Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes
Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes
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DOI：
10.1186/s13287-018-1085-9
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发表时间：
2018-12-07
影响因子：
7.5
通讯作者：
Kurihara, Hidemi
中科院分区：
文献类型：
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作者：
Komatsu, Nao;Kajiya, Mikihito;Kurihara, Hidemi
背景间充质干细胞（mesenchymal stem cell，MSC）/细胞外基质（extracellular matrix，ECM）复合物（C-MSCs）三维漂浮培养块由细胞和自身产生的ECM组成。先前的研究表明，C-MSC可以在没有人工支架的情况下移植到骨病变中以诱导骨再生。此外，骨诱导培养基（OIM）处理的C-MSC（OIM-C-MSC）在体内显示出快速和增加的新骨形成。为了将OIM-C-MSCs应用于新的骨再生细胞治疗，必须在分子水平上阐明其细胞特性。转录共激活因子是与PDZ结合的转录共激活因子（雅普/TAZ），在细胞的机械传导级联反应中起着关键作用，控制着MSCs的细胞谱系定型。与传统的2D培养系统相比，在漂浮条件下培养的3D C-MSC/OIM-C-MSC可以提供不同的微环境，从而诱导独特的机械转导级联，这是合理的。因此，本研究探讨了雅普/TAZ活性在三维培养的C-MSCs/OIM-C-MSCs在floating conditions.Methodshuman bone marrow derived MSCs培养在生长培养基中补充抗坏血酸。为了获得C-MSC，使用微量移液器尖端刮擦在细胞片上形成的汇合细胞，然后撕下。将薄片卷起以形成圆形细胞团。结果C-MSCs在漂浮培养条件下失去肌动蛋白细胞骨架，雅普/TAZ活性下降，导致细胞向脂肪/软骨细胞分化。OIM处理诱导C-MSCs中大量的COL 1沉积，这促进了Int 1依赖的肌动蛋白纤维的形成和雅普/TAZ活性，从而提高了成骨主转录因子runt-related转录因子2（RUNX 2）mRNA的表达水平。重要的是，通过OIM的雅普/TAZ活性的升高与COL 1沉积和F-肌动蛋白的完整性相关，这表明在OIM-C-MSCs.ConclusionThese研究结果表明，OIM-C-MSCs，形成一个独特的微环境，保持高雅普/TAZ活性，可以作为更好的候选人骨再生细胞治疗比C-MSCs。
BackgroundThree-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.MethodsHuman 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.ResultsC-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 Int1-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.ConclusionThese 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.