Background and Motivation

• Human mesenchymal stem cells (hMSCs) are used in therapies for inflammatory and degenerative diseases.
• However, not all hMSCs are the same – they are heterogeneous, which can lead to inconsistent treatment outcomes.
• This research explores a new way to sort hMSCs based on their electrical properties to enrich for cells with better healing potential.

Research Goals

• Develop a method to separate hMSCs into distinct groups using a fluorescent dye (TMRE) that reflects their electrical and ionic states.
• Determine if the cells with lower or higher TMRE signals (indicating different membrane “charges”) show differences in aging, regeneration, and immune regulation.

Materials and Methods

• Cell Culture:
  • hMSCs are thawed from cryopreserved bone marrow samples and grown in a nutrient-rich medium.
  • Cells are expanded until they cover 70–85% of the culture surface and then split to continue growing.
• Fluorescence-Activated Cell Sorting (FACS) with TMRE:
  • TMRE is a fluorescent dye that accumulates in cell membranes based on the cell’s electrical potential, much like a battery indicator showing its charge.
  • Cells are incubated with a low concentration of TMRE to avoid overloading them.
  • Using FACS, cells are sorted into two groups:
    • MSC-DCL: Cells with low TMRE signal (depolarized membranes; akin to a low battery charge).
    • MSC-DCH: Cells with high TMRE signal (hyperpolarized membranes; like a fully charged battery).
• Co-culture with Macrophages:
  • Macrophages (immune cells) are cultured and activated using inflammatory agents.
  • Both groups of hMSCs are co-cultured with these activated macrophages to assess their effect on immune cell behavior.
• Gene Expression Analysis:
  • RNA is extracted from the cells to measure the levels of key genes.
  • Markers for senescence (aging), stemness (regenerative potential), autophagy (cellular recycling), and immunomodulation (inflammation control) are analyzed.
  • Analyses are performed immediately after sorting (0 hours) and after 24 hours in culture.

Results: Enrichment Strategy and Immediate Findings

• Sorting Outcome:
  • The FACS procedure successfully separated the hMSCs into two distinct groups based on TMRE intensity.
  • MSC-DCH cells had a TMRE signal roughly 15 times higher than MSC-DCL cells and were larger and more complex (as seen by forward and side scatter measurements).
  • The yield (number of cells collected) was higher for MSC-DCH, though both groups maintained similar cell viability.
• Immediate Gene Expression (0 Hours Post-Sorting):
  • MSC-DCL (low TMRE) showed:
    • Lower levels of p21, a marker of cell aging (senescence), indicating they are “younger”.
    • Higher levels of DNMT1, which helps maintain the cell’s ability to renew itself (stemness).
    • Increased expression of ULK1, suggesting more active autophagy (the cell’s recycling process).
  • MSC-DCH (high TMRE) generally showed opposite trends for these markers.

Results: 24-Hour Post-Sorting Findings

• Gene Expression Changes After 24 Hours:
  • Some senescence markers became similar between the groups, but MSC-DCL maintained lower levels of GLB1 and FUCA1, further suggesting reduced aging.
  • Stemness Markers:
    • CD44 levels were similar between the groups.
    • CD105 was lower in MSC-DCL, indicating a shift in cell characteristics over time.
  • Autophagy Markers:
    • MSC-DCL increased expression of p62, ULK1, and LC3B, reinforcing that these cells have a stronger self-cleaning and repair mechanism.
  • Immunomodulatory Markers:
    • After 24 hours, MSC-DCL showed higher levels of HO-1 and IL-6, which are linked to anti-inflammatory effects and improved healing.

Results: Functional Effects in Co-Culture with Macrophages

• Co-Culture Experiment:
  • Activated macrophages were co-cultured with MSC-DCL and MSC-DCH cells.
  • Macrophages exposed to MSC-DCL expressed significantly lower levels of several pro-inflammatory markers (both M1 and M2 types), indicating a stronger immunosuppressive effect.
• Interpretation:
  • MSC-DCL cells appear to better suppress inflammation, which is beneficial for therapies aiming to control overactive immune responses.
  • Higher HO-1 expression in MSC-DCL may be a key factor in this anti-inflammatory capability.

