Mesenchymal stromal cells (MSC) have been used in over 800 clinical trials with encouraging results in the field of transplant medicine and chronic inflammatory diseases. Today, Umbilical Cord (UC)-derived MSC are the second leading source used for clinical purposes, mainly due to its easy access and superior immune modulatory effects. Although the underlying molecular mechanisms of immune suppressive activities have not been fully understood, research over the last decade strongly suggests that MSC-mediated benefits are closely related to activation of secretome networks. Nevertheless, recent findings also point to cytokine-independent mechanisms as key players of MSC-mediated immune modulation. Here, we set up a robust in vitro immune assay using phytohemagglutinin-or anti-CD3/CD28treated human peripheral blood mononuclear cells in cell-to-cell interaction or in cellcontact independent format with UC-MSC and conducted integrated transcriptome and secretome analyses to dissect molecular pathways driving UC-MSC-mediated immune modulation. Under inflammatory stimuli, multiparametric analyses of the secretome led us to identify cytokine/chemokine expression patterns associated with the induction of MSC-reprogrammed macrophages and T cell subsets ultimately leading to immune suppression. UC-MSC transcriptome analysis under inflammatory challenge allowed the identification of 47 differentially expressed genes, including chemokines, antiand pro-inflammatory cytokines and adhesion molecules found also in UC-MSCimmunosupressive secretomes, including the novel candidate soluble IL-2R. This study enabled us to track functionally activated UC-MSC during immune suppression and opened an opportunity to explore new pathways involved in immunity control by UC-MSC. We propose that identified immunomodulatory molecules and pathways could potentially be translated into clinical settings in order to improve UC-MSC-therapy quality and efficacy.
Umbilical cord mesenchymal stromal cells (UC-MSC) are promising candidates for cell therapy due to their potent multilineage differentiation, enhanced self-renewal capacity, and immediate availability for clinical use. Clinical experience has demonstrated satisfactory biosafety profiles and feasibility of UC-MSC application in the allogeneic setting. However, the use of UC-MSC for bone regeneration has not been fully established. A major challenge in the generation of successful therapeutic strategies for bone engineering lies on the combination of highly functional proosteogenic MSC populations and bioactive matrix scaffolds. To address that, in this study we proposed a new approach for the generation of bone-like constructs based on UC-MSC expanded in human platelet lysate (hPL) and evaluated its potential to induce bone structures in vivo. In order to obtain UC-MSC for potential clinical use, we first assessed parameters such as the isolation method, growth supplementation, microbiological monitoring, and cryopreservation and performed full characterization of the cell product including phenotype, growth performance, tree-lineage differentiation, and gene expression. Finally, we evaluated bone-like constructs based on the combination of stimulated UC-MSC and collagen microbeads for in vivo bone formation. UC-MSC were successfully cultured from 100% of processed UC donors, and efficient cell derivation was observed at day 14 ± 3 by the explant method. UC-MSC maintained mesenchymal cell morphology, phenotype, high cell growth performance, and probed multipotent differentiation capacity. No striking variations between donors were recorded. As expected, UC-MSC showed tree-lineage differentiation and gene expression profiles similar to bone marrow- and adipose-derived MSC. Importantly, upon osteogenic and endothelial induction, UC-MSC displayed strong proangiogenic and bone formation features. The combination of hPL-expanded MSC and collagen microbeads led to bone/vessel formation following implantation into an immune competent mouse model. Collectively, we developed a high-performance UC-MSC-based cell manufacturing bioprocess that fulfills the requirements for human application and triggers the potency and effectivity of cell-engineered scaffolds for bone regeneration.
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