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

年轻的细胞外基质如何​​使衰老的间充质干细胞恢复活力？

• 批准号: 9561542
• 负责人: XIAO-DONG CHEN
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-10-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9561542
• 关键词: Adenovirus Infections; Aging-Related Process; Animals; Architecture; Autologous; Behavior; Bone Marrow; Cell Culture Techniques; Cell-Matrix Junction; Cells; Clinical; Culture Media; Data; Disease; Elderly; Extracellular Matrix; Funding; Goals; Health; Human; Mesenchymal Stem Cells; Mus; Osteogenesis; Outcome; Patients; Play; Population; Positioning Attribute; Progress Reports; Property; Proteins; Proteomics; RNA Interference; Recombinants; Rejuvenation; Reporting; Role; Safety; Stem cells; Stromal Cells; Structural Protein; Surface; Surveys; System; Testing; Time; Variant; Veterans; age related; aged; bone; cell age; cell bank; clinical application; human old age (65+); improved; in vivo; innovation; knock-down; mechanical properties; monolayer; osteoblast differentiation; osteoprogenitor cell; overexpression; pathogen; protein structure; self-renewal; senescence; stemness; tissue culture

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.

以前，我们报道了骨髓源性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）可以通过细胞外基质培养完全恢复， 由来自年轻供体的BM基质细胞（BM-ECM）合成的基质（ECM）。然而，这种恢复性 当来自年轻或年老供体的细胞在由 来自老年供体的BM基质细胞（老年ECM）。这次更新申请的目的是剖析出有效的 存在于BM-ECM中的组分，其在BM-MSC性质的保持中起关键作用， 再生来自老年供体的BM-MSC（老年MSC）。为了确定蛋白质组成的差异 在两个年轻和年老的ECM之间，我们进行了蛋白质组学分析，发现CCN 1/Cyr 61 是唯一存在于旧ECM中而不存在于旧ECM中的蛋白。虽然CCN 1/Cyr 61已经被证明是 调节成骨细胞的分化，但尚不清楚它是否也能 作为结构蛋白（在BM-ECM合成过程中掺入）发挥作用并促进MSC的保留 特性.在更新申请中，我们假设CCN 1/Cyr 61在EPC-ECM中的存在是 对于MSC特性的保持和老年BM-MSC的再生至关重要。我们能够在 因为我们已经建立了独特的3D天然BM-ECM培养系统，所以我们测试了该假设。来测试我们 假设，我们将评估CCN 1/Cyr 61在控制MSC的行为中的作用， - 在合成EST-ECM（特异性目标1）期间掺入基质中并增加其掺入 在旧ECM的合成过程中进入基质（特定目标2）。为了确定单独的CCN 1/Cyr 61是否 为了使老化的MSC恢复活力，将重组人CCN 1/Cyr 61加入细胞培养基中， 物理吸附到组织培养塑料（TCP）表面上，或并入自组装单层中 （具体目标3）。拟议的研究是创新的，因为我们将首次：a）证明 CCN 1/Cyr 61的功能，作为结构调节剂并入BM-ECM（BM-MSC微环境）中， 在控制真实BM-MSC的命运中，和B）使用 重组CCN 1/Cyr 61，其模拟天然BM-ECM产生高表达的大规模扩增的能力。 高质量的MSC和老年MSC的复壮。如果成功，拟议研究的结果将 重要的是，它们将克服与我们建立的3D原生BM-ECM相关的挑战， 适合临床应用（例如：由不同供体制备的ECM的变化;相关安全问题 未检测到的病原体的潜在污染;以及可能产生 可变/不可预测的临床结果）。根据2015年全国退伍军人健康调查， 和依赖退伍军人事务部，47%的美国退伍军人人口是65岁或以上，创造了一个重大挑战， 治疗了大量的老年病患者。从拟议研究中获得的信息 将是重要的发展一个明确的文化系统，可用于快速扩大大量的 高质量的自体骨髓间充质干细胞用于个人干细胞库，允许连续施用“再生” 自体MSC不仅替代老化细胞，而且逐渐逆转老化的微环境， 延缓与年龄有关的疾病或衰老过程本身的进展。