Aging and Reprogramming of Somatic Cells to Pluripotency

体细胞的衰老和重编程至多能性

• 批准号: 7907777
• 负责人: PETER J HORNSBY
• 金额: $ 15.07万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907777
• 关键词: Abnormal Cell; Adult; Age; Aging; Aging-Related Process; Animals; Back; Basic Science; Biopsy; Callithrix; Cell Aging; Cell Lineage; Cell Therapy; Cell surface; Cells; Characteristics; Chromatin; Coculture Techniques; Defect; Development; Disease; Elderly; Environment; Exhibits; Fibroblasts; Future; Genes; Germ Lines; Gold; Haplorhini; Health; Health Status; Housing; Human; Immunodeficient Mouse; Immunohistochemistry; In Vitro; Individual; Literature; Living Donors; Longevity; Malignant - descriptor; Mesenchymal; Modeling; Monkeys; Morphology; Mus; Nature; Newborn Infant; Organ; Parkinson Disease; Patients; Predisposition; Primates; Property; Skin; Somatic Cell; Subfamily lentivirinae; System; Telomerase; Teratoma; Testing; Therapeutic Effect; Tissues; Transplantation; Work; age effect; age related; c-myc Genes; dopaminergic neuron; embryonic stem cell; frailty; in vivo; induced pluripotent stem cell; insight; neuron development; nonhuman primate; novel; pluripotency; public health relevance; transcription factor

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.

描述（由申请人提供）：老化改变了体细胞的特性，导致组织和器官功能下降。将体细胞重编程为诱导多能干细胞（IPS细胞）提供了测试细胞水平衰老是否可逆的机会，以及是否可以消除由衰老引起的细胞性质的变化，并且细胞返回到完全多能状态。该提案的一个新方面是，我们的重编程研究将使用来自小型非人类灵长类动物Marmoset的细胞进行。摩尔果的寿命最短。因此，它们可以更有效地用于比较早期捐助者与晚期捐助者的重编程。在此处提出的工作的未来发展中，可以将重编程/重新分化的细胞移植回同一单个动物中，从中得出了体细胞，从而对重新分析/重新分析细胞在体内环境中正常功能进行“金标准”测试。具体目的1：我们假设衰老会损害转录因子的重编程，以便从老年捐赠者中得出的细胞会表现出更大的趋势，这些趋势被错误编程，并且能够正确重编程为真正多能状态的能力。这将通过逆转录病毒引入重编程基因（OCT4，SOX2，KLF4，带有或不带C-Myc）的逆转录病毒，从一系列年龄的Marmoset，新生儿到非常古老（约13年）中的皮肤成纤维细胞。我们将检查结果细胞菌落中的重编程；重编程中的缺陷可以观察到与衰老相关的错误编程的增加，产生缺乏多能性并且可能获得的恶性特性的细胞。如果这是正确的，未来的工作将旨在阐明那些干扰重编程的老化过程。但是，我们还预计，即使在非常年龄的时候，有些细胞也会正确地重新编程为多能状态。具体目标2：我们假设已经正确重编程为多能性的细胞，即使是从旧供体中，也能够在暴露于适当的体外环境时沿定义的谱系正确分化。我们建议诱导它们与多巴胺能神经元区分开。通过发展神经元形态和多巴胺能特异性标记的表达来评估适当的分化。如果重新分化或异常细胞特性的缺陷作为由旧供体形成的IPS细胞的特征出现，则进一步的研究将集中在这些与老化有关的变化的性质上，这些变化的性质会干扰重新分化。这些研究提供了支持未来患者特异性细胞疗法的必要基础科学。在Marmoset模型中，我们可以确定是否可以从皮肤活检中得出细胞，受到重编程/重新分化，然后安全地将其移植回供体以发挥所需的治疗作用，包括诸如帕金森氏病等晚期疾病的治疗。公共卫生相关性：项目叙事衰老会导致细胞的许多变化，最终导致身体功能，脆弱和增加对疾病的易感性的下降。最近的研究表明，这些细胞衰老变化是可逆的可能性可逆的，这些研究表明，可以将皮肤细胞从成年人中吸收并将其重新编程为胚胎干细胞样状态。在此提案中，我们将测试衰老对果猴猴子皮肤细胞的能力的影响，其中有可能将植入植入胚胎干细胞样细胞重新回到供体动物中以测试治疗作用。