我们将建立一个细胞如何被限制在不同细胞身份的地图。然后将这些数据与来自小鼠和人类干细胞分化研究的可用数据进行比较，这将使我们能够建立物种之间发育机制的相似性。接下来，我们将使用新的基因组编辑技术来验证这些发现，以破坏胚胎中的蛋白质功能并确定突变胚胎的形态。我们将确定突变胚胎中的细胞身份，并确定这些关键蛋白在发育的关键时期所发挥的作用。研究的最终目的是确定猪/人嵌合胚胎中人类细胞的发育途径。由两个物种产生的胚胎，称为嵌合体，是评估体内细胞发育潜力的绝佳工具。在这里，我们将测试新建立的hESC和其他祖细胞结合化学方法防止细胞死亡以增强嵌合体整合的能力。我们将使用标记技术来跟踪细胞发育并确定猪胚胎中的细胞命运。了解猪/人嵌合体是如何发展的，将是在大型动物中产生用于异种移植的人体器官的重要一步。更好地了解这些方法的生物屏障和技术局限性将提高这些新移植技术的安全性和有效性。

项目成果

Untangling early embryo development using single cell genomics.

• DOI: 10.1016/j.theriogenology.2020.01.062
• 发表时间: 2020-01
• 期刊: Theriogenology
• 影响因子: 2.8
• 作者: R. Alberio

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Nuclear transfer and the development of genetically modified/gene edited livestock.

• DOI: 10.1530/rep-21-0078
• 发表时间: 2021-06-11
• 期刊: Reproduction (Cambridge, England)
• 作者: Alberio R;Wolf E
• 通讯作者: Wolf E