Discussion and Interpretation

• Key Findings:
  • Sorting hMSCs by their electrical properties using TMRE creates two distinct populations.
  • Cells with low TMRE intensity (MSC-DCL) exhibit markers indicating reduced aging, enhanced self-repair (autophagy), and improved immune regulation.
  • These traits suggest that MSC-DCL cells could be more effective in therapeutic applications.
• Metaphors and Analogies:
  • Imagine TMRE as a battery tester – cells with low readings are like batteries that are not fully charged but may be more “youthful” and ready for a recharge.
  • FACS sorting works like a high-tech sieve that separates objects (cells) by color and size, making the groups more uniform.
  • Autophagy is the cell’s own recycling center, cleaning up damaged parts to keep the cell functioning optimally.
• Implications:
  • This enrichment strategy could improve stem cell therapies by selecting cells that are less aged and have a better capacity for repair and immune regulation.
  • Further studies may refine these methods with additional protein and metabolic analyses.

Conclusions

• hMSCs can be enriched into two distinct groups based on their electrical properties as measured by TMRE staining.
• Cells with low TMRE intensity (MSC-DCL) show lower signs of aging, stronger autophagy, and enhanced immunosuppressive potential.
• These findings support the concept of selecting specific stem cell subpopulations to improve the effectiveness of cell-based therapies.

Glossary of Key Terms

• hMSCs: Human mesenchymal stem cells that can differentiate into various cell types to help repair tissues.
• TMRE: A fluorescent dye that indicates the electrical potential of cell membranes, similar to checking a battery’s charge.
• FACS: A technique (Fluorescence-Activated Cell Sorting) used to separate cells based on size, fluorescence, and other properties.
• Membrane Potential: The voltage difference across a cell membrane, analogous to the charge difference in a battery.
• Senescence: The process of cell aging, where cells lose their ability to function optimally.
• Autophagy: The cell’s process of self-cleaning and recycling damaged components.
• Stemness: The potential of a stem cell to differentiate into multiple cell types.
• Immunomodulation: The ability to regulate or modify the immune response, often reducing inflammation.

观察与动机 (背景)

• 人类间充质干细胞 (hMSCs) 被用于治疗多种炎症和退行性疾病。
• 由于细胞群体存在异质性，hMSCs 疗法的效果可能会有所不同。
• 本研究探索了一种基于细胞电学和离子特性富集 hMSCs 的新方法，以选出具有更好修复潜力的细胞。

研究目标

• 开发一种利用荧光染料 TMRE 根据细胞电特性对 hMSCs 进行分选的方法。
• 探讨低 TMRE 信号和高 TMRE 信号细胞是否在衰老、干性及免疫调节方面存在差异。

材料与方法

• 细胞培养：
  • 从人骨髓中获得的 hMSCs 被解冻并在营养丰富的培养基中扩增。
  • 细胞生长至覆盖面积 70–85% 后进行分裂，以便进一步扩增。
• 利用 FACS 和 TMRE 进行分选：
  • TMRE 是一种荧光染料，根据细胞膜电位显示不同亮度，就像检测电池电量一样。
  • 细胞在低浓度 TMRE 下孵育，避免染料过量影响细胞。
  • 使用 FACS 技术，将细胞分为两个群体：
    • MSC-DCL：TMRE 信号较低的细胞（膜去极化，类似于电池电量低）。
    • MSC-DCH：TMRE 信号较高的细胞（膜超极化，类似于电池充满电）。
• 与巨噬细胞共培养：
  • 培养并激活巨噬细胞（免疫细胞），以模拟炎症状态。
  • 将两组 hMSCs 与激活的巨噬细胞共培养，评估其对免疫细胞的调控作用。
• 基因表达分析：
  • 提取细胞 RNA，检测关键基因的表达水平。
  • 测定与衰老、干性、自噬和免疫调节相关的标志物。
  • 分析分别在分选后 0 小时和 24 小时的表达变化。

