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Background: Mesenchymal stromal cells (MSCs) are used in over 800 clinical trials mainly due to their immune inhibitory activity. Umbilical cord (UC), the second leading source of clinically used MSCs, is usually cut in small tissue pieces. Subsequent cultivation leads to a continuous outgrowth of MSC explant monolayers (MSC-EMs) for months. Currently, the first MSC-EM culture takes approximately 2 weeks to grow out, which is then expanded and applied to patients. The initiating tissue pieces are then discarded. However, when UC pieces are transferred to new culture dishes, MSC-EMs continue to grow out. In case the functional integrity of these cells is maintained, later induced cultures could also be expanded and used for cell therapy. This would drastically increase the number of available cells for each patient. To test the functionality of MSC-EMs from early and late induction time points, we compared the first cultures to those initiated after 2 months by investigating their clonality and immunomodulatory capacity., Methods: We analyzed the clonal composition of MSC-EM cultures by umbilical cord piece transduction using integrating lentiviral vectors harboring genetic barcodes assessed by high-throughput sequencing. We investigated the transcriptome of these cultures by microarrays. Finally, the secretome was analyzed by multiplexed ELISAs, in vitro assays, and in vivo in mice., Results: DNA barcode analysis showed polyclonal MSC-EMs even after months of induction cycles. A transcriptome and secretome analyses of early and late MSC cultures showed only minor changes over time. However, upon activation with TNF-α and IFN-γ, cells from both induction time points produced a multitude of immunomodulatory cytokines. Interestingly, the later induced MSC-EMs produced higher amounts of cytokines. To test whether the different cytokine levels were in a therapeutically relevant range, we used conditioned medium (CM) in an in vitro MLR and an in vivo killing assay. CM from late induced MSC-EMs was at least as immune inhibitory as CM from early induced MSC-EMs., Conclusion: Human umbilical cord maintains a microenvironment for the long-term induction of polyclonal and immune inhibitory active MSCs for months. Thus, our results would offer the possibility to drastically increase the number of therapeutically applicable MSCs for a substantial amount of patients.

Unlabelled: Mesenchymal stem (or stromal) cells (MSCs) have been used in more than 400 clinical trials for the treatment of various diseases. The clinical benefit and reproducibility of results, however, remain extremely variable. During the in vitro expansion phase, which is necessary to achieve clinically relevant cell numbers, MSCs show signs of aging accompanied by different contributions of single clones to the mass culture. Here we used multicolor lentiviral barcode labeling to follow the clonal dynamics during in vitro MSC expansion from whole umbilical cord pieces (UCPs). The clonal composition was analyzed by a combination of flow cytometry, fluorescence microscopy, and deep sequencing. Starting with highly complex cell populations, we observed a massive reduction in diversity, transiently dominating populations, and a selection of single clones over time. Importantly, the first wave of clonal constriction already occurred in the early passages during MSC expansion. Consecutive MSC cultures from the same UCP implied the existence of more primitive, MSC culture-initiating cells. Our results show that microscopically homogenous MSC mass cultures consist of many subpopulations, which undergo clonal selection and have different capabilities. Among other factors, the clonal composition of the graft might have an impact on the functional properties of MSCs in experimental and clinical settings., Significance: Mesenchymal stem cells (MSCs) can easily be obtained from various adult or embryonal tissues and are frequently used in clinical trials. For their clinical application, MSCs have to be expanded in vitro. This unavoidable step influences the features of MSCs, so that clinical benefit and experimental results are often highly variable. Despite a homogenous appearance under the microscope, MSC cultures undergo massive clonal selection over time. Multicolor fluorescence labeling and deep sequencing were used to demonstrate the dynamic clonal composition of MSC cultures, which might ultimately explain the variable clinical performance of the cells., (©AlphaMed Press.)

