I 型胶原蛋白的沉积通过成骨诱导改善了间充质干细胞/细胞外基质复合物 3D 悬浮培养团块中的 YAP/TAZ 活性。

BACKGROUND: Three-dimensional (3D) floating culture clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. Previous studies have demonstrated that C-MSCs can be transplanted into bony lesions without an artificial scaffold to induce bone regeneration. Moreover, osteoinductive medium (OIM)-treated C-MSCs (OIM-C-MSCs) have shown rapid and increased new bone formation in vivo. To apply OIM-C-MSCs for novel bone regenerative cell therapy, their cellular properties at the molecular level must be elucidated. The transcriptional co-activators yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) have been recognized as key players in the mechanotransduction cascade, controlling cell lineage commitment in MSCs. It is plausible that 3D C-MSCs/OIM-C-MSCs cultured in floating conditions could provide distinct microenvironments compared to conventional 2D culture systems and thereby induce unique mechanotransduction cascades. Therefore, this study investigated the YAP/TAZ activity in 3D-cultured C-MSCs/OIM-C-MSCs in floating conditions. METHODS: Human bone marrow-derived MSCs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make round clumps of cells. Then, YAP/TAZ activity, filamentous actin (F-actin) integrity, collagen type I (COL1) production, and the differentiation potency in 3D floating culture C-MSCs/OIM-C-MSCs were analyzed. RESULTS: C-MSCs cultured in floating conditions lost their actin cytoskeleton to downregulate YAP/TAZ activity, which directed cells to undergo adipogenesis/chondrogenesis. OIM treatment induced abundant COL1 deposition, which facilitated Intβ1-dependent actin fiber formation and YAP/TAZ activity to elevate the expression levels of osteogenic master transcriptional factor runt-related transcription factor 2 (RUNX2) mRNA in C-MSCs. Importantly, elevation of YAP/TAZ activity via OIM was associated with COL1 deposition and F-actin integrity, suggesting a positive feedback loop in OIM-C-MSCs. CONCLUSION: These findings suggest that OIM-C-MSCs, which form a unique microenvironment that maintains high YAP/TAZ activity, can serve as better candidates for bone regenerative cell therapy than C-MSCs.

背景：三维（3D）悬浮培养的间充质干细胞（MSC）/细胞外基质（ECM）复合物（C-MSCs）由细胞和自身产生的 ECM 组成。先前的研究表明，C-MSCs 可以在没有人工支架的情况下移植到骨病变部位，从而诱导骨再生。此外，用成骨诱导培养基（OIM）处理的 C-MSCs（OIM-C-MSCs）在体内显示出快速和增加的新骨形成。为了将 OIM-C-MSCs 应用于新型骨再生细胞治疗，必须阐明其在分子水平上的细胞特性。Yes 相关蛋白/含 PDZ 结合基序的转录共激活因子（YAP/TAZ）转录共激活因子已被认为是机械转导级联中的关键因子，控制着 MSC 中的细胞谱系分化。合理的是，在悬浮条件下培养的 3D C-MSCs/OIM-C-MSCs 可能提供与传统 2D 培养系统相比的不同微环境，从而诱导独特的机械转导级联。因此，本研究调查了在悬浮条件下培养的 3D C-MSCs/OIM-C-MSCs 中的 YAP/TAZ 活性。

方法：用人骨髓来源的 MSC 在补充有抗坏血酸的生长培养基中培养。为了获得 C-MSCs，用微量移液器尖端刮去已在细胞片上形成的汇合细胞，然后将其撕下。将薄片卷起制成圆形细胞团。然后，分析 3D 悬浮培养 C-MSCs/OIM-C-MSCs 中的 YAP/TAZ 活性、丝状肌动蛋白（F-actin）完整性、I 型胶原（COL1）产生和分化潜能。

结果：在悬浮条件下培养的 C-MSCs 失去了肌动蛋白细胞骨架，从而下调了 YAP/TAZ 活性，使细胞向脂肪形成/软骨形成分化。OIM 处理诱导大量 COL1 沉积，促进 Intβ1 依赖性肌动蛋白纤维形成和 YAP/TAZ 活性，提高 C-MSCs 中成骨主转录因子 runt 相关转录因子 2（RUNX2）mRNA 的表达水平。重要的是，通过 OIM 升高的 YAP/TAZ 活性与 COL1 沉积和 F-actin 完整性相关，表明 OIM-C-MSCs 中存在正反馈环。

结论：这些发现表明，形成维持高 YAP/TAZ 活性的独特微环境的 OIM-C-MSCs 可能比 C-MSCs 更适合骨再生细胞治疗。