结果：分选策略与即时发现

• 分选结果：
  • 利用 FACS 技术，成功根据 TMRE 强度将 hMSCs 分为两个不同的群体。
  • MSC-DCH 组的 TMRE 信号大约比 MSC-DCL 组高 15 倍，同时细胞尺寸和复杂性也更大。
  • 尽管两组细胞的存活率相似，MSC-DCH 的回收率较高。
• 即时基因表达 (分选后 0 小时)：
  • MSC-DCL（低 TMRE）显示出：
    • 较低的 p21 表达，提示细胞衰老程度较低。
    • 较高的 DNMT1 表达，有助于维持细胞自我更新能力（干性）。
    • 自噬标志物 ULK1 表达增加，说明细胞自我回收机制较为活跃。
  • MSC-DCH 组则表现出相反的趋势。

结果：24 小时后的发现

• 24 小时后的基因表达变化：
  • 部分衰老标志物在两组间趋于一致，但 MSC-DCL 组的 GLB1 和 FUCA1 水平仍较低，表明其衰老程度较低。
  • 干性标志物：
    • CD44 表达在两组间相似。
    • CD105 在 MSC-DCL 组中表达降低，显示出细胞特性随时间发生转变。
  • 自噬标志物：
    • MSC-DCL 组在 24 小时时 p62、ULK1 和 LC3B 的表达增加，进一步证明其自我清理和修复能力较强。
  • 免疫调节标志物：
    • 24 小时后，MSC-DCL 组表现出 HO-1 和 IL-6 的表达升高，这与抗炎和促进愈合有关。

结果：与巨噬细胞共培养的功能效应

• 共培养实验：
  • 激活的巨噬细胞与 MSC-DCL 和 MSC-DCH 细胞共同培养。
  • 与 MSC-DCL 共培养后，巨噬细胞中多种促炎标志物的表达显著降低。
• 解读：
  • MSC-DCL 细胞表现出更强的抑制炎症能力，这对需要控制免疫反应的治疗非常有利。
  • 较高的 HO-1 表达可能是 MSC-DCL 抑制炎症作用的关键因素。

讨论与解读

• 主要发现：
  • 利用 TMRE 分选技术可以将 hMSCs 分离为两个具有不同电学特性的群体。
  • 低 TMRE 信号的 MSC-DCL 细胞表现出较低的衰老迹象和更强的自噬能力。
  • 这些细胞在免疫调节方面也表现出更大的潜力。
• 比喻与类比：
  • 把 TMRE 看作电池电量指示器；低 TMRE 的细胞就像电量不足但更“年轻”的电池，具有更高的再生潜力。
  • FACS 分选就像用筛子分拣不同颜色和大小的弹珠，使每一组更加均一。
  • 自噬类似于细胞的回收中心，负责清理和再利用废弃物，确保细胞高效运行。
• 意义：
  • 这种富集策略有望通过选出衰老较少、再生能力更强的细胞，从而改善细胞治疗的效果。
  • 未来的研究将进一步完善这一技术，并结合蛋白和代谢分析以深入理解其机制。

结论

• 研究证明，可以通过 TMRE 染色将 hMSCs 分选为两种不同的群体。
• 低 TMRE 信号的 MSC-DCL 细胞表现出较低的衰老特性、更强的自噬能力以及更佳的免疫抑制潜力。
• 这些结果支持通过选择特定干细胞亚群来提高细胞治疗效果的理念。

关键术语词汇表

• hMSCs: 人类间充质干细胞，具有分化为多种细胞类型和促进组织修复的潜力。
• TMRE: 一种荧光染料，用于检测细胞膜电位，类似于检测电池电量。
• FACS: 荧光激活细胞分选技术，根据细胞大小、荧光等特性对细胞进行分选。
• 膜电位: 细胞膜两侧的电压差，类似于电池正负极之间的电压。
• 衰老 (Senescence): 细胞老化过程，老化细胞功能下降。
• 自噬 (Autophagy): 细胞自我清理和回收废弃物的机制，就像细胞内部的回收站。
• 干性 (Stemness): 指干细胞具备分化为多种细胞类型的能力。
• 免疫调节 (Immunomodulation): 调节免疫系统反应，减少炎症或促进修复的能力。