Preconditioning of BM-MSCs by Innate Immunity Agonists Influences Mesenchymal Stem Cell Properties In vitro

Background and Aim: Mesenchymal stem cells (MSCs) therapy has thusfar been shown for inconsistent outputs, indicating an essential need torectify therapeutic efficacy. Although administration of sufficient cells isnecessary to attain maximal therapeutic benefits, documented MSC trialsrelied on injections of ∼1×106 cells/kg, which appears too low to elicita robust therapeutic response according to published animal studies.However, frequent cell passaging necessary for the scale-up expansionof MSCs causes cellular senescence, remodeling of cells and reducesstem cell potency. We hypothesized that preconditioning with TLR3agonist significantly enhance cell proliferation via the Ras-Raf-MEK-ERKsignaling pathway.Methods: Experiments were performed using human bone marrow MSCs.The hMSCs were cultured in low-glucose DMEM supplemented with10% fetal bovine serum and 100 U/100 μg/mL penicillin/streptomycinat 37°C in a humidified 5% CO2 incubator. The cells were fed withfresh medium every 3–4 days and used at passages 5 and 6. The hMSCswere incubated with poly (I:C) (1, 2 and 5 μM/mL, TLR3 primed) in theculture medium for 4 h. Total RNA was extracted from hMSCs and wasreverse-transcribed and subsequently, the resultant cDNA was amplifiedusing Evagreen mastermix. RT-PCR primer pairs for target genes weresynthesized and quantitative real-time PCR was performed on an ABIstep-one plus real-time PCR system. The amplified PCR products weredetected by agarose gel electrophoresis and ethidium bromide staining.Results: Using the poly(I:C) to engage MSC Toll-like receptor 3 (TLR3),we found that poly(I:C), signaling through multiple mitogen-activatedprotein kinase pathways, induced therapeutically specific trophic factorssuch as interleukin-1,6, TNF-α types cytokines, stromal-derived factor1, and VEGF and slightly influence the proliferation of MSC. Poly (I:C)preconditioning enhanced proliferation of MSCs, stimulated expressionof the Ras-Raf-Mek-Erk pathway. So, Poly (I:C) preconditioning provideda novel strategy in maximizing biologic and functional properties ofMSCs.Conclusion: These molecular quantifications thus establish the utility ofTLR3 engagement for enabling the low-dose MSC therapy that may be translated to more efficacious clinical applications.

کلیدواژه ها:

TLR3 ، Innate immunity ، BM-MSCs ، Proliferation