How Does a Young Extracellular Matrix Rejuvenate Old Mesenchymal Stem Cells?

此前，我们报道了骨髓来源的自我更新和骨形成能力的缺陷 老年人骨髓间充质干细胞(BM-MSCs)细胞外培养可完全修复 基质(ECM)由年轻供者的骨髓基质细胞合成(Young-ECM)。然而，这种恢复剂 将年轻或老年供者的细胞培养在由 老年供者骨髓基质细胞(OLD-ECM)。这次续签申请的目的是为了剖析出有效的 成分(S)存在于年轻的细胞外基质中，在保持BM-MSC性能和 老年供者骨髓间充质干细胞(旧MSCs)的再生。确定蛋白质组成的差异 在两个年轻的和年长的ECM之间，我们进行了蛋白质组分析，发现CCN1/Cyr61 是年轻ECM中唯一不存在于OLD-ECM中的蛋白质。尽管已经显示了CCN1/Cyr61 当外源添加到细胞培养中时，它是否也可以调节成骨细胞的分化，目前还不清楚 作为结构蛋白发挥作用(在BM-ECM合成过程中并入)，并促进MSC的保留 属性。在续订申请中，我们假设在Young-ECM中CCN1/Cyr61的存在是 对保持骨髓间充质干细胞的特性和老年骨髓间充质干细胞的年轻化至关重要。我们处于有利地位，可以 既然我们已经建立了一个独特的3D原生BM-ECM培养系统，那么就可以验证这一假设。测试我们的 假设，我们将评估CCN1/Cyr61通过减少其在控制MSCs行为中的作用 在合成Young-ECM的过程中将其掺入基质(特异性目标1)并增加其掺入 在合成OLD-ECM的过程中进入基质(特定目标2)。确定CCN1/Cyr61是否单独 足以使老年MSCs恢复活力的重组人CCN1/Cyr61将被添加到细胞培养液中， 物理吸附在组织培养塑料(TCP)表面，或结合到自组装单分子膜中 (SAMS)(具体目标3)。拟议的研究具有创新性，因为我们将首次：a)论证 CCN1/Cyr61的功能，被整合到BM-ECM(BM-MSC微环境)中作为结构 蛋白质在合成过程中，控制真正的BM-MSCs的命运，以及b)构建定义的ECM，使用 重组CCN1/Cyr61，模拟天然BM-ECM大规模扩增高- 优质MSCs和老年MSCs的年轻化。如果成功，建议的研究结果将是 意义重大，因为它们将克服与我们现有的3D原生BM-ECM相关的挑战，并将 适用于临床应用(例如：不同供体制备的ECM的变化；相关的安全问题 对未检测到的病原体的潜在污染；以及可能产生的未定义的ECM成分 可变/不可预测的临床结果)。根据2015年全国退伍军人健康状况调查 依赖退伍军人管理局，47%的美国退伍军人年龄在65岁或以上，这给 治疗一大批老年性疾病患者。从拟议研究中获得的信息 对于开发可用于快速扩展大量 为个人干细胞库提供高质量的自体MSCs，允许连续给药 自体MSCs不仅可以替代衰老的细胞，而且可以逐渐逆转衰老的微环境和 延缓衰老相关疾病的进展或衰老过程本身。

Previously, we reported that deficits in self-renewal and bone formation capacity in bone marrow-derived mesenchymal stem cells (BM-MSCs) from elderly could be completely restored by culture on extracellular matrix (ECM) synthesized by BM stromal cells from young donors (young-ECM). However, this restorative effect was substantially diminished when cells from young or old donors were cultured on ECM synthesized by BM stromal cells from old donors (old-ECM). The goal of this renewal application is to dissect out the effective component(s) present in young-ECM that play(s) a critical role in the retention of BM-MSC properties and rejuvenation of BM-MSCs from elderly donors (old MSCs). To identify differences in protein composition between the two young- and old-ECMs, we performed proteomic analyses and discovered that CCN1/Cyr61 was the only protein present in young-ECM that was not in old-ECM. Although CCN1/Cyr61 has been shown to regulate osteoblast differentiation when added exogenously to cell cultures, it is not clear whether it can also function as a structural protein (incorporated during BM-ECM synthesis) and promote the retention of MSC properties. In the renewal application, we hypothesize that the presence of CCN1/Cyr61 in young-ECM is essential for the retention of MSC properties and rejuvenation of elderly BM-MSCs. We are well-positioned to test this hypothesis since we have established a unique 3D native BM-ECM culture system. To test our hypothesis, we will evaluate the role of CCN1/Cyr61 in controlling the behavior of MSCs by decreasing its incorporation into the matrix during synthesis of young-ECM (Specific Aim 1) and increasing its incorporation into the matrix during synthesis of old-ECM (Specific Aim 2). To determine whether CCN1/Cyr61 alone is sufficient to rejuvenate elderly MSCs, recombinant human CCN1/Cyr61 will be added to cell culture media, physisorbed onto tissue culture plastic (TCP) surfaces, or incorporated into self-assembled monolayers (SAMs) (Specific Aim 3). The proposed studies are innovative in that we will for the first time: a) demonstrate the function of CCN1/Cyr61, incorporated into BM-ECM (the BM-MSC microenvironment) as a structural protein during synthesis, in controlling the fate of authentic BM-MSCs, and b) construct a defined ECM, using recombinant CCN1/Cyr61, that mimics the ability of native BM-ECM to produce large-scale expansion of high- quality MSCs and rejuvenation of elderly MSCs. If successful, the results of the proposed studies will be significant because they will overcome challenges related to our established 3D native BM-ECM and be more suitable for clinical applications (e.g: variations in ECM prepared from different donors; safety concerns related to potential contamination with undetected pathogens; and undefined ECM components that may produce variable/unpredictable clinical outcomes). According to the 2015 National Survey of Veteran Enrollees' Health and Reliance on VA, 47% of the US veteran population is 65 years or older, creating a major challenge for treating a large number patients with age-related diseases. The information gained from the proposed studies will be important for developing a defined culture system that can be used to quickly expand large numbers of high-quality autologous MSCs for personal stem cell banks, allowing serial administration of “rejuvenated” autologous MSCs that not only replace aged cells but also gradually reverse the aged microenvironment and delay the progression of age-related diseases or the aging process itself.