ABSTRACT: Mesenchymal stem cells (MSCs) exhibit immunosuppressive properties. However the homing mechanisms underlying MSC trafficking to target tissues remain elusive. Here we report that skin-derived MSCs (S-MSCs) secrete high levels of interleukin-6 (IL-6), inhibit T helper (Th)1- and Th17-cell differentiation, and possess IL-6-dependent immunomodulatory capacity in inflammatory microenvironment. Disruption of the il6 locus or its signaling transducer gp130 blocks voltage-operated calcium (Ca2+) channels (VOCC) critically required for cell contraction involved in the sequential adhesion and de-adhesion events during S-MSC migration. IL-6 directly regulates CaV2.3 encoded by cacna1e of R-type Ca2+ channels through the JAK3/STAT3 signaling pathway. Deletion of il6 or silencing CaV2.3 gene expression leads to a severe defect in S-MSC’s homing and immunosuppressive function in vivo. Thus, this unexpected requirement of autocrine IL-6 for activating CaV2.3 Ca2+ channels uncovers a previously unrecognized link between IL-6 signaling and the VOCC, and provides novel mechanistic insights for the immunomodulatory activities of S-MSCs. A two chip study using total RNA recovered from three mixed S-MSCs derived from WT mice and three mixed S-MSCs derived from IL6KO mice.
Project description:MSCs are inherently tumor-homing and immunosuppressive and can be isolated, cultured, expanded, and transduced, making them viable candidates for cell therapy. MSCs can also be useful in allogeneic transplantation because of their immunocompatibility. MSCs have the capacity to home specifically to tumors including gliomas and breast, colon, ovarian, and lung carcinomas, among many other primary and metastatic tumors. Some discrepancies are however present regarding the mechanism and the involvement of molecules/receptors in MSC homing to tumours. We have used in this study a combination of genome expression profiling and cytokine arrays to screen for candidates mediating MSC homing to two different cancer cell lines: A549 and MDAMB231. We found a variety of interleukines and cytokines already described as players in the process, such as IL6, IL8, CCL2. Additionally, from in vitro migration and invasion assays, we show that CXCR4 is a major player in this mechanism being the essential MSC receptor for the process to occur. For the first time, we have identified in this study a novel axis: MIF-CXCR4, showing a physical interaction between them and validating their essential role in vitro and in vivo. A better understanding of MSC homing players towards tumours will help the development of novel strategies in their use as vehicles in cancer cell therapy. Overall design: Total RNA obtained from mesenchymal stem cells up to passage 5 stimulated or not with conditioned medium from MDAMB231 cell line or A549 cell line for 24h
Project description:Bone marrow mesenchymal stromal cells (MSCs) regulate homeostasis and trafficking of cells of the blood lineage. In response to traumatic injury or infection, MSCs are believed to mobilize from the bone marrow, but it is largely unknown how egress into circulation impacts MSC function. Here we show that biomechanical forces associated with trafficking of MSCs from the bone marrow into the vasculature contribute uniquely to genetic signaling that reinforces MSC repression of immune cell activation. Laminar wall shear stress (LSS) typical of fluid frictional forces present on the lumen of arterioles stimulates increases in antioxidant and anti-inflammatory mediators, as well as an array of chemokines capable of immune cell recruitment. Importantly, LSS promotes a signaling cascade through COX2 that elevates prostaglandin E2 (PGE2) biosynthesis, permitting MSCs to suppress immune cell activation in the presence of inflammatory cues. Pharmacological inhibition of COX2 depleted PGE2 and impaired the ability of MSCs to block tumor necrosis factor-α (TNF-α) production, supporting a key role for PGE2 in the MSC immunomodulatory response to LSS. Preconditioning of MSCs by LSS ex vivo was an effective means of enhancing therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased numbers of apoptotic and M1-type activated microglia in the hippocampus and by retention of endogenous MSCs in the bone marrow. We conclude that biomechanical forces provide critical cues to MSCs residing at the vascular interface which influence MSC immunomodulatory and paracrine functions, thus providing unique opportunities for functional enhancement of MSCs used in therapeutic applications. Overall design: We hypothesized that fluid shear stress caused by vascular flow might alter mesenchymal stromal cell (MSC) signaling and function. To mimic arterial force at the vascular wall, we cultured human bone marrow-derived MSCs in microfluidics capable of producing uniform laminar flow at a wall shear stress (LSS) of 15 dyne/cm^2. Bone marrow MSCs were derived from whole bone marrow from independent human donors (AllCells). Cells were passaged into microfluidic IBIDI channels (μ-Slide VI 0.4) at a density of 3x10^6 cells/ml and permitted to attach to the culture surface for 18 hours. Following attachment, a peristaltic pump (REGLO analog MS4/12, Ismatec) was used to produce LSS of 15 dyne/cm2 for 6 hours. Lysis for RNA isolation was conducted immediately after LSS within the microfluidics.
