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Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo -expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank ( n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics., (Copyright © 2019 Andrzejewska, Catar, Schoon, Qazi, Sass, Jacobi, Blankenstein, Reinke, Krüger, Streitz, Schlickeiser, Richter, Souidi, Beez, Kamhieh-Milz, Krüger, Zemojtel, Jürchott, Strunk, Reinke, Duda, Moll and Geissler.)

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro . For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1β, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

The interaction of human mesenchymal stem cells (hMSCs) and tumor cells has been investigated in various contexts. HMSCs are considered as cellular treatment vectors based on their capacity to migrate towards a malignant lesion. However, concerns about unpredictable behavior of transplanted hMSCs are accumulating. In malignant gliomas, the recruitment mechanism is driven by glioma-secreted factors which lead to accumulation of both, tissue specific stem cells as well as bone marrow derived hMSCs within the tumor. The aim of the present work was to study specific cellular interactions between hMSCs and glioma cells in vitro. We show, that glioma cells as well as hMSCs differentially express connexins, and that they interact via gap-junctional coupling. Besides this so-called functional syncytium formation, we also provide evidence of cell fusion events (structural syncytium). These complex cellular interactions led to an enhanced migration and altered proliferation of both, tumor and mesenchymal stem cell types in vitro. The presented work shows that glioma cells display signs of functional as well as structural syncytium formation with hMSCs in vitro. The described cellular phenomena provide new insight into the complexity of interaction patterns between tumor cells and host cells. Based on these findings, further studies are warranted to define the impact of a functional or structural syncytium formation on malignant tumors and cell based therapies in vivo., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Gas chromatography-mass spectrometry (GC-MS) profiles were generated from U87 glioma cells and human mesenchymal stem cells (hMSC). 37 metabolites representing glycolysis intermediates, TCA cycle metabolites, amino acids and lipids were selected for a detailed analysis. The concentrations of these metabolites were compared and Pearson correlation coefficients were used to calculate the relationship between pairs of metabolites. Metabolite profiles and correlation patterns differ significantly between the two cell lines. These profiles can be considered as a signature of the underlying biochemical system and provide snap-shots of the metabolism in mesenchymal stem cells and tumor cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)