Due to their immunomodulatory property, MSCs have been increasingly used to treat a range of immunopathy [26, 27], and 3D cultured MSCs show enhanced immunomodulatory property compared with those in 2D culture . However, both the regulatory mechanisms remain unclear. Meaningfully, we modulated YAP pathway both of 2D and 3D cultured UC-MSCs and validated its role in colitis and T1DM mouse models, which proved that mechanical stress played an important role in maintaining the immunomodulatory function and therapeutic potential of 2D and 3D cultured UC-MSCs, and PGE2 could be used to up-regulate YAP and improve the immunomodulatory ability of 2D cultured UC-MSCs after extensive expansion.
As a kind of adult stem cells, MSCs possess two characteristics that are distinct from other adult cells. First, they have stemness, as embodied by their self-renewal and tri-lineage differentiation into osteoblasts, chondrocytes and adipocytes. Second, MSCs own immunomodulatory property . Studies have shown that although cell replacement plays an important role in MSCs therapy for specific diseases, the final therapeutic effects are mainly the results of MSCs-derived immunomodulatory ability of inhibiting the proliferation and functions of various immunocytes, including T lymphocytes, B lymphocytes, dendritic cells, macrophages, natural killer cells and neutrophils, suppressing the differentiation of naive CD4+ T cells into proinflammatory Th1 and Th17 cell lineages and promoting the generation of Treg cells [3, 29, 30]. Here, the P5 UC-MSCs did exhibit both features, which were consistent with previous findings.
MSCs release cytokines into the microenvironment through autocrine and paracrine to maintain living conditions. The microenvironment, in turn, conducts variation of the environment in the whole body through changes in metabolism, secretion, immunity and functions, limiting and affecting the occurrence and development of MSCs . However, traditional 2D culture cannot mimic the in vivo architecture and microenvironment well. Therefore, 2D cultured MSCs display many characteristics of replicative senescence compared with in vivo MSCs [1, 32]. Furthermore, senescent cells secrete IL-6 and IL-8, which act in an autocrine and a paracrine way to reinforce senescence . In this study, compared with P5 UC-MSCs, when P15 UC-MSCs were co-cultured with activated T cells, it showed enhanced T cell proliferation, increased IL-6 and decreased IL-10 and TGF-β, which may be the reasons for the decrescent therapeutic effects of 2D cultured UC-MSCs.
Increasing studies have explored drugs that can reverse the replicative senescence of MSCs. For example, reduced glutathione (GSH) and melatonin displayed an anti-senescent effect in MSCs and preserved stem cell functions including cell migration, stemness, and multidirectional differentiation potential through reducing reactive oxygen species (ROS) generation during long-term in vitro expansion . Imperfection of 2D culture has also inspired the emergence of 3D cell culture systems. Up to now, various types of architectures and biomaterials that support 3D cell proliferation have been reported. For one instance, TableTrix™ (Cytoniche) is a commercially available microcarrier based on macroporous and elastic gelatin . Hydrogels, a common material form, have also been successfully used to support 3D cell growth in vitro, such as Extracell™ (Dextran Biological System) . However, existing drugs or products have mostly focused on retarding the senescence phenotype of MSCs and promoting the proliferation and differentiation in the process of long-term expansion, while the improvement of immunomodulatory ability, a hinge of boosting the therapeutic effects of MSCs, has not received much attention.
In a 3D culture system, cells can better communicate with neighboring cells, and the cell-to-cell and cell-to-matrix connections are reflected in the culture environment, which are consistent with the in vivo microenvironment [37, 38]. Moreover, 3D cultured MSCs significantly maintain elevated expression levels of stemness genes, yield high frequencies of colony-forming units and show enhanced osteogenic and adipogenic differentiation efficiency . One research also indicated that the expressions of HLA-G, IDO1, PTGS2 and TGF-β1, which were closely related to immune regulation, were higher in 3D culture than those in 2D culture. Further, 3D cultured MSCs could inhibit the proliferation of activated lymphocytes more effectively . Here, we compared the stemness and immunomodulatory ability between 2D and 3D UC-MSCs and found that 3D culture did preserve the stemness, T cell proliferative inhibition and the expression of anti-inflammatory cytokines such as IL-10 and TGF-β of long-passaged UC-MSCs. Moreover, we observed that 3D cultured UC-MSCs at P5 or P15 was more effective in treating T1DM and colitis mice than those in 2D culture, adding to the current knowledge of 3D cultured MSCs for the treatment of immune disorders.
Most notably, the morphology of MSCs in the 3D cultured systems markedly differ from those on 2D surfaces throughout the cultivation process , and the morphology of MSCs can be affected by a variety of mechanical properties, including elasticity, geometry and adhesion, which will ultimately affect the tension of the cytoskeleton . It has been widely accepted that the transcriptional effector of the hippo pathway-the YAP/transcriptional coactivator with PDZ-binding motif (TAZ) acts as a sensor and mediator of mechanical signals in response to extracellular matrix cues [42, 43] Meanwhile, YAP signaling is strongly activated during the culture of cells in the matrigel-encapsulated 3D culture system and maintains the proliferative capacity of the encapsulated cells . In line with these phenomena, we found the YAP expression both of P5 and P15 UC-MSCs in 3D culture was significantly higher than those in 2D culture. Furthermore, we demonstrated that mechanical stress played a significant role in maintaining immunoregulatory function of 2D and 3D cultured UC-MSCs by modulating YAP pathway, and PGE2 could be used to up-regulate YAP and improve the immunomodulatory ability of long-passaged 2D cultured UC-MSCs, providing theoretical supports for the regulation of cellular immunity and therapeutic efficacy by adjusting mechanical stress.