The Cytobiological Characteristics Of AMSCs And The Inhibitory Effects Of AMSCs Transplantation On Glioma

Mesenchymal stem cells (MSCs) is a kind of mesoderm-derived stem cells with greatpotential of self-renewal and multilineage differentiation. MSCs, with less immunogenicity,tumor targeting and tumor suppression, have become the focus on research in the injuryrepair and tumor therapy. Previous research mainly concentrated on bone marrow-derivedMSCs which can only be obtained by bone marrow aspiration and distribute sparsely withincrease of age. Recently, amniotic membrane of placenta becomes one of the new sourcesof MSCs (amniotic mesenchymal stem cells, AMSCs) because it is rich in all kinds of stemcells, easily procured with rarely pollution, easily performed with good proliferationactivity, which may render these cells as good potential sources for future therapeuticapplications.Glioma is the most common primary malignant tumor of the central nervous system.It possesses extraordinary migratory ability and readily penetrate adjacent normal tissue atthe early period. Tumor cells migrating away from the primary tumor site often form tumormicrosatellites at distal sites. Recent research shows that MSCs possess great migratoryability and glioma tropism, and can be largely distributed at the border zone between tumorand normal parenchyma. They develop a capsule-like structure and also infiltrated into thetumor bed relatively uniformly. Moreover, some MSCs can chase down‘the outgrowingglioma cells. Thus, MSCs may serve as a promising vector for gene-targeted gliomatherapy. MSCs can also exert anti-tumor effect on glioma, via the induction of apoptosis ofglioma cells. Further study should be focused on the mechanism in glioma tropism andanti-tumor effect and what about the relevanted mechanism, pending further study.In our study, the effective amplification system for AMSCs was set up, and theneurobiology characteristics of AMSCs were explored by induction. The migratory ability,glioma tropism and anti-tumor effect of AMSCs were observed in a series of experimentsin vitro. Moreover, the AMSCs were adopted as seed cells, then their survival, migrationafter transplanted into glioma on nude mice were observed, and the possible mechanism inglioma tropism and anti-tumor effect was investigated. Part I Isolation, culture and identification of AMSCsObjective: To establish an effective culture system for AMSCs in vitro, and toexplore the neurobiology characteristics of AMSCs by induction.Methods: The amnion and chorion layers were bluntly separated. The AMSCsisolated from minced amnion and chorion tissues were cultured in vitro. Flow Cytometrywas used to detect the surface marker of AMSCs. The morphology was observed underinverted microscope. Then under specific induction conditions, AMSCs were induced tochondrocytes, osteocytes, adipocytes and neurocytes, and specific dye was adopted todetermine their differentiation potential. The specific genes expression of adipocytes,chondrocytes, and osteocytes and neural-specific protein were detected by RT-PCR afterthe AMSCs were treated by different induction conditions for different mesodermallineages.Results: The AMSC showed plastic adherence and typical fibroblastic morphology bylight microscopy, and the Flow Cytometry analysis showed that AMSCs highly expressedCD73、CD90、CD105, negative for CD14、CD34、CD45、HLA-DR; Exposure to osteogenicinductive medium resulted in secretion of extracellular calcium crystals, identified by vonKossa staining, indicating osteogenic differentiation. When cells were cultured inadipogenic inductive medium, intracytoplasmic lipid vacuoles were observed, andconfirmed by oil red O staining. After been cultured in chondrogenic inductive medium,chondrogenic differentiation was identified demonstrated by toluidine blue staining. Whencells were cultured in neural inductive medium, glial cell-like or/and neuron-likestructures were observed, and, most of the cells showed GFAP positive byimmunofluorescence. RT-PCR results showed, after exposed to different inducingconditions, the specific genes expression of PLIN (adipose cells), ACAN(chondrocytes)and RUNX2(osteocytes). AMSCs showed the specific genes expression of nestin, GFAP,mushashi-1and β-tubulin III before expored to neural tissue induing condition. NF wasexpressed positively after induction for2days. The expression level of nestin decreased5days after induction.Conclusions: Human placenta amnion was proved to be rich in