ABSTRACT
Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.
ABSTRACT
Bone formation during embryogenesis is driven by interacting osteogenesis and angiogenesis with parallel endothelial differentiation. Thence, all in vitro bioengineering techniques are aimed at pre-vascularization of osteogenic bioequivalents to provide better regeneration outcomes upon transplantation. Due to appearance of cell-cell and cell-matrix interactions, 3D cultures of adipose-derived stromal cells (ADSCs) provide a favorable spatial context for the induction of different morphogenesis processes, including vasculo-, angio-, and osteogenesis and, therefore, allow modeling their communication in vitro. However, simultaneous induction of multidirectional cell differentiation in spheroids from multipotent mesenchymal stromal cells (MMSCs) was not considered earlier. Here we show that arranging ADSCs into spheroids allows rapid and spontaneous acquiring of markers of both osteo- and angiogenesis compared with 2D culture. We further showed that this multidirectional differentiation persists in time, but is not influenced by classical protocols for osteo- or angio-differentiation. At the same time, ADSC-spheroids retain similar morphology and microarchitecture in different culture conditions. These findings can contribute to a better understanding of the fundamental aspects of autonomous regulation of differentiation processes and their cross-talks in artificially created self-organizing multicellular structures. This, in turn, can find a wide range of applications in the field of tissue engineering and regeneration.
