Retinoic Acid Receptor Beta Mediates The Facilitation For Mesenchymal Stem Cells Neuronal Differentiation Induced By All-trans Retinoic Acid

Part I Effects of all-trans retinoic acid pre-induction onmesenchymal stem cells neuronal differentiation andexploration of optimal pre-induction concentrationObjective: To observe the effects of different concentrations ATRA onneuronal differentiation efficiency, morphology, maturity, proliferation,apoptosis and function of neural-like cells derived from rMSCs and toexplore optimal pre-induction concentration of ATRA. And then, wedetected the expressions of neural specific marks and function ofneural-like cells derived from rMSCs with optimal concentration of ATRApre-induction and the changes of retinoic acid signaling pathway whenrMSCs were induced by MNM and ATRA.Methods: rMSCs were isolated from4-week-old SD rat femursaccording to the reported method and identified by flow cytometry. rMSCswere pre-induced by0,0.01,0.1,1,10,100μM ATRA respectively and then induced by MNM. Cells exhibiting protruding cell bodies with axonsor dendrites were counted over total cell counts. Neural-like cell bodydiameter and axons length were measured by using Nikon Elements Dsoftware. Cells death rates were counted with the Trypan blue exclusionassay; survival and growth ability of different groups were detected byMTS assay; cells apoptosis rates were counted with Hoechst staining. Andthen, the expressions of neural specific marks Nestin, NSE and MAP-2ofneural-like cells derived from rMSCs with different concentration ATRApre-induction were determined by real time PCR. After optimalpre-induction concentration of ATRA was determined, four different groups,control, ATRA, MNM, ATRA+MNM were set. The mRNA and proteinexpression level of neuron specific marks including Nestin, NSE andMAP-2and gliocyte specific marks including GFAP, CD68and GDNF ofdifferent groups were detected by RT-PCR and immunofluorescencestaining, respectively. We analyzed Nestin and NSE expression during a36h span of MNM induction by western blotting. And then, the function ofcells of different groups were measured by whole-cell patch clamp andcalcium detection system. At last, the expression changes of retinoidsignaling pathway, RARs, of different groups were determined by real-timePCR and immunofluorescence staining.Results:(1) Isolation and identification of rMSCs: rMSCs adhered tothe surface of plastic culture dishes and exhibited a spindle-shaped fibroblast-like morphology as cells approached confluence. rMSCs wereidentified by flow cytometry. As expected, more than99%of the cells wereCD29, CD44, CD90and CD106positive, and vast majority of the cellswere CD34and CD45negative.(2) The effects of different concentrationsATRA pre-induction on rMSCs neuronal differentiation: After induced byMNM, rMSCs were changed into neural-like cells presenting distinctneuronal morphologies including simple bipolar, large and extensivelybranched multipolar appearance. Neuronal differentiation efficiency wasincreased with ATRA pre-induction, which was shown in a dosagedependent manner ranging from0.01to1μM and decreased a little bit inthe10μM ATRA group. Neural-like cells derived from ATRA pretreatedrMSCs exhibited bigger cell body diameter, longer axonal length and moredendrites as ATRA concentration increased ranging from0.01to10μM(p<0.05). The death rate and apoptosis rate of1μM ATRA group were thelowest (p<0.01/p<0.05). There were no influence of0.01to1μM ATRApre-induction on the survival and growth ability of cells induced by MNM,but ATRA up10μM suppressed the survival and growth ability significantly.Using real-time PCR, we found that expression of three neural relatedmarkers, Nestin, NSE, and MAP-2in1μM ATRA group were all thehighest. As a result,1μM was optimal pre-induction concentration.(3)ATRA pre-induction can improve the neuronal differentiation of rMSCs:RT-PCR analysis showed that ATRA alone only upregulated Nestin, a neural stem cell marker, expression. The ATRA pre-induced cells exhibitedhigher expression of Nestin, NSE and MAP-2but lower expression ofGFAP, CD68than that of the cells treated with MNM alone, indicating thatATRA may improve differentiation of MSCs into neurons but notneuroglial cells. Immunofluorescence staining confirmed the aboveRT-PCR results. Western blotting result displayed that NSE expressionincreased at3h post MNM induction and continued to increase for up to36h, whereas Nestin increase peaked at18h, and then decreased sharply.