| Mesenchymal stem cells (MSCs) have the activities of immunoregulation andcytokine secretion, endowing themselves with great values in cell therapy and tissueengineering. MSCs, like hematopoietic stem cells, are a kind of adult stem cells thathave been extensively investigated at bench and clinically. Meanwhile, the frequencyof MSCs in a variety of tissues is extremely low and ex vivo expansion is needed fortheir clinical application. Fetal bovine serum (FBS) is usually used as nutrients andstimuli for cell culture, and has been applying in the isolation and culture-expansion ofMSCs. However, MSCs cultured with this protocol may not only cause the recipientsexposed to the potentially contaminated pathogens in FBS, but also make thexenogeneic proteins be engulfed into the cytoplasm of MSCs, resulting in the risk ofsensitization reaction. Further, the use of FBS from batch to batch in the expansion ofMSCs may affect the reproducibility. Therefore chemically defined media withoutanimal serum for clinical grade MSC expansion is required to insure the safety andstability of MSC in vitro culture. But MSCs can not grow well in serum-free mediumwith the lack of multiple cell growth factors which are rich in FBS. And the centralproblem is that MSCs are of adherent cells, which need various substrates such asfibronectin, laminin, and collagen for MSC adhesion to the plastic. Generally, acocktail of growth factors and extracellular matrix molecules including fibronectin,collagen and fetuin should be added to support MSC proliferation and attachment.Meanwhile, MSCs themselves can secrete various cytokines, growth factors and aseries of extracellular matrix molecules including collagens and FN. This phenomenoninduced us to search some stimuli that could promote the secretion of quantity ofmatrix molecules by MSCs. If it does work, it will be a new way to develop a serumfree medium for MSC expansion.According to the published literatures and our preliminary experiments, we foundthat clinical-grade thrombin may be a useful stimulus. Thrombin is a serine proteasewhich has a variety of biological activities in addition to its key role in haemostasis. Itwas reported that thrombin could stimulate collagen synthesis in mesangial cells and could enhance FN production by human proximal tubular epithelial cells. It was alsoreported that it could induce mast cell attachment and could induce glioblastoma cellsproliferation and secretion of nervous growth factors. In this study, the stimulatoryeffect of thrombin on MSCs was investigated, including the effect of thrombin on FNsecretion by MSC and its mechanism. Further, the effects of thrombin on the biologicalcharacters of MSCs were also investigated.First of all, bone marrow MSCs were isolated from healthy donors by classicdensity gradient centrifuge and adherent culture method. Then MSCs were identifiedby cytometry method and multi-directional induction differentiation assays. To observethe potential effect of thrombin on MSCs, PCR was performed to detect whether MSCexpress the receptors for thrombin, protease activated receptors, PARs. The resultsshowed that MSCs expressed PAR-1and PAR-2, suggesting that thrombin mightexhibit its activity through PAR-1and PAR-2.To observe whether thrombin could promote FN secretion by MSCs and improveMSCs adherent to plastic in serum free medium, MSCs were treated with thrombin,then PCR and immunofluorescence staining were performed to detect the FNexpression on mRNA and cellular structure levels. The secretion of FN by MSCs wasmeasured by ELISA and the attachment of MSCs to plastic plates was measured by1hr spontaneous adhesion assay. Cellular adherent molecules ITGA were detected byPCR method. The results showed that FN expression on mRNA level was greatlyelevated by thrombin treatment (nearly3fold compared with control group, p<0.05)and the quantity of FN secretion into culture supernatants was remarkably increased(p<0.01) at a time-and dose-dependence manners. Meanwhile, the results were furthersupported by immunohistology and immunofluorescence staining, the FN level incellular plasma and structure was enhanced. On the other hand, the adhesion activity ofMSC was increased markedly after thrombin pretreatment compared with controlgroup (p<0.01). Quantitative RT-PCR showed that the expression of integrin alpha-5subunit in MSC was also greatly enhanced. The results above suggested that thrombincould promote FN expression and secretion by MSCs, and improve adhesion activityof MSC in serum free medium.To further investigate the underlying mechanisms, Western blotting wasperformed to detect the activation of signal pathways after thrombin treatment. Theresult indicated that treatment of MSCs with thrombin resulted in a rapidphosphorylation of ERK1/2, reaching its maximum at5minutes post treatment and lasting at least for60min. Thrombin also induced phosphorylation of NFKB, however,in sharp contrast to ERK1/2activation, NFKB p65phosphorylation