Preparation Of Monoclonal Antibodies Against Human Mesenchymal Stem Cells(hMSCs) And Its Analysis Of Biological Characteristics

Mesenchymal stem cells(MSCs), Which reside within the stromal compartment of marrow, is one kind of stem cells which have the capacity of renewal and polypotential,the research of these cells had becoming an hotspot of the stem cells research presently. These cells have shown a favorable application in cell and gene engineering.With the breakthough of stem cells research in last decade,the biological characteristics of hMSCs were well known,however,untill now,the specific surface markers have not been found yet.The general isolation method is based on its culture characteristics,isotype character and potential proliferation abilities,and so many factors can influence the result.However,it is hard to manipulate the isolation method which costs a lot. The cells gained in this way are not fully homogeneous at all,and the comparison of the isolated cells among each laboratories is not perfect.Therefore,finding out the specific molecule is a significant work.In this study,BALB/C mice were Immunized with hMSCs,McAbs were prepared by hybridoma technique.Its biological characteristics were analysed by indirect immunofluorescence ,immunohistochemistry and flow cytometricanalysis,the application in MSCs isolation and the research of the membrane specific molecule are in progress.Methodsl.Isolation,culture and identification of hMSCs: marrows which was from healthy donors isolated by Ficoll-paque and the middle layer cells were cultured in basal medium(LG-DMEM) in the presence of 10% fetal bovine serum, while the cells which did not adhere to the culture bottle were discarded,and their surface markers were measured by flow cytometric analysis.2.The preparation of the anti-MSCs McAbs: the cells that we used to immunize BALB/C mice were from at least 2 different donors.The mice were injected intraperitoneally with 0.2ml(1.0x106cells/ml) every week.After the fifith immunization,hybridoma cells were prepared.Then the supernatant fluid was detected by indirect immunofluorescence to select the positive cells which were cloned by limited dilution method until the positive rates was up to 100%. These hybridomas were recultrued and injected to BALB/C mice which had already injected paraffine. All the ascites were collected and purified by ion exchange chromatography,the purification was checked by SDS-PAGE.3. Identification of the characteristics of these 5 McAbs: the relative affinities of 5 McAbs were determinated by ELISA, while the Phenotypes analysis was finished in the same way,and its biological characteristics were analysed by indirect immunofluorescence and immunohistochemistry.4.application:an immunomagnetic selection method was established and the McAbs were maked with FITC successfully.Resultsl.Isolation,culture and identification of hMSCs: hMSCs were cultured and expanded and their Phenotypes were analysed by flow cytometric : CD29+% CD44\ CD166\ CD34\ CD45\ HLA-DR'.2. The preparation of the anti-MSCs McAbs: McAbs against hMSCs were produced by using conventional cell fusion method.After fusion,the hybridomas were selected by indirect immunofluorescence.In initial screening by indirect immunofluorescence,27 McAbs can reactive with the hMSCs, 10 McAbs were then cloned.Among these,5 McAbs showing strong reaction with hMSCs(named ZUB1,ZUB4,ZUC3, ZUE12,and ZUF10). After 6 months culture in vitro,these five McAbs could stably secrete McAbs agaist hMSCs,the cells showing good state after freezing and anabiosis3.Identification of the characteristics of these McAbs: The relative affinities of 5 McAbs : ZUB1:1X1O6> ZUB4 : 1X105. ZUC3:1X1O\ ZUE12: 1X1O5^ ZUF10: 1X 105.Phenotypes analysis: ZUBK ZUE12^ ZUF10 recognize the same epitope on hMSCs while ZUC3 and ZUB4 can recognize other two different epitopes,respectively.The specificity of these five McAbs were Identified by indirect immunofluorescence and immunohistochemistry,these five McAbs were found to react to hMSCs but no-reaction with human cells lines and tissues.4 .application: an immunomagnetic selection method was established , the human marrow monocytes were selected in this way,and 4-5 X105 MSCs were gained from 7-10 X107 monocytes,the phnotype of these cells is CD29"\ CD44"\ CD105+> CD166V CD34" and CD45".these cells absolutely have the polypotential proliferation abilities.ConclusionIn this study,5 McAbs which can specially react to hMSCs were successfully prepared, These McAbs were identified in the present study might be useful for the isolation and characterization of MSCs,and the anti-MSC McAbs could be used in stem cell studies and developing more sensitive and specific diagnostic assays.