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

项目概要/摘要 在各种多能干细胞中，诱导性多能干细胞（iPSC）-通过异位诱导产生。 四种胚胎重编程基因在体细胞中的表达-提供了患者特异性干细胞的来源 没有与胚胎干细胞（ESC）相关的伦理限制的细胞。然而，我们和其他人 报道iPSC是不完全重编程的，我们发现重编程的程度 随着供体组织的老化而减少。我们的总体研究目标是改善 iPSC以产生用于移植的高质量的患者特异性组织相容性组织。因为 老年患者更有可能从iPSC的临床应用中受益，因此， 全面评估来源于较老供体细胞的iPSC（A-iPSC），并鉴定额外的多能干细胞， 这些重编程因子可以逆转衰老对A-iPSC重编程的负面影响。多 有报道提出，p53是体细胞重编程的负调节因子， p53增加iPSC重编程效率。然而，基础p53水平在ESC和iPSC中升高， 与体细胞相比，导致我们提出一个创新的假设-iPSC重编程是 由于p53抑制因子对升高的p53的抑制不足而不完全。我们进一步假设 A-iPSC可能无法激活这些假定的p53抑制因子来克服升高的p53 在iPSC重编程过程中结果，p53不能被完全抑制，并且残留的p53活性 刺激组成性DNA损伤反应，该反应模拟通常仅在响应于DNA损伤时发生的反应。 遗传毒性的侮辱慢性DNA损伤反应（通过p53依赖性和非依赖性途径） 在体细胞的正常老化过程中观察到，并可导致DNA损伤的完全丧失。 癌症发展过程中的反应。我们假设整个DNA损伤的表观遗传破坏 反应将改善A-iPSC重编程和质量。在初步研究中，我们检查了 两个假定的p53抑制因子，并使用这些因子来鉴定多能调节因子， 纠正衰老相关的表观遗传景观并提高A-iPSC的质量。在本提案中，我们将 通过进行全面的比较，检查这些多能调节因子的功能， 分析这些iPSC类型。在目标1中，我们将确定多能调节性细胞的机制。 这些因素逆转了异常DNA损伤反应和衰老对A-iPSC表型的负面影响。在 目的2，我们将确定多能调节因子的功能作用，通过比较， 在几个体外和体内测试中，每个细胞系的多能性和分化潜力。最终，我们 这些发现将为我们如何提高患者特异性干细胞的质量提供重要的见解。 用于组织再生和移植。 公共卫生相关性 使用个体专利衍生的iPSC的再生医学具有产生 组织相容性可移植组织;这种方法将特别有利于老年人， 可能患有退行性疾病最终，我们的发现和拟议的研究将提供关键的见解， 我们如何提高从老年患者中提取的患者特异性干细胞的质量， 再生和移植。

Project Summary/Abstract 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. Public Health Relevance Regenerative medicine using individual patent-derived iPSC has enomous potential to generate histocompatible transplantable tissue; this approach will particuarly benefit the aged population who is more likely to have degenerative disease. Ultimately, our findings and proposed research 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.