TLR4Signaling Pathway In Mesenchymal Stem Cells Derived From Umbilical Cord And The Study On HWJ-MSCs Co-culture With BV-2

BackgroundsThere is no timely and effective treatment on neonatal brain injury. MSCs are the mostpromising tool of cell therapy for the treatment of neonatal brain injury. The clinicalapplication of MSCs on brain injury treatment is still in the research stage, more evidencesand detailed understood were needed for the MSCs therapy in brain injury.ObjectiveOn the basis of successfully isolated and identified mesenchymal stem cells from humanumbilical cord (hWJ-MSCs), we observed the cell morphology, amplification efficiency ofhWJ-MSCs in vitro, and analyzed subsequently the biological phenotype and functionalproperties changes in mimicked inflammatory environment. For evaluated the infusedhWJ-MSCs interacted with the injuried microenvironment, an immortalized microglia celllines drived from mouse, BV-2cells, was used and co-cultured with hWJ-MSCs. Thecytokine profiles of BV-2cells or hWJ-MSCs were analyzed by qRT-PCR analysis andELISA. The results would provide research foundation for the intracranial transplantationin the clinical application of MSCs in the future.MethodsThis study is divided into the following three parts.1. Isolation, identification and amplification of HWJ-MSCs in vitroFive umbilical cords from full-term cesarean section babies were collected from theGeneral Hospital of Beijing Military Region. All umbilical cord tissues were immediatelypreservated into sterile complete culture medium and dealt with within1-6hours to ensurecell viability of hWJ-MSCs. The umbilical artery and vein were removed in a sterileenvironment and then cut into1-2mm2segments for the following hWJ-MSCs isolationprocesses. The hWJ-MSCs were isolated by enzyme digestion method and adherentmethod. For enzyme digestion method, the segments were digested with0.075%collagenase II combined with0.125%trypsin to obtain single cell suspension; for adherentmethod, the the segments were placed in a petri dish to allow the migration of adherenthWJ-MSCs. The primary adherent cells were passaged at80-90%confluence to obtainpure hWJ-MSCs. The cell morphology was observed at different time and different passages under an inverted microscope. hWJ-MSCs cell surface markers CD14, CD31,CD73, CD105, CD90, HLA-ABC and HLA-DR were identified by flow cytometryanalysis. The differentiation potential of hWJ-MSCs was analyzed using an adipogenicdifferentiation kit and a chondrogenic differentiation kit. The cell morphology, apoptosisand cell cycles of hWJ-MSCs isolated by enzyme digestion method and adherent methodwere observed respectively.2. TLR4expression profile analysis in hWJ-MSCs and TLR4engagement impactedon hWJ-MSCs by LPSThe expression profiles of TLR4and CD14in hWJ-MSCs were analyzed by Flowcytometry analysis and qRT-PCR analysis. We used LPS, a classic TLR4activator, totrigger the hWJ-MSCs to mimick a cell model in inflammatory milieu. The cellmorphology, proliferation, apoptosis and cell cycle were observed, the condition mediumwere collected and the total mRNA were extracted after hWJ-MSCs exposing to LPS atdifferent period (24h,48h or72h). The expression profiles of IL-1β, IL-1α, IL-2, IL-4,IL-6, IL-8, IL-10, IL-12, IL-13, indoleamine2,3-dioxygenase [IDO]-1, TNF-α, IFN-γ,MMP-2and MMP-9were analyzed by qRT-PCR analysis and the protein concentration ofIL-1β, IL-6, IL-8, IL-12, TNF-α, IFN-γ and MMP-2in condition medium from LPSexposing groups or control group were determined by ELISA.3. hWJ-MSCs co-culture with BV-2cells line in vitroTo evaluate the mutual impact of BV-2cells direct contact with hWJ-MSCs, weco-cultureed hWJ-MSCs and BV-2at different ratios, and collected the cellculture supernatants by centrifugation and cell lysates using trizol reagent after varioustime points exposing to LPS or not. The total mRNAs were extracted with trizol reagentand cDNA were synthesized using a commercial kit. The human gene