Aging and Reprogramming of Somatic Cells to Pluripotency

描述（由申请人提供）：衰老改变体细胞的特性，导致组织和器官功能下降。体细胞重编程为诱导多能干细胞（iPS细胞）提供了一个机会来测试细胞水平的衰老是否可逆，以及由衰老引起的细胞特性的变化是否可以被消除，细胞是否可以恢复到完全的多能状态。这个提议的一个新颖之处在于，我们的重编程研究将使用一种小型的非人类灵长类动物——狨猴的细胞进行。狨猴是所有类人猿灵长类动物中寿命最短的；因此，它们可以更有效地用于比较早期和晚期捐献者的重编程。在本研究的未来发展中，重编程/再分化细胞可以被移植回体细胞来源的同一动物个体，从而为重编程/再分化细胞在体内环境中正常工作的能力提供“金标准”测试。具体目标1：我们假设衰老会损害转录因子的重编程，因此来自老年供体的细胞将表现出更大的错误编程倾向，更少的能力被正确重编程为真正的多能状态。这将通过将重编程基因（Oct4, Sox2, Klf4，含或不含c-Myc）逆转录病毒引入不同年龄的狨猴皮肤成纤维细胞来进行测试，从新生儿到非常大的（~13岁）。我们将检查产生的细胞菌落中的重编程；重编程缺陷可以观察到与年龄相关的错误编程增加，产生缺乏多能性的细胞，并可能获得恶性特性。如果这是正确的，未来的工作将致力于阐明那些干扰重编程的衰老过程。然而，我们也预计，即使在非常年老的时候，一些细胞也会被正确地重新编程为多能状态。具体目标2：我们假设，即使是来自老供体的细胞，经过正确的重编程，也能在适当的体外环境中沿着定义的谱系正确地再分化。我们建议诱导它们向多巴胺能神经元分化。适当的分化将通过神经元形态的发展和多巴胺能神经元特异性标志物的表达来评估。如果再分化缺陷或细胞特性异常成为老年供体形成的iPS细胞的特征，那么进一步的研究将集中在这些干扰再分化的衰老相关变化的性质上。这些研究为支持未来患者特异性细胞治疗提供了必要的基础科学。在狨猴模型中，我们可以确定是否可以从皮肤活检中提取细胞，进行重编程/再分化，然后安全地移植回供体，以发挥所需的治疗效果，包括治疗老年疾病，如帕金森病。衰老导致细胞发生许多变化，最终导致身体功能下降、虚弱和对疾病的易感性增加。最近的研究表明，有可能从成人身上提取皮肤细胞，并将其重新编程为胚胎干细胞样状态，从而提出了这些细胞衰老变化是可逆的可能性。在这个提议中，我们将测试衰老对狨猴皮肤细胞重编程能力的影响，其中有可能将胚胎干细胞样细胞重新移植回供体动物以测试治疗效果。

DESCRIPTION (provided by applicant): Aging alters the properties of somatic cells, resulting in declines in tissue and organ function. Reprogramming of somatic cells to induced pluripotent stem cells (iPS cells) provides the opportunity to test whether aging at the cell level is reversible and whether the changes in cell properties that are caused by aging can be erased and the cell returned to a completely pluripotent state. A novel aspect of this proposal is that our reprogramming studies will be performed using cells from a small nonhuman primate, the marmoset. Marmosets have the shortest life span of any anthropoid primate; therefore, they can more efficiently be used to compare reprogramming in early- versus late-life donors. In a future development of the work proposed here, reprogrammed/re-differentiated cells can be transplanted back into the same individual animal from which the somatic cells were derived, thus providing a "gold standard" test of the ability of reprogrammed/redifferentiated cells to function normally in an in vivo environment. Specific Aim 1: We hypothesize that aging will impair reprogramming by transcription factors, so that cells derived from older donors will exhibit a greater tendency to be misprogrammed and less ability to be correctly reprogrammed to a true pluripotent state. This will be tested by the retroviral introduction of reprogramming genes (Oct4, Sox2, Klf4, with or without c-Myc) into skin fibroblasts from marmosets of a range of ages, newborn to very old (~13 years). We will examine reprogramming in resultant cell colonies; defects in reprogramming may be observed as an aging-related increase in misprogramming, producing cells that lack pluripotency and which may have acquired malignant properties instead. If this is correct, future work will aim to elucidate those aging processes that interfere with reprogramming. However, we also anticipate that even at very old age some cells will be correctly reprogrammed to a pluripotent state. Specific Aim 2: We hypothesize that cells that have been correctly reprogrammed to pluripotency, even from old donors, will be able to properly redifferentiate along defined lineages when exposed to appropriate in vitro environments. We propose to induce them to differentiate to dopaminergic neurons. Proper differentiation will be assessed by development of neuronal morphology and expression of dopaminergic neuron-specific markers. If defects in redifferentiation or abnormal cell properties emerge as characteristics of iPS cells formed from old donors, further studies will focus on the nature of these aging-related changes that interfere with redifferentiation. These studies provide the necessary basic science that supports future patient-specific cell therapy. In the marmoset model, we can determine whether cells can be derived from skin biopsies, subjected to reprogramming/redifferentiation, and then safely transplanted back into the donor to exert a desired therapeutic effect, including therapies of late-life diseases such as Parkinson's disease. PUBLIC HEALTH RELEVANCE: Project Narrative Aging causes many changes in cells that eventually result in declines in body function, frailty, and increased susceptibility to diseases. The possibility that these cellular aging changes are reversible has been raised by recent studies that show that it is possible to take skin cells from adults and reprogram them to an embryonic stem cell-like state. In this proposal we will test the effects of aging on the ability to reprogram skin cells from marmoset monkeys, in which there is the potential of retransplanting embryonic stem cell-like cells back into the donor animal to test a therapeutic effect.