The immunosuppressive activity of mesenchymal stromal cells (MSC) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSC are undetectable following administration. Therefore, understanding the fate of infused MSC could help to predict clinical responses. Using a murine model of graft-versus-host disease (GvHD) we demonstrate that MSC are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSC we found a striking parallel, whereby only those with high cytotoxic activity against MSC responded to MSC infusion whereas those with low activity did not. Importantly, the need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSC generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSC and produce indoleamine 2,3-dioxygenase (IDO) that is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSC or that all patients could be treated with ex vivo apoptotic MSC.

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

Bone marrow microenvironment is fundamental for hematopoietic homeostasis. Numerous efforts have been made to reproduce or manipulate its activity to facilitate engraftment after hematopoietic stem cell transplantation but clinical results remain unconvincing. This probably reflects the complexity of the hematopoietic niche. Recent data have demonstrated the fundamental role of stromal and myeloid cells in regulating hematopoietic stem cell self-renewal and mobilization in the bone marrow. In this study we unveil a novel interaction by which bone marrow mesenchymal stromal cells induce the rapid differentiation of CD11b+ myeloid cells from bone marrow progenitors. Such an activity requires the expression of nitric oxide synthase-2. Importantly, the administration of these mesenchymal stromal cell-educated CD11b+ cells accelerates hematopoietic reconstitution in bone marrow transplant recipients. We conclude that the liaison between mesenchymal stromal cells and myeloid cells is fundamental in hematopoietic homeostasis and suggests that it can be harnessed in clinical transplantation.