Background and aims: Adipose tissue is a source of multipotent adipose stromal/stem cells (ASCs). ASCs have excellent proliferation and multilineage differentiation capacity, low immunogenicity and promising immunosuppressive capacity. The immunosuppressive properties of ASCs have been previously studied but detailed intracellular mechanisms are still unknown. The aim of this study was to investigate the suppressive potential of ASCs in direct versus indirect co-culture with peripheral blood mononuclear cells (PBMCs) and to identify the signaling pathways (STAT3, STAT1, NF-κB and Smad1/5) that are activated during ASC-mediated immunosuppression.

Methods: Four different ASC donors were used to study the immunosuppressive capacity of ASCs. Mixed lymphocyte reactions (MLR) using direct and indirect co-cultures of ASCs and PBMCs were used to study the immunosuppressive capacity of ASCs. Immunosuppression was analyzed using BrdU-ELISA. Activation of intracellular signaling pathways were analyzed using Western Blot analysis from direct and indirect MLR samples.

Results: Strong immunosuppression on PBMC proliferation was obtained with three ASC donors. ASCs in direct co-culture with PBMCs had stronger immunosuppressive capacity compared to indirect co-cultures. ASCs cultured with MLR2 combination possessed stronger immunosuppressive capacity compared to MLR1 combination. There was variation in the activation of intracellular signaling pathways between different donors. Two signaling pathways (NF-κB and Smad1/5) were activated only in direct reactions compared to pathways STAT3 and STAT1, which were activated also in indirect reactions. NF-κB phosphorylation was inhibited in ASCs in direct reactions. Activation of Smad1/5 was donor-specific and it was activated with other ASC donor and inhibited with another ASC donor. In direct reactions STAT3 phosphorylation was inhibited and in indirect reactions STAT3 was mainly activated. STAT1 was phosphorylated in PBMCs with three ASCs donors and in two ASC donors in indirect reactions.

Conclusion: ASCs have immunosuppressive capacity when co-cultured with PBMCs in both direct and indirect cultures. More studies with more ASC and PBMC donors are needed to obtain more detailed information about intracellular signaling behind the immunosuppression of ASCs.