Background: Mesenchymal stromal cells (MSCs) are excessively investigated in the context of inflammation-driven diseases, but the clinical results are often moderate. MSCs are naturally activated by inflammatory signals, which lead to the secretion of immune inhibitory factors in inflamed tissues. Many work groups try to improve the therapeutic outcome of MSCs by genetic modification and the constitutive overexpression of immune modulatory transgenes. However, the ectopic secretion of immune inhibitory transgenes increases the chances of infections, and constitutive transgene expression is not necessary for chronic diseases undergoing different inflammatory stages. Methods: We designed and tested inflammation-induced promoters to control transgene expression from integrating lentiviral vectors in human umbilical cord MSCs. Therefore, we investigated different combinations of general transcription factor elements to achieve a minimal promoter with low basal activity. The best candidates were combined with interferon-induced GAS or ISRE DNA motifs. The constructs with the highest transgene expression upon addition of pro-inflammatory cytokines were compared to vectorized promoters from inflammation-induced genes (CD317, CXCL9, CXCL10, CXCL11 and IDO1). Finally, we investigated IL10 as a potential immune inhibitory transgene by transcriptome analyses, ELISA and in an acute lung injury mouse model. Results: The synthetic promoters achieved a high and specific transgene expression upon IFN-γ addition. However, the CXCL11 promoter showed synergistic activity upon IFN-γ, TNF-α and IL1-β treatment and surpassed the transgene expression height of all tested promoters in the study. We observed in transcriptome analyses that IL10 has no effect on MSCs and in ELISA that IL10 is only secreted by our genetically modified and activated CXCL11-IL10-MSCs. Finally, transplanted CXCL11-IL10-MSCs increased CD19+ and CD4+ lymphoid cells, and decreased CD11b+ Ly6g myeloid cells in an ALI mouse model. Conclusion: These results provide new insights into MSC inflammatory activation and the subsequent translation into a tool for a tailored expression of transgenes in inflammatory microenvironments. The newly developed promoter elements are potentially interesting for other inflamed tissues, and can be combined with other elements or used in other cell types. [ABSTRACT FROM AUTHOR]

Objectives: Preeclampsia is one of the main contributers to maternal and fetal morbidity and mortality during pregnancy. A history of preeclampsia puts mother and offspring at an increased cardiovascular risk in later life. We hypothesized that at the time of birth functional impairments of fetal endothelial cells can be detected in pregnancies complicated by preeclampsia and that a therapeutic intervention using 1,25 (OH)2 vitamin D3 can reverse the adverse effects of preeclampsia on cell function. Methods: Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords obtained from preeclamptic (N = 12) and uncomplicated pregnancies (N = 13, control). Placental villous tissue fragments from uncomplicated term pregnancies were incubated in explant culture for 48 h at 2% (hypoxia), 8% or 21% O2. Explant conditioned media (CM) was collected and pooled according to oxygen level. We compared the ability of preeclampsia vs. control HUVEC to migrate, proliferate, and form tubule-like networks in a Matrigel assay, in the presence/absence of CM and 1,25(OH)2 vitamin D3. Results: HUVEC from preeclamptic pregnancies showed reduced migration (P = 0.04) and tubule formation (P = 0.04), but no change in proliferation (P = 0.16) compared to healthy pregnancies. Placental villous explant CM derived from 2% O2 incubations significantly reduced HUVEC migration, when compared to non-CM (P = 0.04). Vitamin D3 improved HUVEC function in neither of the groups. There was no significant difference in VEGF gene expression between healthy and preeclamptic pregnancies and no effect of Vitamin D3 on VEGF expression. Conclusions: Reduced functional abilities of fetal endothelial cells from preeclamptic pregnancies suggests that disease pathways, possibly originating from the dysfunctional placenta, negatively impact fetal endothelium. The neutral effect of 1,25(OH)2 vitamin D3 contrasts with previous findings that vitamin D rescues the poor migration, proliferation and tubule formation exhibited by cord blood fetal endothelial progenitor cells from preeclamptic pregnancies. Further investigations to distinguish pathways by which offspring exposed to preeclampsia are at risk for cardiovascular disease are needed. [ABSTRACT FROM AUTHOR]

Following allogeneic hematopoietic stem cell transplantation (HSCT), interferon (IFN)-γ levels in the recipient's body can strongly influence the clinical outcome. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are lucrative as biological toleranceinducers in HSCT settings. Hence, we studied the molecular mechanism of how UC-MSCs influence natural killer (NK) cell-mediated IFN-γ production. Allogeneic NK cells were cultured in direct contact with UC-MSCs or cell-free supernatants from mesenchymal stem cell (MSC) cultures (MSC-conditioned media). We found that soluble factors secreted by UC-MSCs strongly suppressed interleukin (IL)-12/IL-18-induced IFN-γ production by NK cells by reducing phosphorylation of STAT4, NF-κB, as well as T-bet activity. UC-MSCs secreted considerable amounts of activin-A, which could suppress IFN-γ production by NK cells. Neutralization of activin-A in MSC-conditioned media significantly abrogated their suppressive abilities. Till date, multiple groups have reported that prostaglandin (PG)-E2 produced by MSCs can suppress NK cell functions. Indeed, we found that inhibition of PGE2 production by MSCs could also significantly restore IFN-γ production. However, the effects of activin-A and PGE2 were not cumulative. To the best of our knowledge, we are first to report the role of activin-A in MSC-mediated suppression of IFN-γ production by NK cells. [ABSTRACT FROM AUTHOR]

Background Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells. Results UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via the soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity. Conclusion Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell -MSC crosstalk. [ABSTRACT FROM AUTHOR]