A new hypothesis: role of p53 inhibitory factors in cellular reprogramming

新假说：p53抑制因子在细胞重编程中的作用

• 批准号: 9268543
• 负责人: Kitai Kim
• 金额: $ 46.62万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9268543
• 关键词: ATM promoter; Age; Aging; Alpha Cell; Apoptotic; Binding; Cell Line; Cell physiology; Cells; Centromere; Chromosomal Instability; Chronic; Clone Cells; DNA Damage; Degenerative Disorder; Development; Ectopic Expression; Embryo; Epigenetic Process; Ethics; Fibroblasts; Genes; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Histocompatibility; Human; Immune system; In Vitro; Individual; Lead; Legal patent; MAPK9 gene; Malignant Neoplasms; Molecular Abnormality; Mutation; Oncogenic; Oocytes; PLK1 gene; Pathway interactions; Patients; Phenotype; Phosphorylation; Pluripotent Stem Cells; Regenerative Medicine; Replacement Therapy; Reporting; Research; Residual state; Role; SIRT1 gene; Somatic Cell; Source; Stem cells; Systems Biology; TP53 gene; Testing; Tissue Donors; Tissue Transplantation; Tissues; age effect; aged; aging population; cell type; clinical application; comparative; embryonic stem cell; genome integrity; genome-wide; genotoxicity; histone modification; improved; in vivo; induced pluripotent stem cell; innovation; insight; mouse model; normal aging; older patient; pluripotency; pressure; public health relevance; response; tissue regeneration; tool

DESCRIPTION (provided by applicant): Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)-generated by ectopic expression of four embryonic reprogramming genes in somatic cells-provide a source of patient-specific stem cells without the ethical limitations associated with embryonic stem cells (ESC). However, we and others have reported that iPSC are incompletely reprogrammed, and we found that the degree of reprogramming decreases with the age of the donor tissue. Our overall research goal is to improve the reprogramming of iPSC in order to produce high-quality patient-specific histocompatible tissues for transplantation. Because older patients are more likely to benefit from the clinical applications of iPSC, there is a significant need to comprehensively evaluate iPSC derived from older donor cells (A-iPSC) and identify additional pluripotent reprogramming factors that can reverse the negative impact of aging on A-iPSC reprogramming. Multiple reports have proposed that p53 is a negative regulator of somatic cell reprogramming, and that elimination of p53 increases iPSC reprogramming efficiency. However, basal p53 levels are elevated in ESC and iPSC compared with somatic cells, leading us to propose an innovative hypothesis-that iPSC reprogramming is incomplete due to insufficient inhibition of elevated p53 by p53 inhibitory factors. We further hypothesized that A-iPSC may be unable to activate these putative p53 inhibitory factors to overcome the elevated p53 during iPSC reprogramming. As a result, p53 cannot be completely restrained, and the residual p53 activity stimulates a constitutive DNA damage response that mimics what normally occurs only in response to a genotoxic insult. A chronic DNA damage response (via p53-dependent and -independent pathways) has also been observed during normal aging of somatic cells, and can lead to the complete loss of the DNA damage response during cancer development. We posited that epigenetic destruction of the entire DNA damage response would improve A-iPSC reprogramming and quality. In preliminary studies, we examined the function of two putative p53 inhibitory factors and used these factors to identify pluripotent regulatory factors that can correct the aging-related epigenetic landscape and improve the quality of A-iPSC. In this proposal, we will examine the function of these pluripotent regulatory factors by conducting a comprehensive comparative analysis of these iPSC types. In Aim 1, we will determine the mechanism by which the pluripotent regulatory factors reverse the aberrant DNA damage response and negative effects of aging on the A-iPSC phenotype. In Aim 2, we will determine the functional effects of the pluripotent regulatory factors by comparing the pluripotency and differentiation potential of each cell line in several in vitro and in vivo tests. Ultimately, our findings will provide critical insight into how we can improve the quality of patient-specific stem cells derived from aged patients for use in tissue regeneration and transplantation.

描述（由申请人提供）：在各种多能干细胞中，诱导多能干细胞(iPSC)-通过体细胞中四种胚胎重编程基因的异位表达产生-提供了一种没有胚胎干细胞（ESC）相关伦理限制的患者特异性干细胞来源。然而，我们和其他人已经报道了iPSC不完全重编程，我们发现随着供体组织的年龄，重编程的程度降低。我们的总体研究目标是改善iPSC的重编程，以生产用于移植的高质量患者特异性组织相容性组织。由于老年患者更有可能从iPSC的临床应用中获益，因此有必要全面评估来自老年供体细胞的iPSC (a -iPSC)，并确定能够逆转衰老对a -iPSC重编程的负面影响的额外多能重编程因子。多篇报道提出p53是体细胞重编程的负调控因子，消除p53可提高iPSC重编程效率。然而，与体细胞相比，ESC和iPSC的基础p53水平升高，这使我们提出了一个创新的假设——iPSC重编程是不完整的，这是由于p53抑制因子对升高的p53的抑制不足。我们进一步假设A-iPSC可能无法激活这些假定的p53抑制因子来克服iPSC重编程过程中p53的升高。因此，p53不能被完全抑制，残余的p53活性刺激了组成性DNA损伤反应，模仿了通常只发生在基因毒性损伤反应中的反应。在体细胞的正常衰老过程中也观察到慢性DNA损伤反应（通过p53依赖和不依赖的途径），并可能导致癌症发展过程中DNA损伤反应的完全丧失。我们假设整个DNA损伤反应的表观遗传破坏将改善A-iPSC重编程和质量。在初步研究中，我们检测了两种推测的p53抑制因子的功能，并利用这些因子确定了能够纠正衰老相关表观遗传景观并提高A-iPSC质量的多能调控因子。在本提案中，我们将通过对这些iPSC类型进行全面的比较分析来研究这些多能性调控因子的功能。在Aim 1中，我们将确定多能调控因子逆转异常DNA损伤反应和衰老对A-iPSC表型的负面影响的机制。在目标2中，我们将通过比较几种细胞系的多能性和分化潜力来确定多能性调节因子的功能作用