expression profilesof IL-1β, IL-6, IL-8and MMP-2and the mouse gene expression profiles of Arginase1,Arginase2, Mrc1, Ym1and Fizz1were analyzed by qRT-PCR analysis. The concentrationof NO in cell culture supernatants was determined using a commercial Griess reagent kit.Results1. hWJ-MSCs were isolated and amplificated successfully in vitroThe fibroblast-like cells were isolated from umbilical cord tissues by enzyme digestionmethod or adherent method and amplificated successfully in vitro. The pure fibroblast-like cells were obtained based on the migratory and adhesive properties of MSC. The cellswere subsequntly identified as hWJ-MSCs by the unique characteristics of mesenchymalstem cells: adhesive properties, cell surface markers and induced differentiation potentialin vitro. We analyzed the surface markers of the cells isolated by enzyme digestion methodand adherent method by flow cytometry analysis and the results showed CD14, CD31andHLA-DR were negative and CD73, CD105, CD90and HLA-ABC were positive. Bothchondrogenic and adipogenic differentiation were successfully induced in vitro. Theseresults suggested both two isolation methods couled be used to isolate MSCs fromumbilical cord. The obtained hWJ-MSCs could maintain the fibroblast-like morphologyuntil it was passaged to the20th generation. The cell cycles of hWJ-MSCs (P3to P7andrecovery P5) were analyzed by PI staining and showed few deaths, late apoptotic cellswere observed and most cells were in G1/G2phas, which indicated a good proliferativepotential of hWJ-MSCs.2.TLR4was identified as a functional receptor in hWJ-MSCs and TLR4engagementmodulate the biological and immunosuppressive properties of hWJ-MSCsThe morphology of hWJ-MSCs was not affected by LPS exposing, as observed bymicroscopy using a Nikon eclipse TE2000-S Microscope. In addition, the proliferation andapoptosis of hWJ-MSCs primed with LPS, as analyzed by MTT assay and PI stainingrespectively, showed no differences in contrast to control group at different time points.The expression profiles of the TLR4and CD14, as the receptors of LPS, were analysed atthe mRNA and protein levels by qRT-PCR analysis and flow cytometry. Flow cytometryanalysis showed that hWJ-MSCs were marginally positive for TLR4(approximately3%)but negative for CD14(below1%) in comparison to the isotype control, which bothshowed no obvious changes response to LPS stimuli. However, qRT-PCR analysis revealedthat TLR4mRNA was expressed at relatively higher levels compared to that of GAPDH,and both TLR4and CD14were markedly up-regulated when the cells were primed withLPS for up to72h (4.79-and5.34-fold, respectively). qRT-PCR analysis was utilised toanalyse the Th1(IL-2, IFN-γ, and TNF-α) and Th2cytokine (IL-4, IL-10, and IL-13),inflammatory cytokines (IL-1β, IL-1α, IL-6, and IL-12), and chemokine (IL-8) expressionprofile in hWJ-MSCs. The results demonstrated the constitutive expression of IL-1β, IL-1α,IL-6, and IL-8at very high levels, whereas the expression levels of IL-2, IL-4, IL-10,IL-13, and TNF-α were undetectable. LPS had no effect on the expression of IL-1β, IL-1α, IL-6, and IL-8in hWJ-MSCs at48h though a marked and delayed increase was observedafter72hours. Very low IL-12and IFN-γ mRNA levels were detected by qRT-PCR;overall, IL-12was inhibited and IFN-γ was not induced by LPS after the entire treatment.To confirm the qRT-PCR results, we assessed the cytokines’ levels in the conditionedmedium from hWJ-MSCs treated with LPS. Of all the tested cytokines, only IL-6and IL-8were detectable, showing a significant increase accompanying changes in mRNAtranscription during LPS stimulation. As shown by qRT-PCR analysis, MMP-2andMMP-9were constitutively expressed in hWJ-MSCs, and MMP-2was expressed at highlevels; in contrast, MMP-9was expressed at a relatively low level. hWJ-MSCs responcedto LPS stimulation and resulted in the up-regulation of the expression of MMP-2andtransiently down-regulated the expression of MMP-9. The