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Background/Aim: Microglia, the resident macrophages in the central nervous system, secrete various proinflammatory cytokines and undergo proliferation upon activation in various neurodegenerative diseases. Activation of microglia has been implicated in exacerbation of various neurodegenerative diseases. Recently, it has been proposed that mesenchymal stem cells (MSC) have immunosuppressive properties and the potential to moderate inflammation. This study aimed to elucidate the effects of MSC-conditioned medium (MSC-CM) in modulating microglial activation by analyzing microglial proinflammatory and anti-inflammatory factors [interleukin (IL)-6, tumor necrosis factor (TNF)-a, inducible nitric oxide synthase (iNOS) and IL-10], signaling pathway molecules [NFκB, c-Jun N-terminal kinase (JNK) and MKP-1) and NO production. Methods: Immortalized murine microglia cell line, BV2 microglia and primary microglia isolated from C57BL/6 mouse pup brains were used in this study. Mouse MSC were isolated from the male C57BL/6 mouse tibia and fibula. The effects of MSC-CM on the expression of inflammatory cytokines and signaling molecules in microglia were elucidated using RT-PCR, immunofluorescence analysis and Western blot analysis. NO production in microglia was assessed using a Griess kit. Results: MSC-CM significantly reduced the mRNA and protein expression levels of proinflammatory cytokines (IL-6 and TNF-a) in microglia activated by lipopolysaccharide (LPS). In addition, MSC-CM significantly reduced the protein expression of NFκB, JNK and c-Jun, but increased the expression levels of IL-10 and MKP-1 in activated BV2 microglia. NO production and iNOS expression by BV2 microglia in MSC-CM were increased. Conclusions: Overall, our findings suggest that MSC immunomodulate microglial activities through paracrine effects.

BackgroundProgression of neurodegenerative diseases occurs when microglia, upon persistent activation, perpetuate a cycle of damage in the central nervous system. Use of mesenchymal stem cells (MSC) has been suggested as an approach to manage microglia activation based on their immunomodulatory functions. In the present study, we describe the mechanism through which bone marrow-derived MSC modulate the proliferative responses of lipopolysaccharide-stimulated BV2 microglia.MethodsBV2 microglia were cultured with MSC and stimulated with 1 μg/ml lipopolysaccharide. Using an inducible nitric oxide synthase inhibitor, tritiated thymidine (3H-TdR) incorporation assay was performed to determine the role of nitric oxide in the anti-proliferative effect of MSC. We also studied apoptosis and the cell cycle of both cell types using flow cytometry and explored their cytokine profile using protein and cytometric arrays. Moreover, the role of IL-6 and TNF-α in immunomodulation was deduced using specific blocking antibodies and recombinant proteins.ResultsMSC reduces microglia proliferation upon lipopolysaccharide stimulation by 21 to 28% and modulates the levels of nitric oxide, IL-6 and TNF-α. The role of nitric oxide in conferring the anti-proliferative effect of MSC was ruled out. Furthermore, we found that MSC exert their anti-proliferative effect by restoring the percentage of BV2 cells at S and G2/M phase to levels similar to unstimulated cells. MSC undergo a G0/G1 arrest while exerting this effect. We have also identified that MSC-mediated modulation of microglia is independent of IL-6, whilst reduction of TNF-α in co-culture is critical for inhibition of microglia proliferation.ConclusionsOur study demonstrates that MSC inhibit microglia proliferation independent of nitric oxide and IL-6, although reduction of TNF-α is critical for this effect. The inhibition of proliferation is through cell cycle modulation. These findings shed light on the mechanisms of microglial immunomodulation by MSC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0149-8) contains supplementary material, which is available to authorized users.

AIM:To assess the capacity to isolate and expand mesenchymal stem cells (MSC) from bone marrow of CBA/Ca, ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed, cultured and maintained in supplemented Dulbecco's modified Eagle's medium. MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106, Sca-1 and CD44 and negativity to CD45, CD11b and MHC class Ⅱ. Differentiation capacity of MSC towards osteogenic and adipogenic lineages were also assessed. RESULTS

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