Human tendon stem progenitor cell aging and regeneration

描述(申请人提供)：老化的肌腱变得更容易受伤，愈合损伤的能力更差。归根结底，肌腱功能随年龄的下降必然反映了维持组织的腱细胞和补充组织的常驻干细胞的变化。 整个成年期的腱细胞数量。肌腱细胞总数随着年龄的增长而下降，这表明肌腱细胞群补充能力的进行性丧失可能是组织功能受损的原因之一。这项建议的目标是：i)定义人类肌腱组织驻留干细胞中与年龄相关的变化；ii)确定干细胞维持的机制；iii)测试这些机制是否可以被操纵以促进肌腱修复。在初步研究中，我们发现在大鼠和人的肌腱中，与年轻肌腱相比，肌腱干细胞/祖细胞(TSPC)的频率显著减少，这与整个肌腱细胞密度的减少一致。此外，来自老化肌腱的TSPC增殖缓慢，并显示细胞周期停滞在G2期的证据。我们的初步研究表明，转录因子CITED2(CREB结合蛋白/p300-与富含ED的尾巴相互作用的反式激活因子)是一种细胞生长调节因子和细胞衰老的有效抑制因子，在衰老的TSPC中显著下调，并且它被shRNA敲除后增加了细胞周期停滞。我们的初步数据还表明，CITED2调控着几个控制成体干细胞自我更新的基因。基于这些发现(目标1)，我们将确定不同年龄的人肌腱中TSPC的含量和功能(增殖率、细胞周期状态、分化潜能)。下一步(目标2)，我们将通过获得和丧失功能的方法来确定CITED2在TSPC随年龄增长的维持中的作用，以确定CITED2在TSPC增殖、细胞周期进展和细胞凋亡中的作用。我们还将评估CITED2在控制细胞增殖和存活的通路中调节关键步骤的能力。最后(目标3)，我们将 测定CITED2促进TSPC体内肌腱修复的能力。我们将比较年轻和老年肌腱来源的TSPC在体内修复髌腱损伤的能力，然后测试CITED2在这些细胞中的过度表达是否会促进细胞的再生和愈合。我们将使用从相同捐赠者分离的骨髓来源的干细胞(MSCs)进行类似的实验，因为MSCs被认为是再生肌腱和其他组织的优秀干细胞来源。这些研究将确定人类TSPC随年龄发生的功能变化(目标1)，建立涉及CITED2的拟议新机制是否可以解释与年龄相关的人类TSPC功能变化(目标2)，并测试操作CITED2是否可以增强干细胞在体内修复肌腱损伤的能力(目标3)。这些发现可能为年龄相关性肌腱疾病的基础以及肌腱修复和再生的新策略的发展提供新的见解。

DESCRIPTION (provided by applicant): Aging tendons become more susceptible to injury and less able to heal damage. Ultimately, declines in tendon function with age must reflect changes in the tenocytes that maintain the tissue, and in the tissue-resident stem cells that replenish the tenocyte population throughout adult life. Declines in overall tendon cell numbers with age have been reported, suggesting that a progressive loss in the ability to replenish the tenocyte population may contribute to impaired tissue function. The goals of this proposal are i) to define age-related changes in human tendon tissue-resident stem cells, ii) to identify mechanisms responsible for stem cell maintenance, and iii) to test whether those mechanisms can be manipulated to enhance tendon repair. In preliminary studies, we found that the frequency of Tendon Stem/Progenitor Cells, or TSPCs is markedly reduced in aged vs. young tendons of rats and humans, consistent with reduced overall tendon cellularity. Furthermore, TSPCs from aged tendons proliferate slowly and show evidence of cell cycle arrest in the G2 phase. Our preliminary study indicates that the transcription factor CITED2 (CREB-binding protein/p300-interacting transactivator with ED- rich tail), a cell growth regulator and potent suppressor of cel senescence, is strongly downregulated in aged TSPCs, and its knockdown by shRNA increases cell cycle arrest. Our pilot data also indicate that CITED2 regulates several genes governing adult stem cell self-renewal. Based on these findings (Aim 1), we will determine TSPC content and functionality (proliferation rate, cell cycle status, differentiation potential) in human tendos at different ages. Next (Aim 2), we will determine the role of CITED2 in TSPC maintenance with age using gain and loss of function approaches to determine the role of CITED2 in TSPC proliferation, cell cycle progression and apoptosis. We will also assess the ability of CITED2 to regulate key steps in pathways controlling cell proliferation and survival. Lastly (Aim 3), we will determine the ability of CITED2 to enhance tendon repair by TSPCs in vivo. We will compare the ability of TSPCs from young and old tendons to repair patellar tendon damage in vivo, then test whether overexpression of CITED2 in these cells will improve cell repopulation and healing. We will carry out similar experiments using bone marrow-derived stem cells (MSCs) isolated from the same donors, as MSCs are considered an excellent source of stem cells for regeneration of tendons and other tissues. These studies will identify functional changes that occur in human TSPCs with age (Aim 1), establish whether a proposed novel mechanism involving CITED2 can account for age-related changes in human TSPC function (Aim 2), and test whether manipulation of CITED2 can enhance the ability of stem cells to repair tendon damage in vivo (Aim 3). These findings may provide new insights into the basis for age-related tendon disorders, and the development of new strategies for tendon repair and regeneration.