Markers of cellular reprogramming potential in fibroblast cell lines

成纤维细胞系中细胞重编程潜力的标志物

• 批准号: RGPIN-2021-02675
• 负责人: Mastromonaco, Gabriela
• 金额: $ 2.71万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746258
• 关键词: Markers; cellular; reprogramming; potential; fibroblast

The biodiversity crisis is one of the most pressing environmental problems of our time, yet after >30 years of research, there has been limited progress on the implementation of assisted reproductive technologies for endangered mammals. Species-specific requirements for successful embryo development have impacted the advancement of in vitro-based embryo technologies resulting in a lack of effective protocols for key conservation species. The ability to preserve and access both female and male genetic material is of fundamental importance when developing strategies for the long-term sustainability of threatened wildlife species. As such, somatic cells are highly valued for their ease of collection, culture and cryopreservation from all animals, as well as their reprogramming capabilities and potential use in somatic cell-based technologies (somatic cell nuclear transfer, SCNT; induction of pluripotent stem cells, iPSCs). However, the production of living offspring from SCNT- and iPSC-derived embryos remains an inefficient and variable process. Our studies have resulted in the first-ever production of bison blastocysts by interspecies SCNT (iSCNT) in American (plains, wood) and European bison. Nevertheless, bison iSCNT embryos differed significantly from cattle SCNT controls in developmental potential, including cell number, apoptosis, and gene expression. Most importantly, multiple cell lines were assessed for SCNT with some cell lines being unable to produce blastocysts after repeated attempts. Recently, there has been a growing body of evidence indicating that the individual cell line is the greatest determinant of outcome following reprogramming for reasons beyond traditional cell health metrics. Metabolism plays a key role in cellular processes, and thus, in the regulation of the epigenome, a principal driver of reprogramming success. Our research lies in understanding the cellular and molecular factors influencing the pluripotency potential of somatic cells for the production of embryos by SCNT and iPSC techniques. The proposed research will focus on the characterization and modification of cellular metabolism in an effort to alter the epigenetic profile of somatic donor cells, and positively impact reprogramming and downstream embryo production events. The first phase of the study will characterize the metabolic profiles of bovine donor cells and identify metaboloepigenetic markers related to reprogramming outcomes. In the second phase, modification of the metaboloepigenetic status of the cells will be carried out in an effort to improve reprogramming capabilities. This study will increase our understanding of cellular determinants of reprogramming success, specifically the role of metabolic and epigenetic status of the donor cell, and provide us with potential methods for improving somatic cell-based technologies. This is a necessary step towards the application of SCNT and iPSC strategies in the recovery of threatened species.

生物多样性危机是我们这个时代最紧迫的环境问题之一，但经过30多年的研究，在濒危哺乳动物辅助生殖技术的实施方面进展有限。成功胚胎发育的物种特异性要求影响了体外胚胎技术的发展，导致缺乏关键保护物种的有效协议。在为受威胁野生物种的长期可持续性制定战略时，保护和获取雌性和雄性遗传材料的能力至关重要。因此，体细胞因其易于从所有动物收集、培养和冷冻保存，以及其重编程能力和在基于体细胞的技术（体细胞核转移，SCNT;诱导多能干细胞，iPSC）中的潜在用途而受到高度重视。然而，从SCNT和iPSC衍生的胚胎生产活后代仍然是一个低效和可变的过程。我们的研究首次在美洲（平原，木材）和欧洲野牛中通过种间SCNT（iSCNT）生产野牛囊胚。然而，野牛iSCNT胚胎与牛SCNT对照组在发育潜力方面存在显著差异，包括细胞数量、凋亡和基因表达。最重要的是，对多个细胞系进行了SCNT评估，其中一些细胞系在重复尝试后无法产生囊胚。最近，越来越多的证据表明，单个细胞系是重编程后结果的最大决定因素，其原因超出了传统的细胞健康指标。代谢在细胞过程中起着关键作用，因此，在表观基因组的调节中，表观基因组是重编程成功的主要驱动力。我们的研究在于了解影响体细胞多能性潜力的细胞和分子因素，以通过SCNT和iPSC技术生产胚胎。拟议的研究将侧重于细胞代谢的表征和修饰，以改变体细胞供体细胞的表观遗传特征，并对重编程和下游胚胎生产事件产生积极影响。研究的第一阶段将描述牛供体细胞的代谢特征，并确定与重编程结果相关的代谢表观遗传标记。在第二阶段，将对细胞的代谢表观遗传状态进行修饰，以提高重编程能力。这项研究将增加我们对重编程成功的细胞决定因素的理解，特别是供体细胞的代谢和表观遗传状态的作用，并为我们提供改进基于体细胞的技术的潜在方法。这是将SCNT和iPSC战略应用于受威胁物种恢复的必要步骤。