(4)Differentiated neural-like cells with ATRA pre-induction possessedadvantages in neuroelectrophysiological function: Neural-like cells withATRA pre-induction exhibited a much higher RMP than that of the MNMgroup (p<0.05). Neurons induced by MNM alone showed an apparentincrease over baseline, but the fold increase of intracellular free Ca2+wassignificantly lower than that in neurons with ATRA pre-induction (p<0.05).(5) The expression changes of retinoid signaling pathway, RARs, of rMSCsinduced by ATRA and MNM: Using RT–PCR analysis, we found that theendogenous expression of RARα and RARγ was readily detected. However,RARβ was almost undetectable under the same conditions. Throughreal-time PCR, we found that the expression of all RARs isoforms,especially RARβ, in MNM treated cells was statistically higher than that incontrol cells, suggesting that retinoid signaling was indeed activated inMNM-induced cells. Furthermore, we found that RARβ was the only isotype that exhibited significant increase in cells treated with only ATRAinduction. And RARβ was significant higher in rMSCs with ATRApre-induction than that of rMSCs without ATRA pre-induction afterneuronal differentiation. What is more, immunofluorescence stainingshowed that the redistribution of RARβ near the nuclear membrane in thedifferentiated neurons indicating that RARβ may be involved in neuronaldifferentiation of rMSCs.Conclusions:(1) rMSCs identified by flow cytometry were obtainedsuccessfully.(2) rMSCs could be induced into neural-like cells by MNM.The neural-like cells not only expressed neural related makers but alsopossessed a certain nerve excitability.(3) ATRA pre-induction facilitatedrMSCs neuronal differentiation and promoted cells survival, whereasinhibited its apoptosis. And optimal ATRA pre-induction concentration was1μM.(4) ATRA pre-induction improved differentiation of rMSCs intoneurons and inhibited neuroglial differentiation. Differentiated neural-likecells with ATRA pre-induction possessed advantages inneuroelectrophysiological function.(5) In the process of neuronaldifferentiation of rMSCs, the retinoid signaling pathway, especially RARβ,was indeed activated. Subcellular location of RARβ also changed whenrMSCs differentiated into neural-like cells. Maybe, the facilitation effectsof ATRA on neuronal differentiation of rMSCs was associated withpre-activation of retinoid signaling pathway and especially RARβ signaling. Part II Construction and identification of recombinantadenovirus carrying rat retinoic acid receptor α, β and γ geneand small interfering RNA against rat retinoic acid receptor βChapter I Construction and identification of recombinantadenovirus carrying rat retinoic acid receptor α, β and γ geneObjective: To construct three recombinant adenovirus carrying ratretinoic acid receptor α, β and γ gene respectively, with the purpose toresearch the function of RARα, RARβ and RARγ in the process ofdifferentiation of mesenchymal stem cells (MSCs) into neuronal-like cells.Methods: Rat RARα, RARβ and RARγ gene were amplified in vitroand cloned into pAdTrace-TOX vector orientationally to obtainpAdTrace-RARα, pAdTrace-RARβ and pAdTrace-RARγ. The recombinantvector pAd-RARα, pAd-RARβ and pAd-RARγ were gained byhomologous recombination between pAdTrace-RARα, pAdTrace-RARβ,pAdTrace-RARγ and backbone vector pAdEasy-1in E.coli BJ5183.pAd-RARα, pAd-RARβ and pAd-RARγ were linearized by Pac I and transfected into HEK293cell lines to package recombinant adenovirusAd-RARα, Ad-RARβ and Ad-RARγ. Rat MSCs were infected byAd-RARα, Ad-RARβ and Ad-RARγ respectively, and the expressions ofRARα, RARβ and RARγ were detected by real-time PCR and Westernblotting.Results: All results from PCR, endonuclease cutting and genesequanceing confirmed that RARα, RARβ and RARγ gene were clonedinto adenovirus vector correctly. Infection efficiency of Ad-RARα,Ad-RARβ and Ad-RARγ in rMSCs achieved60-70%. Both the RARα,RARβ and RARγ mRNA and protein expression level of rMSCs wasincreased significantly by Ad-RARα, Ad-RARβ and Ad-RARγ infection.Conclusions: The recombinant adenovirus carrying rat RARα, RARβand RARγ gene were created successfully, and the adenovirus have thefunction of upregulating corresponding gene expression in rMSCs. Chapter II Construction and identification of smallinterfering RNA against rat retinoic acid receptor β byrecombinant adenovirusObjective: To construct small interfering RNA against rat retinoic acid receptor β (RARβ) by recombinant adenovirus, with the purpose to providepractical technique and material for researching the function of RARβ inthe process of the facilitation effects of all-trans retinoic acid (ATRA) onneuronal differentiation of rMSCs.Methods: Four pairs of