Reprogramming Of HEK-293T Cells Into IPSCs With A Single Gene Oct4 Mediated By Superparamagnetic Iron Oxide Nanoparticles

The strategy of reprogramming somatic cells into induced pluripotent stem cells(iPSCs)has attracted much attention because of its ability to avoid the ethical controversy caused by embryonic stem cells(ESCs).However,the problems such as low efficiency,long time consuming and poor safety limit its clinical applications.In view of those problems,in this study,superparamagnetic iron oxide nanoparticles coated with polyethyleneimide(SPION-PEI),a novel non-viral gene carrier,were used to mediate the overexpression of a single transcription factor(Oct 4)in combination with the use of small molecular substances including CHIR99021?PD0325901 and A-83-01(3i)to reprogram human embryonic kidney293T(HEK-293T)cells,in order to establish a new method for obtaining iPSCs with high safety and considerable efficiency.To effectively increase the amount of SPION-PEI carrying Oct4 into HEK-293T cells and improve the efficiency of cell transfection,the concentration of SPION-PEI with good biocompatibility was first screened to determine the carrying capacity of SPION-PEI binding transcription factor Oct4 and the possible mechanism of SPION-PEI carrying Oct4 into HEK-293T cells.The EGFP positive clones were proliferated after screened with G418,and then the positive clones were treated with3i to reprogram the HEK-293T cells.The strategy can effectively avoid the potential tumorigenic risk caused by the intervention of virus vectors and oncogenes including c-Myc and Klf4,and accelerate the clinical application process of iPSCs.Part 1:Study on the cytotoxicity effects of SPION-PEI on HEK-293T cellsIn order to screen an appropriate concentration of SPION-PEI with good biocompatibility and further use it as a non-viral vector to effectively carry transcription factors Oct 4 into HEK-293T cells,different concentrations of SPION(10 ng/?l?50 ng/?l?100 ng/?l?200ng/?l?400ng/?l)or different concentrations of SPION-PEI(10 ng/?l?50 ng/?l?100 ng/?l?200ng/?l?400ng/?l)were used to be incubated with HEK-293T cells.The effects of SPION or SPION-PEI on HEK-293T cells were studied by comparing the differences of cell adhesion morphology,survival rate,proliferation toxicity,oxidative damage and DNA damage.The results showed that there was no significant difference between 10 ng/?l SPION-PEI treatment group and 10ng/?l SPION treatment group(P>0.05).Under other same concentration,the effects of SPION-PEI treatment on adhesion morphology of HEK-293T cells were smaller than that of SPION treatment group,and the survival rate,proliferation toxicity,oxidative damage and DNA damage of HEK-293T cells were lower than those of SPION treatment group.With the increase of treatment concentration of SPION or SPION-PEI,the effects of morphological changes,survival rate,proliferation toxicity,oxidative damage and DNA damage of HEK-293T cells increased.Compared with the control group,the effects of 10 ng/?l or 50 ng/?l SPION-PEI on the adherent morphology,survival rate,proliferation toxicity,oxidative damage and DNA damage of HEK-293T cells were not significant(P>0.05).Therefore,it is advisable to select the appropriate concentration of SPION-PEI carrying Oct 4 in the range of less than 50 ng/?l for transfection of HEK-293T cells.Part 2:Optimization of conditions for SPION-PEI as a gene vector to carry Oct 4 into HEK-293T cellsBy the combination efficiency test of SPION-PEI and Oct 4,and DNase?protection test,the carrying capacity of SPION-PEI to Oct 4 plasmid DNA and its protective effect against DNase?were investigated.The results showed that the binding efficiency of Oct 4 reached 97.13±0.24%when the mass ratio of SPION-PEI to Oct4 plasmid DNA was 1:4,and the binding efficiency was almost no longer increased when the amount of SPION-PEI was increased,indicating that the binding ability was close to saturation.When the mass ratio of SPION-PEI to Oct 4 is 1:1,SPION-PEI and Oct 4 are fully combined.After digesting and eluting the Oct 4 plasmid DNA on SPION-PEI,the brightness of agarose gel band was basically the same as