Induced Pluripotent Stem Cells (iPSC) From Patients With Acute Myeloid Leukemia Based On Cellular Reprogramming

Object: To establish the stable induced pluripotent stem cells derived frommyeloid blasts based on cellular reprogramming, for the further study of pathogenesisof acute myeloid leukemia and the potential target molecules needed forcarcinogenesis.Methods:1. Commercial lentiviral vectors expressed with4reprogrammingfactors: OCT4, SOX2, C-MYC, KLF4were packaged with two helper plasmidspCMV-dR8.91, pCMV-VSV-G,293T package cells. The construction was confirmedby RT-PCR and restriction enzyme digestion anaysis and the lentivirus titer wasdetermined using GFP/DAPI based on cell count by Image J softwrae.2. Primarymouse embryo fibroblast (PMEF) feeder cells were isolated and prepared from CF-1inbred mouse strain to help maintain pluripotency and to provide a cellular matrix forstem cells growing.3. HL60cells were infected for24hours by the lentiviralcocktails carrying4factors (OCT4, SOX2, C-MYC and KLF4) with the multiplicityof infection (MOI) of2:1,5:1,10:1respectively, afterwards transferred on feederlayers of mitomycin-C-treated CF-1cells in6-well plates in hES medium withbFGF(10μg/μl) for7days, and then transferred to condition medium until the cellclones growed to appropriate size and appeared ES-like morphology4. Mononuclearcells (MNC) from venous blood of a patient with acute myeloid leukemia M4isolatedby Ficoll gradient. Primary AML blasts were selected from the MNC with the depletion of CD3+T cells using CD3microbeads, and then incubated in RPMI1640medium with the cytokines including hSCF, hFLT3L and IL-3for4days forproliferation. Using the same protocol as mentioned above, the proliferated AMLcells were infected for24hours by the lentiviral cocktails and transferred on feederlayers until the cell clones formation.5. The cells acquired from the infiltrated skintissue of a patient with AML(M6) were maintained in DMEM with10%FBS for2generations expansion. The cell phenotype like CD13, CD34, CD71, CD235a,etc.were analyzed by flow cytometry. Cells were then infected by the lentivirus cocktailswith Multiplicity of infection (MOI) of5:1,10:1,20:1respectively. The procedure ofcells induced by lentivirus cocktail was repeated with the AML infiltrated cells.6hES-like cell clones were picked between day30and day35after lentivirus infectionand were preliminarily confirmed stem cells characteristic by alkalinephosphatase(AP) staining.6. Two of five iPS cell clones were selected for furtheridentification the biological features: cell phenotype like antigen CD13, CD34, CD71,CD235a,etc.by flow cytometry and STR allele change by capillary electrophoresiswith fluorescence detection fragment analysis were used to determine what the iPScells originated; The stem cell properties were tested by immunostaining of AP,NANOG, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81; mRNA expression of OCT4,SOX2, C-MYC，KLF4,Nanog, Lin28, FOXD3,etc. by RT-PCR or QT-PCR and thedemethylation change in the promoter regions of OCT4gene by bisulfite genomicsequencing. Cell pluripotency was verified by in vitro experiments EB formationassay and in vivo experiments teratoma formation test. The possibility of iPS cellsaberration was excluded by karyotype and STR analysis.Results:1.lentiviral vectors expressing4factors OCT4, SOX2, C-MYC andKLF were successfully packaged with the expected titer over106TU/ml.2. Primarymouse embryo fibroblast (PMEF) feeder cells were successfully isolated andprepared from CF-1inbred mouse strain3. Cell clones derived from HL60cell linesappeared like hESC at d14after infection. The expression of CD34+on the cellsurface increased from1.77%to98.42%after cell reprogramming.4. Cell clones derived from MNC from the patient with AML M4appeared like hESc with welldefined borders and prominent mucleoli at d13after lentivirus cocktail infection, butthe further functional identification was not performed due to the rapid celldifferentiation.5. AP staining confirmed the iPS cell clones were successfullyacquired from the leukemia cells in infiltrated skin tissue of the patient withAML(M6). The phenotyping analysis shown the expression of the myeloid antigenCD13,CD117and erythroid antigen CD71,CD235a on cells befored thereprogramming, while,their expressions decreased or disapperaed on the cell surfaceafter the reprogramming. The iPS cells appeared AP staining positive in the initialidentification, the further anaysis suggested they had similar cell antigen markerswith hES cells，for example NANOG, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. Thegene expressions of OCT4, SOX2indicated that the exogenous gene weredown-regulation or silence while the endogenous gene were up-regulation. Bisulfitegenomic sequencing analyses of the promoter regions of OCT4gene showed highlyunmethylated in iPS cells compared to the parental cells. EB formation assay in vitroand teratoma formation test in vivo indicated that the iPS cells have the potential todifferentiate to three germ layers. STR analysis and karyotype anaysis shown noaberration during the reprogramming.Conclusion:1. Lentivirus vector expressing OCT4, SOX2, C-MYC and KLF4gene weresuccessfully packaged. Primary mouse embryo fibroblast (PMEF) feeder cells weresuccessfully isolated.2. ES-like cell clones were successful induced from both HL-60and AMLblasts. However the stable iPS cell lines were not established.3. The iPS cell lines derived from a patient with M6were successfullyestablished without the aberration.