AMSCs, which could be obtained from the amnion by specific separation digestion. The results of FlowCytometry and differentiation induction indicated that the cultured AMSCs maintained thesurface markers, cytobiological properties and the multi-directional differentiationpotential as MSCs.Part Ⅱ Experimental research on the inhibitory effect of AMSCson glioma cells in vitroObjective: To observe the directional migration ability of AMSCs on human gliomacells and its effect on glioma cell growth, and to explore the mechanism on its gliomatropism and anti-tumor effect in vitro.Methods: A transwell cell migration assay was used to investigate the impact ofU251cells on migratory ability of AMSCs cells after different density of U251cells wereplaced in the lower transwell chamber. Using a microconcentrator, the protein in theAMSCs medium supernatant were concentrated and were added into the U251cell culturemedium. Subsequently, the invasion of U251cells was detected by transwell matrigelinvasion assay; the ultrastructure morphological changes of glioma cells were observedunder transmission electron microscope; the cell apoptosis was detected byAnnexinV-FITC-PI double staining; the mRNA expression of Casepase-3, Bax and Bcl-2was detected by RT-PCR.Results: Transwell assay results showed that the migration of AMSCs was stimulatedin a dose-dependent manner varying with U251cell density. AMSCs could inhibit theU251cells invasiveness in Matrigel in vitro. Under electron microscope, cell shrinkagewith apoptosis and high dense masses scattered in nucleuses were observed and pyknosisof nucleuses and apoptotic bodies could be found. Annexin V-FITC-PI double stainingshowed an apoptosis rate of9.34±4.27%after24h treated by AMSCs, while42.93±11.54%after48h, which display time-dependent manner. RT-PCR showed that comparedwith control, the AMSCs treated group expressed lower mRNA levels of Casepase-3andBax, but higher mRNA levels of Bcl-2, which also displayed a time-dependent manner. Allof the differences were statistically significant (P<0.05).Conclusions: The soluble chemotatic factors released from glioma cells couldmediate the activation of AMSCs migration in a dose-dependent manner. AMSCs can inhibit glioma cells growth and invasiveness and induce apoptosis of glioma cells in vitrowhich possible related to the decreased ratio of Bcl-2/Bax would ultimately led to theactivation of caspases-3and an induction of apoptosis.Part Ⅲ Experimental research on the inhibitory effect of AMSCson glioma in vivoObjective: To observe the effect of AMSCs survival, migration and transplantation onglioma in vivo, and to explore the mechanism of inhibition effect of AMSCs on gliomafurtherly.Methods: The models were used to induce human glioma in nude mice, which weresubsequently randomly divided into blank, control and experimental groups for treatmentwith empty, PBS and BrdU-labeled AMSCs respectively. Two weeks after transplantation,glioma tissues were processed for hematoxylin-eosin staining and immunofluorescence toobserve survival and spatial distribution of the transplanted AMSCs. The ultrastructuremorphological changes of glioma cells were observed under transmission electronmicroscope. The mRNA expression of Casepase-3, Bax and Bcl-2was detected byRT-PCR in tumor tissue.Results: After the mice were implanted intratumorally with empty, PBS and AMSCs,the tumor volume was measured. PBS-treated group was consistent with contrl, whileAMSCs-treated group significantly reduced tumor size on14d as compared to control(P<0.05). Under light microscope, it revealed that the tumor cells arranged densely withinvasive growth into striated muscle. Nuclear atypia, hemorrhage, necrosis and capillaryaccrementition were visible. BrdU-labeled AMSCs were found uniformly distributed in theglioma under fluorescence microscope. Under electron microscope, cell shrinkage and highdense masses scattered in nucleuses were observed and pyknosis of nucleuses andapoptotic bodies could be found. RT-PCR showed that compared with control, the AMSCstreated group expressed lower mRNA levels of Casepase-3and Bax, but higher mRNAlevels of Bcl-2, which also display a time-dependent manner. All of the differences werestatistically significant (P<0.05).Conclusions: Intratumoral injection of AMSCs may inhibit glioma growth byinducing an apoptosis of glioma in vivo, which demonstrates that that AMSCs transplantation may be applied as a new effective therapeutic approach for the treatment ofgliomas.