Unraveling TNFalpha‐stimulated gene 6 (TSG‐6) function in switching stemness and biological properties of mesenchymal stem cells
Authors: Romano Barbara , Petti Luciana , Mascaraque Cristina , Lucchetti Donatella , Sgambato Alessandro , Danese Silvio , Vetrano Stefania 
Published: November 2023

Biological and functional features of mesenchymal stem cells (MSCs) are not fully depicted to date, despite their promising use for several immune diseases. TSG‐6 is a crucial protein responsible of the anti‐inflammatory properties of MSCs in several models of in vivo inflammation. However, whether and how TSG‐6 affects MSC biology and activities has never been investigated. Aim of this study was to unravel the biological role of TSG‐6 in MSCs. MSCs were isolated from bone marrow of wild type (WT) or TSG‐6 deficient mice, cultured for 4 weeks and then sorted for Sca‐1+, CD31‐, cocktail lineage‐surface markers. WT and TSG‐6 MSCs deficient cells were successfully obtained to compare their morphological features as well as their ability in: proliferation, forming colonies, migration, differentiation and in maintaining their immunosoppressive activity. To these purposes, cell morphology was assessed by confocal F‐actin immunostaining and extracellular vesicles were extracted by ultracentrifugation and characterized by combining dynamic light scattering and Transmission Electron Microscopy. Cell proliferation was analyzed by [3H]thymidine incorporation assay, MSC colonies were revealed by Giemsa staining and cell migration by using scratch assay analysis. The adipogenic and osteogenic differentiation was revealed by the Oil Red O staining and van Kossa staining as well as by the quantification, using RT‐PCR, of the transcription factors for the differentiation genes (PPARg2 and RUNX). Finally the immunosuppressive activity was evaluated by using [3H] thymidine after co‐culture of MSCs with activated CD3‐CD28 primary splenocytes.

MSCs lacking of TSG‐6 displayed different cell shape characterized by an enlarged cytoplasm and dissimilar actin‐cytoskeleton organization compared to MSC WT cells. Furthermore, the analysis of extracellular vesicles showed a significant reduced size of extracellular vesicles in MSC TSG‐6 deficient cells. Interestingly, the loss of TSG‐6 decreased dramatically cell proliferative rate, survival, colony forming unit capacities and wound healing compared to WT cells. On the other hand, despite the positivity to stemness markers such as Sca+1, MSCs lacking of TSG‐6 failed to differentiate into adipocytes and osteocytes. In fact, the levels of PPARg2 and RUNX were undetermined after 14 days of differentiation in MSCs lacking of TSG‐6, whereas highly expressed in MSC WT. Finally, MSC TSG‐6 deficient cells maintained, even though with less efficiency compared to WT cells, immunosuppressive activity. Taken together, these data demonstrated that TSG‐6 has a crucial role in maintaining stemness and regulating biological properties of MSCs. The loss of TSG‐6 promoted the differentiation of MSC into fibroblast‐like cells with reduced immunosuppressive properties and loss of regenerative capacities.

Support or Funding Information

GR‐2009 to SV