reached maximumat60min. To further determine which pathway took the main effect on FN expressionand secretion, the specific inhibitors ethyl pyruvate which inhibits NFKB signaling bydirectly targeting p65subunit, and PD98059which blocks ERK1/2signaling pathwaywere used separately. FN secretion by MSCs was measured by ELSIA. Result showsthat EP and PD pretreatment suppressed thrombin elicited FN secretion by MSCs.Especially when ERK signaling pathway was blocked, the FN secretion was inhibitedgreatly (p<0.01), though FN secretion in EP pre-treated MSCs was still significantlyincreased compared with the SFM group.To further observe how thrombin elicited NFKB p65and ERK1/2activation,PAR1specific antagonist SCH79797and PAR2specific blockade agent FSLLRY wereadded in the culture of thrombin-treated MSCs, and the activation status of NFKB p65and ERK1/2was revisited. The results showed that after PAR1was blocked, the earlyphosphorylation of ERK1/2at the time point of5min was not inhibited while itscontinuous activation was suppressed. Concomitantly, FN secretion was suppressedcompared with thrombin treated alone group (p<0.05), but still higher than SFMcontrol group (p<0.05). When PAR2was inhibited, the phosphorylation of ERK1/2was greatly inhibited. And thrombin effect on MSCs of FN secretion was significantlyinhibited (p<0.01). However, blockage to PAR1and PAR2had little effect on thephosphorylation status of NFKB p65when they were use respectively. So it can beinfer that thrombin promote FN secretion by MSCs via PARs mediated ERK1/2signaling pathway, and NFKB signaling pathway induced indirectly may be alsoinvolved.In order to investigate the potential application of thrombin in the development ofserum-free media for MSC expansion, the biological features of thrombin-treatedMSCs were observed in details. MSC proliferation was measured by MTT test and afluorescent dye dilution assay. The surface markers of MSCs including CD31, CD34,CD45, CD44, CD73, CD90, CD105and HLA-DR were analyzed and the cellularmicrotube structure was revealed by alpha-tubulin immunofluorescence staining. Theabilities of differentiation and immunoregulation were also detected. The MTT resultshows that, thrombin stimulated MSC proliferation in a dose-dependent manner. Theminimal effective concentration was0.5U/ml (p<0.05), and the promoting effectreached its maximum when thrombin was employed at a dose of8U/ml (p<0.01). Proliferation effect was further supported by generation analysis, the generation indexwas higher than control (p<0.01). Meanwhile, the cellular surface markers andmicrotube structure of MSCs after treated by thrombin were not obviously changed.Further, the thrombin-treated MSCs, similar to the parent MSCs, could be induced todifferentiate into osteoblasts and adipoblasts and could inhibit lymphocyte proliferationelicited by PHA.To clarify the potential pathway(s) that thrombin takes to enhance MSCproliferation, PARs were inhibited separately by specific blockades and theproliferation status was revisited. The results showed that when PAR1was blocked, theeffect of thrombin was inhibited (p<0.05), while when PAR2was inhibited, thrombinstill played a positive effect on MSC proliferation. The AKT signal pathway wasactivated after treated with thrombin, after which was blocked, cell proliferation wasinhibited. When ERK1/2phosphorylation which can also be activated by thrombin viaPAR1was inactivated by inhibitor, MSC proliferation was inhibited, proving thatERK1/2was contributed to MSC proliferation. Some genes linked to cell proliferationwere detected by PCR method, and the results showed that c-MYC and EGR1geneexpression were greatly up-regulated by thrombin treatment, and could be revesed byAKT inhibitor and ERK1/2inhibitor separately. Thus, it can be inferred that thrombinmay exert its activity on cell proliferation through PAR1mediated AKT and ERK1/2signal pathway, up-regulating c-MYC and EGR1genes expression and eliciting MSCproliferation.According to the results above, some conclusions might be drawn as follow:(1)Thrombin could promote FN expression and secretion by MSCs via PARs-mediatedERK1/2phosphorylation, and indirect activation of NFKB p65signaling path waymight be also involved.(2) Thrombin-treated MSCs exhibit stronger attachment abilityin serum free medium probably via the up-regulation of ITGA5expression.(3)Thrombin enhances MSCs proliferation through PAR1-mediated AKT and ERK1/2signaling pathway, and c-MYC and EGR1might be the effector molecules. Thecharacters of thrombin treated-MSCs are similar to the parent MSCs, with the ability ofmultiple differentiations into osteoblasts and adipoblasts and the capacity to regulateallogeneic lymphocyte activation. Our results suggest that thrombin may be used as anadditive for serum free medium for clinical grade MSC culture, though further detailedinvestigations are needed to evaluate the safety of thrombin-treated MSCs. |
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