Domnina Alisa, Ivanova Julia, Alekseenko Larisa, Kozhukharova Irina, Borodkina Aleksandra, Pugovkina Natalia, Smirnova Irina, Lyublinskaya Olga, Fridlyanskaya Irina, Nikolsky Nikolay
Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia.
Front Cell Dev Biol. 2020 Jun 16;8:473. doi: 10.3389/fcell.2020.00473. eCollection 2020.
Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin - skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external HO-induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF (vascular endothelial growth factor) than monolayer cells. Taken together, these findings indicate that enhanced secretory activity can promote wound healing potential of eMSC spheroids and that cultivation in the 3D cell environment alters eMSC vital programs and therapeutic efficacy.
间充质干细胞目前正作为一种有前景的工具，用于治疗多种人类疾病。众多研究已证明，由这些细胞形成的球体具有增强的治疗潜力，然而，这种效应的基本原理仍在讨论中。在这项工作中，我们表明，与单层培养的细胞相比，聚集在球体中的子宫内膜间充质干/基质细胞（eMSCs）在治疗对eMSC组织来源无特异性的缺损——皮肤伤口时，具有更高的治疗效果。为了阐明球体效力更高的可能原因，我们比较了在球体和单层中培养的eMSC对应激损伤的耐受性。使用基因编码的过氧化氢生物传感器HyPer，我们发现三维结构（3D）有助于保护球体的内部细胞层免受外部HO诱导的氧化应激。然而，球体中氧化损伤的eMSC的活力似乎远低于单层细胞。一项广泛的分析，包括热休克和辐射应激处理，揭示了受应激因素损伤的球体中的细胞激活了凋亡程序，而单层细胞中则出现了应激诱导的早衰。我们发现抗凋亡和自噬相关基因的基础下调为3D培养的eMSC对凋亡的高易感性提供了可能的分子基础。我们得出结论，凋亡易感性导致受损细胞的程序性清除，并有助于球体的移植安全性。此外，为了研究旁分泌在球体伤口愈合效力中的作用，我们利用了伤口模型（划痕试验），发现eMSC球体条件培养基加速了贴壁细胞的迁移。我们表明，3D eMSC上调转录激活因子缺氧诱导因子（HIF）-1，并且比单层细胞分泌的HIF-1诱导的促血管生成因子血管内皮生长因子（VEGF）多十倍。综上所述，这些发现表明增强的分泌活性可促进eMSC球体的伤口愈合潜力，并且在3D细胞环境中培养会改变eMSC的生命程序和治疗效果。