expression of MMP-2was notaltered in the first48h but increased3.25-fold in the next24h. This finding is in sharpcontrast to MMP-9, which was inhibited by LPS in the first48h and increased to arelatively normal lever after72h. The expression of IDO-1, IDO2, Cox2, and IFN-β wasdetected by qRT-PCR analysis. The expression of IDO-1and IFN-β at the mRNA levelshowed a bimodal increase after LPS exposure over a period of72h, whereas the inductionof Cox2only appeared after a72-h LPS exposure. Regarding the expression of IDO2, nochange was detected at different exposure times to LPS.3. The mutual regulation between hWJ-MSCs and BV-2cells when direct contact invitroThe morphology of BV-2cells could maintain the resting morphology under LPS stimulicondition when co-cultured with hWJ-MSCs, which suggested that hWJ-MSCs couldinhibit BV-2cells polarization to a classic M1-activated morphology induced by LPS, asobserved under microscopy.The NO secreted by BV-2cells was detected with Griess reagent. The reaults showed theNO secrertion of BV-2cells induced by LPS was obviously inhibited when thetwo co-culture cells ratio was at1:1, but the inhibitory effect decreased quickly whenthe co-culture cell ratios were higher than1:1(BV-2cells:hWJ-MSCs). Moreover,hWJ-MSCs induced the expression of arginase1in resting BV-2cells. In the precence ofLPS, the expression level of arginase1in BV-2was equal to that in resting BV-2cells,which mean hWJ-MSCs could attenuate the inductive effect of LPS on BV-2cells. ForhWJ-MSCs, the expression of IL-1β, IL-6, IL-8and MMP-2were also analyzed by qRT-PCR and showed obvious changes in pace with the changes of co-culture cell ratio orLPS exposing. LPS exposing was the most important factor shaping the expression profileof IL-1β of hWJ-MSCs in co-culture system. BV-2cells indued IL-1β expression instandard medium but inhibited it in the precence of LPS. The MMP-2expression profilewas only induced in standard medium after72h co-culturing with BV-2cells, the IL-6expression profile was induced only within24h under LPS exposing. The IL-8expressionprofile was induced only within24h no matter what LPS exposing, and the inducedexpression increased with the ascent of co-culture cell ratio (BV-2:hWJ-MSCs) butdecreased with the declines of co-culture cell ratio.Conclusion1. Both the enzyme digestion method and adherent method could be used to isolatehWJ-MSCs in vitro. hWJ-MSCs could maintain the normal cell morphology and bepassaged continuously only twenty times in vitro.2. The TLR4expression profile in hWJ-MSCs was identified by qRT-PCR analysis andflow cytometry. hWJ-MSCs reacted to the LPS stimuli and resulted in the up-regulation ofinflammatory cytokines (IL-1β, IL-1α, IL-6, and IL-12), chemokine (IL-8), matrixmetalloproteinase (MMP-2) and some immunosuppressive molecules (IDO-1, Cox2andIFN-β), which confirmed that TLR4is a functional receptor in hWJ-MSCs. The changesprovoked by LPS in hWJ-MSCs showed a delayed and “dull” response compared to theresponses observed in MSCs come from adult tissues but showed the similar characteristicsto another fetal tissue MSCs who derived from umbilical cord blood. These findings maybe new evidence that supports the notion that the hMSCs derived from foetal tissue aremore primitive than the MSCs derived from adult tissues.3. hWJ-MSCs could induce the up-regulated expression of arginase1, a typical M2cellphenotypic marker of mouse microglia, and attenuated the NO secretion of BV-2inducd byLPS, which suggested BV-2could be induced M2polarization in vitro by hWJ-MSCs. onthe other hand, some important biological functional molecules of hWJ-MSCs, such asIL-1β, IL-6, IL-8and MMP-2were shaped by the LPS exposing combined with BV-2.These data suggested hWJ-MSCs direct contact with BV-2could induce the immortalizedmicroglia cell line polarization to an anti-inflammatory state, which would be a goodexplanation for the beneficial effects of MSCs in ischemic models of brain injury in vivo.