Establishment Of A New BCR-ABL Positive Leukemia Cell Line Based On The Microvesicle-induced Malignant Transformation Of CD34+ Hematopoietic Stem/progenitor Cells

Biological study of microvesicles derived from chronic myeloid leukemia cells line K562Aim:To study the biological characterization of microvesicles(MVs)released by the chronic myeloid leukemia cell line,K562 cells(K562-MVs).Methods:Through differential centrifugation,we isolated K562-MVs from culture supernatant of K562 cells.By BCA protein concentration assay,we evaluated and compared the K562-MVs secreting levels under the cell culture condition of different concentration of febal bovine serum(FBS).It was worthy to note that cell culture condition were exquisitely maintained,as well as the centrifugation processes.Transmission electron microscopy(TEM),scanning electron microscopy(SEM),flow cytometry analysis(FCM)were performed to observe the morphology and size distribution of K562-MVs.Semi-quantitative manners,western blot(WB)and qRT-PCR were used to analyze the components of K562-MVs.Furthermore,Application of RNA sequencing compared the highly expressed mRNA contents in K562-MVs and multiple myeloma cell line RPMI8226 derived MVs(RPMI8226-MVs),which helped us to analyze the gene expression characteristics in K562-MVs.Results:The FBS-free cell culture condition enhanced the secretion of K562-MVs.TEM,SEM and FCM analysis illustrated the spherical bilayer structure of K562-MVs,with a diameter less than 1 um.Besides,K562-MVs expressed CD63,CD81,TSG101 and ALIX,which were MV-enriched proteins and expected to be present in MVs.Importantly,K562-MVs could regularly harbor the BCR-ABL fusion gene despite the different extraction processes.The highly expressed RNA in K562-MV and RPMI8226-MV showed a high degree of commonality.The major constituents were ribosome proteins and pseudogenes(most of which are genes of the ribosomal protein family).The second were the redox reaction related genes.The third was cytoskeleton consisting of actin,dragline silk protein,and myosin,which constitute the cytoskeleton and were responsible for the cell contractions.Conclusion:The differential centrifugation method conducted in our laboratory was feasible and efficient to harvest K562-MVs.Stress altered the K562-MV secretion levels,thus it was necessary to establish and maintain a stable cell culture condition.K562-MVs harbored the general characterization of MVs,but also K562 specific features.Establishment and characterization of the new BCR-ABL positive leukemia cell lineAim:To establish the microvesicle-induced malignant transformation experimental system with CD34+ hematopoietic stem/progenitor cells(HSPCs)in vitro,and to characterize the malignant transformed leukemia cells.Methods:1)Normal bone marrow aspirate,cord blood and mobilized peripheral blood samples were collected from healthy volunteer,and processed with Ficoll gradient centrifugation and magnetic beads to harvest CD34+ hematopoietic stem/progenitor cells(HSPCs).The enriched CD34+ HSPCs were cultured in vitro with StemSpanTM H3000 medium supplemented with recombinant cytokines including the stem cell factor(SCF),thrombopoietin(TPO),Flt3-ligand(FltL3)and interleukin-6(IL-6).By changing target cells types,cell culture density,concentration and induction frequency of microvesicles(MVs),we determined the optimal parameters of the malignant transformation system,realizing the malignant transformation of CD34+ HSPC.2)The MV-induced leukemia cells(MiLC)were characterized and analyzed by inverted microscopy,Wright-Giemsa staining,transmission electron microscopy,chemical straining,flow cytometry,chromosome karyotype analysis,fluorescence in situ hybridization,western blot,qRT-PCR,cell viability and cell cycle analysis,whole genome sequencing,transcriptome sequencing and phosphorylation quantitative proteomics.Consistently,we performed the above detections of K562 cells.Results:1)We established a specific MV-induced experimental system,which complete the malignant transformation of CD34+ HSPC.The HSPC culture density was 4*10^4/ml,K562-MV treatment concentration maintained 1 ug/ml and K562-MV treatment frequency kept two times per day,which were the optimal transformation condition.2)MiLC differed with K562 cells on cell morphology,cell biological behavior,molecular biology,cytogenetics,genome,transcriptome and quantitative proteomic.Up till now,MiLC had been propagated to P40 and for 6 months in vitro.More importantly,it still retained the original biological characteristics and carried Ph chromosome and BCR-ABL fusion gene.Conclusion:K562-MVs could induce the malignant transformation of normal HSPCs.We successfully harvested a novel leukemia cell line,which stably carried the Ph chromosome and BCR-ABL gene.