Project description:Mesenchymal stromal cells (MSCs) with regenerative and immunomodulatory potential are being investigated as a potential therapeutic tool for cartilage lesions. MSCs express a wide variety of bioactive molecules including cytokines, trophic factors, and proteases, which act in a paracrine fashion to modulate the tissue microenvironment. Yet, little is known about the divergence of these signalling molecules between MSCs populations from adult or young tissues. This makes it challenging to decide the optimal source of MSCs for a specific clinical application. In this study, we investigated cell secretomes from cultured human stromal cells harvested from Hoffa’s fat pad (HFPSCs), synovial membrane (SMSCs), umbilical cord (UCSCs) and cartilage (ACs) by quantitative LC-MS/MS proteomics. We also performed multiplex protein arrays and functional assays to compare the constitutive immunomodulatory capabilities of different MSCs. Proteins involved in extracellular matrix degradation and inflammation such as MMPs, IL-17, and complement factors were significantly downregulated in UCSCs compared to other cell types. Additionally, we found enhanced expression of TGF-β1 and PGE2 in UCSCs supernatants. UCSCs were superior in inhibiting peripheral blood mononuclear cells proliferation, migration and TNF-α and IFN-γ secretion compared to ACs, HFPSCs and SMSCs. Although all cell types could repress HLA-DR surface expression and cytokine release by activated macrophages, only UCSCs significantly blocked IL-6 and IL-12 production. Our data demonstrate that stromal cells from umbilical cords display superior anti-inflammatory and immunosuppressive properties than stromal cells from adult tissues. This Allogeneic cell source could potentially be considered as an adjuvant therapy for articular cartilage repair.
Project description:Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton's jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared to BM-MSCs. One human BM-MSC (pooled sample of 4 independent donors), one human WJ-MSC (pooled sample of 3 independent donors), and differentiated osteocytes and adipocytes derived from BM-MSCs after 2 weeks induction.
Project description:Multipotent mesenchymal stromal cells (MSCs) have been shown to promote tissue repair and inhibit inflammatory/autoimmune responses in preclinical and human clinical trials A major problem for MSC-based therapies is the need to expand MSCs in vitro due to the low frequency of MSCs in tissues and the large numbers of cells needed/patient (1-10 x 10^6 cells/kg). The expansion of MSCs is associated with several problems including loss of homing capacity, onset of cellular senescence, decrease in differentiation capacityand susceptibility to genomic instability and malignant transformation, limiting the utility of MSCs as a cell-based therapy. We have recently reported that murine adipose tissue-derived MSCs (mASCs) and human ASCs (hASCs) express glycoprotein A repetitions predominant (GARP)/leucine-rich repeat-containing 32 (LRRC32). GARP binds LAP/TGF-β1 to the surface of mASCs and hASCs, regulating their secretion and activation of TGF-β1 and promoting their immunomodulatory capacity. Interestingly, silencing of GARP in ASCs significantly decreased their proliferative capacity but the mechanisms remain unknown. As the proliferative capacity of MSCs is fundamental for their success in therapy, we have aimed at understanding how GARP modulates the expansion of MSC. Overall design: Human adipose-tissue derived multipotent mesenchymal stromal cells (hASCs) obtained from three unrelated healthy donors were transduced with two distinct LVs, each encoding for a distinct human GARP-specific shRNA (MISSION® shRNA plasmid DNAs, RefSeq: SHCLND-NM_005512; Sigma-Aldrich), referred to as LV#18 (TRCN0000005218) and LV#19 (TRCN0000005219). A MISSION® pLKO.1-puro non-mammalian shRNA control plasmid (Sigma-Aldrich, MO, USA) was used as control (LV-CTRL). Non-transduced (NT) hASCs were also used as control.
