Epigenetic Barriers to Cell Fate Reprogramming

在胚胎发育过程中，细胞越来越倾向于某种特定的细胞命运。它们很少，如果有的话，会变成另一种类型。例如，皮肤细胞不会自然转变为脑细胞，也不会产生脑细胞。然而，细胞的这种稳定的承诺可以通过某些实验程序来恢复，例如当特定细胞的细胞核被移植到去核的卵子上时。在这种重新编程过程中，分化细胞的基因表达模式可以改变为胚胎细胞的基因表达模式。这个过程很有趣，因为识别重新编程是如何发生的，可以帮助我们理解细胞是如何保持其身份的。这一点很重要，因为许多病理情况都是由于细胞特性的丧失引起的。其次，通过对一种特殊细胞(例如皮肤细胞)进行重新编程而获得的胚胎干细胞，然后可以生产出另一种健康的特殊细胞，例如脑细胞。这在细胞替代疗法中有潜在的应用，这些细胞可以补偿不可逆转的受损或有缺陷的细胞的损失。从同一个体的另一种细胞类型中分离出替代的心脏、神经元或胰腺细胞是可能的，从而避免了免疫抑制治疗的需要。尽管这种方法具有巨大的潜力，但其有效性受到其低效率的限制，因为只有一小部分细胞可以重新编程。像皮肤这样的成人身体细胞对核重新编程没有反应的原因可能与一种细胞转变为另一种细胞的固有抵抗力有关。事实上，通过将特定细胞的细胞核移植到卵子中产生的细胞通常会继续表现出它们所来自的特定细胞的特征，比如基因的表达。例如，重新编程的皮肤细胞继续表达皮肤基因，但无法完全激活新细胞类型的基因。这表明在特化过程中，细胞获得了对其细胞身份的记忆，这防止了在正常发育或重新编程期间细胞类型的不必要变化。我们项目的目的是阐明细胞记忆现象。我们使用非洲爪哇卵的特殊细胞核移植作为一个模型系统来理解这一过程。与人类卵子形成鲜明对比的是，非洲爪哇的卵子很容易大量获得，而且很容易操作。由于细胞记忆的机制在脊椎动物之间很可能是保守的，而且考虑到使用人类材料进行这类研究的伦理问题，非洲爪哇是进行这项研究的理想模型系统。该项目包括三个阶段。首先，我们将调查通过核移植产生的胚胎中有多少表现出对它们来源的细胞类型的强烈记忆。我们将确定核移植后显示细胞记忆的基因。其次，我们将研究与细胞记忆基因相关的分子，因为这些分子可能会在正常发育和核重新编程期间抑制细胞命运的改变。最后，我们建议在将特定细胞的核移植到卵子之前改变其记忆激发因素，以提高新细胞类型的生成效率。通过这种方式，我们将了解核的哪些修饰可以提高可用于细胞替代的高质量胚胎细胞的生成。此外，我们将更好地了解在正常发育和疾病期间细胞记忆和细胞分化稳定性的重要机制。

During embryonic development, cells become increasingly committed to a certain cell fate. They rarely, if ever, change to another type. For example, as skin cell do not naturally change to, or give rise to a brain cell. However, this stable commitment of a cell can be reverted by certain experimental procedures, as for example when the nucleus of a specialised cell is transplanted to an enucleated egg. During this reprogramming, the gene expression pattern of the differentiated cell can be changed to that of an embryonic cell. This process is of interest because identifying how reprogramming takes place can help us to understand how cells maintain their identity. This is important because many pathological conditions arise from loss of cell identity. Second, the embryonic stem cells that can be obtained by reprogramming a specialised cell of one kind, e.g. a skin cell, can then be made to produce healthy specialized cells of an other kind, e.g. brain cells. This has potential application in cell-replacement therapy, where these cells can compensate the loss of irreversibly damaged or defective cells. It might be possible to derive replacement heart, neuronal or pancreatic cells from another cell type of the same individual, thereby avoiding the need for immunosuppression treatments.Despite its enormous potential, the usefulness of this approach is limited by its low efficiency, as only a small number of cells can be reprogrammed. The reason why adult body cells like skin do not respond to nuclear reprogramming is likely related to an inherent resistance of one kind of cell to change to another kind. Indeed, cells produced by transplanting the nucleus of a specialised cell to an egg often continue to show characteristics, such as expression of genes, of the specialised cell they were derived from. For example, a reprogrammed skin cell continues to express skin genes and fails to fully activate genes of the new cell type. This suggests that during specialisation, cells have acquired memory of their cellular identity, which prevents unwanted changes in cell type during normal development or during reprogramming.The aim of our project is to elucidate the phenomenon of cellular memory. We use nuclear transplantation of specialised cell nuclei to Xenopus eggs as a model system to understand this process. In stark contrast to human eggs, Xenopus eggs are readily available in large quantities and easy to manipulate. Since the mechanisms underlying cellular memory are most likely conserved between vertebrates, and given the ethical concerns of using human material for this kind of research, Xenopus is the ideal model system to perform this study. The project consists of three stages. First we will investigate how many embryos produced by nuclear transfer show a strong memory of the cell type they were derived from. We will identify the genes that show cellular memory after nuclear transplantation. Second, we will investigate the molecules associated with cellular memory genes, since these molecules likely inhibit a change in cell fate during normal development and during nuclear reprogramming. Lastly, we propose to change the memory-conferring factors in specialised cells before transplanting their nuclei to eggs in order to increase the efficiency of new cell type generation. In this way, we will understand which modifications of nuclei can improve the generation of high quality embryonic cells that can be used for cell replacement. Furthermore, we will gain a better insight into the mechanisms important for cellular memory and the stability of cell differentiation during normal development and disease.