Human tendon stem progenitor cell aging and regeneration

描述（由申请人提供）：老化的肌腱变得更容易受伤，更难愈合损伤。最终，肌腱功能随年龄的下降必须反映维持组织的肌腱细胞的变化，以及补充细胞的组织驻留干细胞的变化。 整个成年期的肌腱细胞群。据报道，随着年龄的增长，总体肌腱细胞数量下降，这表明补充肌腱细胞群的能力逐渐丧失可能导致组织功能受损。该提案的目标是：i）定义人类肌腱组织驻留干细胞中与年龄相关的变化，ii）确定负责干细胞维护的机制，以及iii）测试是否可以操纵这些机制以增强肌腱修复。在初步研究中，我们发现肌腱干/祖细胞或TSPC的频率在大鼠和人类的老年肌腱与年轻肌腱中显着降低，与整体肌腱细胞结构的减少一致。此外，来自老化肌腱的TSPCs增殖缓慢，并显示出细胞周期停滞在G2期的证据。我们的初步研究表明，转录因子CITED 2（CREB结合蛋白/p300相互作用的反式激活因子与艾德-丰富的尾巴），细胞生长调节剂和细胞衰老的有效抑制剂，强烈下调老化TSPCs，并通过shRNA敲低增加细胞周期阻滞。我们的试验数据还表明，CITED 2调节了几个控制成体干细胞自我更新的基因。基于这些发现（目的1），我们将确定TSPC含量和功能（增殖率，细胞周期状态，分化潜力）在不同年龄的人肌腱。接下来（目的2），我们将使用功能获得和丧失方法确定CITED 2在TSPC增殖、细胞周期进展和凋亡中的作用，从而确定CITED 2在TSPC维持中随年龄的作用。我们还将评估CITED 2调节控制细胞增殖和存活的途径中的关键步骤的能力。第三个目标（Aim 3）： 确定CITED 2在体内增强TSPC的肌腱修复的能力。我们将比较来自年轻和老年肌腱的TSPC在体内修复髌腱损伤的能力，然后测试CITED 2在这些细胞中的过表达是否会改善细胞再生和愈合。我们将使用从相同供体分离的骨髓源性干细胞（MSC）进行类似的实验，因为MSC被认为是肌腱和其他组织再生的干细胞的极好来源。这些研究将确定人类TSPC随年龄发生的功能变化（目标1），确定是否提出了一种涉及CITED 2的新机制可以解释人类TSPC功能的年龄相关变化（目标2），并测试CITED 2的操作是否可以增强干细胞修复体内肌腱损伤的能力（目标3）。这些发现可能为与年龄相关的肌腱疾病的基础提供新的见解，并为肌腱修复和再生提供新的策略。

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.