Project description:Mesenchymal stromal cells (MSCs) are multipotent stem cells with potent immunosuppressive and trophic support functions. Although bone marrow is considered the golden standard to isolate classical MSCs (BM-MSC), MSC-like cells are currently also derived from other, more easily accessible extra-embryonic tissues such as the umbilical cord. In this study we compared the gene expression profile of human Wharton's jelly explant-derived MSC cultures with two adult MSC populations derived from bone marrow, namely BM-MSC and multipotent adult progenitor cells (MAPC). Here we demonstrate, by using genome wide gene expression analysis, that WJ-MSCs intrinsically have a differential gene expression profile compared to the adult MSCs. Gene ontology analysis revealed an increased expression of genes associated with cell-adhesion, proliferation, and immune system functioning. Furthermore, in comparison to adult MSC, stem cells from the Wharton’s jelly highly express genes involved in neurotrophic support (e.g. LIF, BDNF, NTF3). Such enhanced signatures make WJ-MSC an attractive candidate for cell-based therapy in neurodegenerative and immune-mediated CNS disorders such as multiple sclerosis or amyotrophic lateral sclerosis. Overall design: Umbilical cord Wharton's jelly-derived MSCs (n=5) were obtained after explant tissue culturing and harvested after 3 passages. Bone marrow-derived MSCs (n=5) were monolayer cultured until their third passage and harvested. MAPC (n=5) were cultured as previously described [Reading et al. J Immunol 2013; 190:4542-4552].
Project description:Mesenchymal stromal cells (MSCs) sense and modulate inflammation and represent potential clinical treatment for immune disorders. However, many details of the bidirectional interaction between MSCs and the innate immune comaprtment are still unsolved. Here we describe an unconventional but functional interaction between pro-inflammatory classically activated macrophages (M1MФ) and MSCs, with CD54 playing a central role. CD54 was upregulated and enriched specifically at the contact area between M1MФ and MSCs. Moreover, the specific interaction induced calcium signaling and increased the immunosuppressive capacities of MSCs dependent on CD54 mediation. Our data demonstrate that MSCs can detect an inflammatory microenvironment via a direct and physical interaction with innate immune cells. This finding opens new perspectives for MSC-based cell therapy. Overall design: Human bone-marrow derived mesenchymal stromal cells were co-cultivated with pro (M1MФ) or anti (M2MФ) -inflammatory macrophages. After 24h of co-culture, cells were detached and MSCs and MФ were magnetically separated based on the CD45 expression using the AutoMACS pro separator.
Project description:Activated T cells polarize mesenchymal stromal cells (MSCs) to a proinflammatory Th1 phenotype which likely has an important role in amplifying the immune response in the tumor microenvironment. We investigated the role of interferon gamma (IFN-g) and tumor necrosis factor alpha (TNF-a), two factors produced by activated T cells, in MSC polarization. Gene expression and culture supernatant analysis showed that TNF-a and IFN-g stimulated MSCs expressed distinct sets of proinflammatory factors. The combination of IFN-g and TNF-a was synergistic and induced a transcriptome most similar to that found in MSCs stimulation with activated T cells and similar to that found in the inflamed tumor microenvironment; a Th1 phenotype with the expression of the immunosuppressive factors IL-4, IL-10, CD274/PD-L1 and indoleamine 2,3 dioxygenase (IDO). Single cell qRT-PCR analysis showed that the combination of IFN-g and TNF-a polarized uniformly to this phenotype. The combination of IFN-g and TNF-a results in the synergist uniform polarization of MSCs toward a primarily Th1 phenotype. The stimulation of MSCs by IFN-g and TNF-a released from activated tumor infiltrating T cells is likely responsible for the production of many factors that characterize the tumor microenvironment. Overall design: BMSCs (passage 4) were stimulated with 6.5 or 65 ng/ml of IFN-g (R&D System Minneapolis, MN, USA), and with 1.5 or 15 ng/ml of TNF-a (R&D System Minneapolis, MN, USA), or the combination of IFN-g and TNF-a (1.5 ng/ml of TNF-a + 6.5 ng/ml of IFN-g or 15 ng/ml of TNF-a + 65 ng/ml of IFN-g). After the adherent BMSCs were incubated with the cytokines for 48 hours, the BMSCs were harvested using trypsin and they were then evaluated by gene expression profiling.
Project description:We have studied the plasma membrane protein phenotype of human culture-amplified and native Bone Marrow Mesenchymal Stem Cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in Colony-Forming Units-Fibroblasts was low for CD49b, CD90 and CD105, but high for CD73, CD130, CD146, CD200 and integrin alphaV/beta5. Additionally, the expression of CD73, CD146 and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in depth studies. Keywords: cell type comparison Overall design: Expression profiles of bone marrow MSC were compared with different lineages of purified hematopoietic cells from bone marrow to identify characteristic markers for human BM-MSC
Project description:The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years. However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs. Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function. Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow. We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells. Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern. These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility. Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether. Examination was performed using single-cell RNA-seq of sixteen mouse MSCs (mMSC1-mMSC16), non MSC single cell controls (HL1cm1-HL1cm5), and the population controls (mMSC-PC and HL1cm-PC).