Effects of Atrazine exposure on human bone marrow-derived mesenchymal stromal cells assessed by combinatorial assay matrix.

Introduction: Mesenchymal Stromal/Stem cells (MSCs) are an essential component of the regenerative and immunoregulatory stem cell compartment of the human body and thus of major importance in human physiology. The MSCs elicit their beneficial properties through a multitude of complementary mechanisms, which makes it challenging to assess their phenotype and function in environmental toxicity screening. We here employed the novel combinatorial assays matrix approach/technology to profile the MSC response to the herbicide Atrazine, which is a common environmental xenobiotic, that is in widespread agricultural use in the US and other countries, but banned in the EU. Our here presented approach is representative for screening the impact of environmental xenobiotics and toxins on MSCs as an essential representative component of human physiology and well-being. Methods: We here employed the combinatorial assay matrix approach, including a panel of well standardized assays, such as flow cytometry, multiplex secretome analysis, and metabolic assays, to define the phenotype and functionality of human-donor-derived primary MSCs exposed to the representative xenobiotic Atrazine. This assay matrix approach is now also endorsed for characterization of cell therapies by leading regulatory agencies, such as FDA and EMA. Results: Our results show that the exposure to Atrazine modulates the metabolic activity, size, and granularity of MSCs in a dose and time dependent manner. Intriguingly, Atrazine exposure leads to a broad modulation of the MSCs secretome (both upregulation and downmodulation of certain factors) with the identification of Interleukin-8 as the topmost upregulated representative secretory molecule. Interestingly, Atrazine attenuates IFNγ-induced upregulation of MHC-class-II, but not MHC-class-I, and early phosphorylation signals on MSCs. Furthermore, Atrazine exposure attenuates IFNγ responsive secretome of MSCs. Mechanistic knockdown analysis identified that the Atrazine-induced effector molecule Interleukin-8 affects only certain but not all the related angiogenic secretome of MSCs. Discussion: The here described Combinatorial Assay Matrix Technology identified that Atrazine affects both the innate/resting and cytokine-induced/stimulated assay matrix functionality of human MSCs, as identified through the modulation of selective, but not all effector molecules, thus vouching for the great usefulness of this approach to study the impact of xenobiotics on this important human cellular subset involved in the regenerative healing responses in humans.

Contrariety of Human Bone Marrow Mesenchymal Stromal Cell Functionality in Modulating Circulatory Myeloid and Plasmacytoid Dendritic Cell Subsets.

Mesenchymal Stromal Cells (MSCs) derived from bone marrow are widely tested in clinical trials as a cellular therapy for potential inflammatory disorders. The mechanism of action of MSCs in mediating immune modulation is of wide interest. In the present study, we investigated the effect of human bone-marrow-derived MSCs in modulating the circulating peripheral blood dendritic cell responses through flow cytometry and multiplex secretome technology upon their coculture ex vivo. Our results demonstrated that MSCs do not significantly modulate the responses of plasmacytoid dendritic cells. However, MSCs dose-dependently promote the maturation of myeloid dendritic cells. Mechanistic analysis showed that dendritic cell licensing cues (Lipopolysaccharide and Interferon-gamma) stimulate MSCs to secret an array of dendritic cell maturation-associated secretory factors. We also identified that MSC-mediated upregulation of myeloid dendritic cell maturation is associated with the unique predictive secretome signature. Overall, the present study demonstrated the dichotomy of MSC functionality in modulating myeloid and plasmacytoid dendritic cells. This study provides clues that clinical trials need to investigate if circulating dendritic cell subsets in MSC therapy can serve as potency biomarkers.

Conglomeration of T- and B-Cell Matrix Responses Determines the Potency of Human Bone Marrow Mesenchymal Stromal Cells.

Cell manufacturing facilities need to define the potency of mesenchymal stromal cells (MSCs) as cellular therapeutics in advanced clinical trials or marketing approval. Since MSCs' mechanism of action in humans is not well defined, more than a single functional property of MSCs needs to be captured as a surrogate measure of potency utilizing assay matrix technologies. However, the current limitation is the sole investigation of MSC-mediated T-cell suppression as a surrogate measure of potency. We investigated the effect of MSCs on B-cell matrix responses to be incorporated into the assay matrix potency analytical system. Our results demonstrate that MSCs inhibit B-cell differentiation and block pan-antibody secretion upon activation of B cells in the PBMCs. In contrast, MSCs are inferior in blocking B-cell matrix responses when purified B cells are used. Mechanistic analysis has demonstrated that MSC-mediated inhibition of B-cell matrix responses is non-contact dependent and Tryptophan metabolic pathway plays a major role, akin to the mechanism of MSC-mediated T-cell suppression. MSCs also inhibit both T-cell and B-cell responses when both of these lymphoid populations are concurrently activated in the PBMCs. Secretome analysis of MSC and T/B cell-activated PBMC cocultures identified direct and inverse correlative matrix signatures between humoral antibody isotypes and secretory molecules. The current analysis of the combined and concomitant investigation of T-cell and B-cell matrix responses fulfills the potency assay matrix strategy by incorporating MSCs' interaction with more than a single inflammatory immune responder.