Cell fate regulation during gastrulation in humans and pigs

著名胚胎学家刘易斯·沃尔珀特（Lewis Wolpert）曾说过：“不是出生、结婚或死亡，而是原肠胚形成，这才是你生命中真正最重要的时刻。”原肠胚期是胚胎发育的时期（大约在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决定。这是一个关键时期，因为如果这种发育受到干扰，就可能导致不可逆转的异常，或对以后的健康产生长期有害后果。人类的原肠胚形成过程是如何发生的，人们知之甚少，因为出于伦理原因，人类胚胎在这一时期不能直接研究。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有很大的不同。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。小鼠胚胎发育为一个锥形结构，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，这与胎盘组织建立了不同的关系。我们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成扁平的圆盘。我们的研究表明，调节精子和卵子前体细胞形成的基因在猪和老鼠之间是不同的。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细胞的分子特性是不同的。此外，这表明猪胚胎可以作为非啮齿动物哺乳动物的模型，并用于帮助了解人类的发育。其他细胞和器官，如肌肉、肝脏、肠道和胰腺的形成过程中是否也存在差异，还需要进一步研究。这种新的认识最终将有助于为人类干细胞向具有治疗潜力的特定细胞类型的驱动提供方法。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到不同的细胞身份的地图。然后，这些数据将与小鼠和人类干细胞分化研究的现有数据进行比较，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这一关键发育时期所起的作用。研究的最终目的是确定人细胞在猪/人嵌合胚胎中的发育途径。由两个物种产生的胚胎，被称为嵌合体，是评估细胞在体内发育潜力的极好工具。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/人嵌合体如何发育将是在大型动物体内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障碍和技术限制将提高这些新移植技术的安全性和有效性。

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.