double-stranded DNA fragments for silencingrat RARβ were designed and cloned into pSES-HUS vector to obtainpSES-HUS-siRARβ. The recombinant vector pAd-siRARβ was gained byhomologous recombination between pSES-HUS-siRARβ and backbonevector pAdEasy-1in E.coli BJ5183. pAd-siRARβ was linearized by Pac Iand transfected into HEK293cell lines to package recombinant adenovirusAd-siRARβ. rMSCs were infected by Ad-siRARβ-1,-2,-3,-4for48h, andthe expressions of RARβ were detected by real-time PCR and westernblotting.Results: All results from PCR, endonuclease cutting and genesequanceing confirmed that double-stranded DNA fragments having RNAinterference sequance were cloned into adenovirus vector correctly. A largenumber of high-titer Ad-siRARβ-1,-2,-3,-4were obtained by packagedand amplified in HEK293cell lines. Among these four Ad-siRARβ,Ad-siRARβ-1,-2,-3were funtional in down-regulating RARβ expression.Whereas, Ad-siRARβ-4had no function. Both the RARβ mRNA andprotein expression level of rMSCs were down-regulated byAd-siRARβ-pool infection. Conclusions: The recombinant adenovirus Ad-siRARβ-1,-2,-3,-4were created successfully, and the adenovirus has the function of silencingRARβ expression in rMSCs. Part III Investigate about the role of retinoic acid receptors onneuronal differentiation of mesenchymal stem cellsObjetive: To investigate the effects of each subtype retinoic acidreceptor on neuronal differentiation of mesenchymal stem cells by usingAd-RARα, Ad-RARβ and Ad-RARγ infecting rMSCs respectively beforeinduced by MNM. To investigate the role of RARβ on the facilitation forrMSCs neuronal differentiation induced by ATRA, Ad-siRARβ and LE135,a specific RARβ inhibitor, were used to block the function of RARβ.Methods: Six different groups, Ad-RFP, Ad-RFP+MNM,Ad-RFP+RA+MNM, Ad-RARα+MNM, Ad-RARβ+MNM andAd-RARγ+MNM, were set to investigate the effects of each subtyperetinoic acid receptor on neuronal differentiation of rMSCs. A certainamount of Ad-RARα, Ad-RARβ and Ad-RARγ were added inAd-RARα+MNM, Ad-RARβ+MNM and Ad-RARγ+MNM grouprespectively before induced by MNM. The Ad-RFP (Ad-null) was used as negative control. Ad-RFP and ATRA were added simultaneously inAd-RFP+RA group. After24h adenovirus infection, cells of all groupswere rinsed by D-Hank's buffer and then induced by MNM. Further more,another four groups, Ad-RFP+MNM, Ad-RFP+RA+MNM,Ad-siRARβ+RA+MNM and LE135+RA+MNM were set to investigate therole of RARβ on the facilitation of rMSCs neuronal differentiation。Afterinfected with Ad-RFP or Ad-siRARβ for24h, ATRA were added andinduced for another24h. LE135and ATRA were added simultaneously andinduced for24h in LE135+RA+MNM group. And then, cells of all groupswere rinsed by D-Hank's buffer and then induced by MNM. Real-time PCR,western blotting and immunofluorescence staining detection methods wereused to determine the expression level of neuron related makers of cells indifferent groups. And then, the function of cells of different groups weremeasured by whole-cell patch clamp and calcium detection system.Results:(1)The expression level of Nestin, NSE, MAP-2, Tau andβ-III-tubulin in both Ad-RARα+MNM group and Ad-RARγ+MNM groupequaled to Ad-RFP+MNM group. But they were significantly higher inAd-RARβ+MNM group than that of Ad-RFP+MNM group. And theexpressions level of these neuron related makers of Ad-RARβ+MNMgroup were similar to that of Ad-RFP+RA+MNM group. In the functionaltests, RMP and calcium excitability in both Ad-RARα+MNM group andAd-RARγ+MNM group equaled to Ad-RFP+MNM group. However, they were significantly higher in Ad-RARβ+MNM group than that ofAd-RFP+MNM group. What is more, both RMP and calcium excitability inAd-RARβ+MNM group were comparable with those inAd-RFP+RA+MNM group.(2) After activation of RARβ induced byATRA was blocked by Ad-siRARβ or LE135, the expression level ofNestin, NSE, MAP-2, Tau and β-III-tubulin became lower compared withAd-RFP+RA+MNM group. Similarly, both RMP and calcium excitabilitywere lower than that of Ad-RFP+RA+MNM group after RARβ activationblocked by Ad-siRARβ or LE135.Conlusions:(1) There was no apparent promoting effect for neuronaldifferentiation of rMSCs after activating RARα or RARγ signal previously.(2) Activating RARβ signal previously improved differentiation of rMSCsand the function maturity of neural-like cells. The effects of RARβpre-activation were almost equal to ATRA pre-induction. The facilitationfor rMSCs neuronal differentiation induced by ATRA could be inhibited byAd-siRARβ or LE135.(3) Maybe, RARβ mediated the facilitation formesenchymal stem cells neuronal differentiation induced by ATRA.