Extracellular Vesicles From Liver Progenitor Cells Downregulates Fibroblast Metabolic Activity and Increase the Expression of Immune-Response Related Molecules

Extracellular vesicles are an important part of cell to cell crosstalk, and help to the acceptor cells to prepare for changes in their environment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with cell fate signaling. In the current work, we have fed 3T3-L1 mouse fibroblast with those EVs, and then analyzed the cellular response by proteomics and transcriptomics. Results showed a downregulation of both proteins and transcripts related to proliferative and metabolic routes dependent on TGFb, and an increase on the ERBB2 inhibitor protein (ERBIN). An increase in the transcription of CXCL2, as well as the increase of ribosome biogenesis and interferon response molecules points toward the activation of immune system signaling, compatible with a response against tissue damage. Overall design: Approximately 2 million of 3T3-L1 cells were treated with the EVs obtained from 200 million of MLP29 cell line. On average, this represents 20 ug of protein (measured by Bradford) coming from 8.9*E10 particles (measured with Nanosight LM10) for treatment. In total 6 plates were treated, with three different preparations of EVs and 3 preparations of control (the very same procedure of EV isolation over a non conditioned-media, to rule out effects associated to the culture media itself), during 24 hours, in 25 mM HEPES-containing complete DMEM medium (contaminating vesicles were first removed by overnight centrifugation at 110000×g). At the end, plates were washed twice with PBS, raised with Tryple (GIBCO) and then divided equally for proteomic and transcriptomics analysis. An aliquot was employed to measure cell viability, higher than 90% in all the cases. The trypsin was removed after pellet the cells 1000xg 5 min, frozen in dry ice and stored at -80ºC until processing.