Improving the therapeutic profile of MSCs: Cytokine priming reduces donor-dependent heterogeneity and enhances their immunomodulatory capacity

Jaris Valencia ^1,2,3 Rosa M Yanez ^2,4,5 SANDRA MUNTION OLAVE ^2,6,7* Maria Fernandez-Garcia ^2,4,5 Jorge Diego Martín-Rufino ^8,9 Agustin G Zapata ^10,11,2 Juan A Bueren ^2,4,5 Angeles Vicente ^11,2,3 Fermin Sanchez-Guijo ^2,6,7

• ¹ Health Research Institute of the Hospital Clínico San Carlos (IdISSC), Madrid, Madrid, Spain
• ² RICORS TERAV, Spanish Network for Advanced Therapies, Carlos III Health Institute (ISCIII), Madrid, Asturias, Spain
• ³ Department of Cell Biology, School of Medicine, Complutense University of Madrid, Madrid, Asturias, Spain
• ⁴ Health Research Institute Foundation Jimenez Diaz (IIS-FJD), Madrid, Madrid, Spain
• ⁵ Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Asturias, Spain
• ⁶ Regenerative Medicine and Cellular Therapy Network Center of Castilla y León, Salamanca, Spain
• ⁷ Department of Medicine, University of Salamanca and Cell Therapy Area and Hematology Department, IBSAL-University Hospital of Salamanca, Salamanca, Spain
• ⁸ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, United States
• ⁹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, United States
• ¹⁰ Department of Cell Biology, Faculty of Biology, Complutense University of Madrid, Madrid, Spain
• ¹¹ (I+12), Research Institute Hospital 12 de Octubre, Madrid, Catalonia, Spain

MSCs exhibit regenerative, anti-inflammatory and immunomodulatory properties due to the large amount of cytokines, chemokines and growth factors they secrete. MSCs have been extensively evaluated in clinical trials, however, in some cases their therapeutic effects are variable. Therefore, strategies to improve their therapeutic potential, such as preconditioning with proinflammatory factors, have been proposed. Several priming approaches have provided non-conclusive results, and the duration of priming effects on MSC properties or their response to a second inflammatory stimulus have not been fully addressed. In the current study, we have investigated the impact of triple cytokine priming in MSCs on their characterization and viability, their transcriptomic profile, the functionality of innate and acquired immune cells, as well as the maintenance of the response to priming over time, their subsequent responsiveness to a second inflammatory stimulus. Our results show that priming MSCs with proinflammatory cytokines (CK-MSCs) do not modify the differentiation capacity of MSCs, nor their immunophenotype and viability. Moreover, cytokine priming enhances the antiinflammatory and immunomodulatory properties of MSCs against NK and dendritic cells, while maintaining the same T cell immunomodulatory capacity as unstimulated MSCs. Thus, they decrease T-lymphocytes and NK cell proliferation, inhibit the differentiation and allostimulatory capacity of dendritic cells and promote the differentiation of monocytes with an immunosuppressive profile.In addition, we have shown for the first time that proinflammatory priming reduces the variability between different donors and MSC origins. Finally, the effect on CK-MSC is maintained over time and even after a secondary inflammatory stimulus. In conclusion, cytokine-priming improves the therapeutic potential of MSCs and reduces inter-donor variability.