Reversing the effects of donor aging on adult stem cell potential

描述（申请人提供）：由于全球心血管疾病的增加，对小直径血管作为替代移植物的需求越来越大。原则上，成体间充质干细胞（MSC）可以满足大量自体细胞填充这些组织的需要，MSC具有显著的增殖和分化潜力。然而，包括我们在内的几个实验室的报告显示，来自老年供体的MSC增殖能力有限，分化潜力显著降低。这是一个主要的问题，因为大多数需要血管移植的患者是老年人。目前的提案试图使用一种高度创新的方法来解决这一挑战，该方法基于我们实验室的初步数据，该数据表明衰老的影响可以通过表达单一转录因子（即Nanog）来逆转。基于这些结果，我们提出以下具体目标。在新的目标1中，我们将研究Nanog的短暂表达是否足以逆转供体衰老对MSC增殖和成肌分化的影响。在目标2中，我们将研究一个新的假设，以揭示Nanog增强肌生成基因转录和收缩功能的机制。我们还将采用寡核苷酸微阵列以及高通量表观遗传分析来确定Nanog如何重组染色质并揭示由于Nanog表达而差异表达的新基因。在目标3中，我们将开发一种新的策略，将Nanog蛋白递送到MSC中，以恢复其失去的功能。将提出新的假设来设计具有增强转录活性的Nanog蛋白变体，以便最大限度地发挥其逆转衰老对MSC肌分化潜力的影响的效力。最后，在目标4中，我们将研究nanog处理的干细胞在工程小直径、功能性血管方面的潜力，这些血管可以植入我们实验室开发的临床前羊动物模型的动脉系统中。该项目的成功完成将大大提高成人供体MSC的临床治疗潜力，而无需进行基因修饰或重编程以达到多能状态。

DESCRIPTION (provided by applicant): Due to rise in cardiovascular disease throughout the world, there is increasing demand for small diameter blood vessels as replacement grafts. In principle, the need for large number of autologous cells to populate those tissues can be met by adult mesenchymal stem cells (MSC), which show significant proliferation and differentiation potential. However, reports from several laboratories including ours showed that MSC originating from older donors suffer from limited proliferative capacity and significantly reduced differentiation potential. This is a major concern, as the patients mostly in need for vascular grafts are elderly. The current proposal seeks to address this challenge using a highly innovative approach that is based on preliminary data from our laboratory showing that the effects of aging can be reversed by expression of a single transcription factor, namely Nanog. Based on these results we propose the following specific aims. In the new aim 1 we will examine whether transient expression of Nanog is sufficient to reverse the effects of donor aging on proliferation and myogenic differentiation of MSC. In aim 2 we will investigate a novel hypothesis to uncover the mechanism through which Nanog enhances transcription of myogenic genes and contractile function. We will also employ oligonucleotide microarrays as well as high throughput epigenetic analysis to determine how Nanog restructures the chromatin and unravel novel genes that are differentially expressed due to Nanog expression. In aim 3 we will develop a novel strategy to deliver the Nanog protein into MSC in order to restore their lost function. Novel hypotheses will be addressed to engineer Nanog protein variants with enhanced transcriptional activity in order to maximize their potency in reversing the effects of senescence on MSC myogenic differentiation potential. Finally, in aim 4 we will examine the potential of the Nanog-treated stem cells for engineering small-diameter, functional blood vessels that could be implanted into the arterial system of a pre-clinical ovine animal model that was developed in our laboratory. Successful completion of this project will significantly enhance the therapeutic clinical potential of MSC from adult donors without genetic modification or reprogramming to the pluripotent state.