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

描述(申请人提供)：在各种多能干细胞中，诱导多能干细胞(IPSC)--通过在体细胞中异位表达四个胚胎重编程基因而产生--提供了一种患者特有的干细胞来源，没有与胚胎干细胞(ESC)相关的伦理限制。然而，我们和其他人已经报道了IPSC不完全重新编程，我们发现重新编程的程度随着捐赠者组织的年龄而降低。我们的总体研究目标是改进IPSC的重编程，以产生高质量的患者特定的组织相容组织用于移植。由于老年患者更有可能从IPSC的临床应用中受益，因此有必要对老年供体细胞来源的IPSC(A-IPSC)进行全面评估，并确定其他多能重编程因子，以逆转衰老对A-IPSC重编程的负面影响。已有多篇报道提出，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，这导致我们提出了一个创新的假说-IPSC的重新编程是不完整的，这是由于P53抑制因子对升高的P53抑制不足所致。我们进一步假设，在IPSC重编程过程中，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。因此，P53不能完全被抑制，残留的P53活性刺激一种结构性的DNA损伤反应，这种反应模仿通常只在对遗传毒性侮辱的反应中发生的情况。在体细胞的正常老化过程中，也观察到了慢性DNA损伤反应(通过P53依赖和独立的途径)，并可能导致在癌症发展过程中DNA损伤反应的完全丧失。我们假设，表观遗传破坏整个DNA损伤反应将改善A-IPSC的重新编程和质量。在初步研究中，我们检测了两个假定的P53抑制因子的功能，并使用这些因子来确定可以纠正与衰老相关的表观遗传格局和提高A-IPSC质量的多功能调节因子。在这项建议中，我们将通过对这些IPSC类型进行全面的比较分析，来研究这些多功能调控因子的功能。在目标1中，我们将确定多功能调节因子逆转异常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，我们将通过比较每个细胞系在几个不同的细胞系中的多能性和分化潜能来确定多潜能调节因子的功能效应。 体外和体内试验。最终，我们的发现将为我们如何提高从老年患者中提取的用于组织再生和移植的患者特有干细胞的质量提供关键的见解。

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.