that of control group,which indicated that the protective efficiency of SPION-PEI on plasmid Oct 4 was close to 100%.The effects of different concentrations of SPION-PEI/Oct4 complexes on the transfection efficiency of HEK-293T cells were compared in the presence of external magnetic field for20 min.The results showed that when the concentration of SPION-PEI/Oct4 complex was 1.5 ng/?l,the cell transfection efficiency was the highest,up to 76.47±2.82%.In order to explore the mechanism of SPION-PEI entering into HEK-293T cells and explore the possibility of further improving the transfection efficiency of SPION-PEI/Oct4,this experiment was carried out under the condition of exogenous magnetic field.The effects of the different concentration of clathrin mediated endocytosis inhibitors—chlorpromazine(CPZ,the concentration was 1?g/ml,2?g/ml,4?g/ml,8?g/ml,16?g/ml,respectively),caveolin mediated endocytosis inhibitors—nystatin(the concentration was 5?g/ml,10?g/ml,20?g/ml,40?g/ml,80?g/ml,respectively),ATP synthesis inhibitor—2-deoxy-D-glucose(2-D-G,the concentration was 1 mM,5 mM,10 mM,20 mM,40 mM,respectively),macropinocytosis inhibitor—amiloride(the concentration was 0.5 mM,1mM,2mM,4mM,8mM,respectively),macropinocytosis activator—phorbol ester(the concentration was 50nM,100nM,200nM,400nM,800nM,respectively)pretreatment on SPION-PEI/Oct4 transfection efficiency were compared.The transfection efficiency of CPZ and nystatin treatment groups was not significantly different from that of control group(75.3±2.36%,75.12±1.55%,75.43±1.76%,73.95±2.18%,74.17±2.2%vs 76.47±2.82%;74.94±1.83%,74.47±1.17%,73.49±1.47%,73.75±1.39%,74.06±1.42%vs 76.47±2.82%)(P>0.05).The transfection efficiency in the 1mM 2-DG treatment group was significantly lower than that of control group(4.63±0.56%vs 76.47±2.82%,P<0.01),and the transfection efficiency was close to 0(0.3±0.58%)when the 2-DG concentration was 10mM.The transfection efficiency in 1mM amiloride treatment group was significantly lower than that of control group(33.8±2.31%vs 76.47±2.82%,P<0.01).With the increase of amiloride concentration,the transfection efficiency was not significantly different from that of 1mM treatment group(P>0.05).The transfection efficiency in the 50nM PMA treatment group was not significantly different from that in the control group(76.29±2.04%vs 76.47±2.82%,P>0.05).When the PMA concentration was 100 nM,200 nM,400 nM,800 nM,the transfection efficiency was significantly higher than that of the control group(P<0.05),where the transfection efficiency was the highest when the PMA concentration was 100 nM,up to 83.44±2.16%.Therefore,it is advisable to select the suitable condition that the mass ratio of SPION-PEI to Oct 4plasmid DNA is 1:1,the concentration of SPION-PEI/Oct 4 complex is 1.5 ng/?l,and the external magnetic field is applied for 20 minutes.The transfection efficiency of HEK-293T cells can be improved by adding 100nM PMA complex.Part 3:Reprogramming of HEK-293T cells into iPSCs with a single gene Oct 4 mediated by SPION-PEIIn order to improve the safety of iPSCs in clinical application from the aspect of transgene vector and transcription factor,we used non-viral transgenic vector SPION-PEI to mediate the overexpression of single transcription factor Oct4.At the same time,the combination of small molecule substances CHIR99021,PD0325901 and A-83-01(3i)was used to avoid introducing tumor-causing genes such as c-Mycf,Klf 4 to reprogram HEK-293T into iPSCs.Their pluripotent characteristics were identified by morphological observation,alkaline phosphatase(AKP)staining,immunocytochemistry staining of Oct-4and SSEA-3,formationand differentiation of embryoid body(EB).The results showed that the number of iPSCs-like colonies reached 78.23±8.46 from EGFP~+cells after 3 weeks of continuous culture on 3i medium,but no iPSCs-like colony was formed from EGFP~+cells cultured on normal stem cell culture medium without 3i at the same time.iPSCs-like colonies induced by Oct 4 overexpression combined with the treatment of small molecule 3i from HEK-293T cells showed"island"shaped,with clear boundaries and strong refraction.Under high power microscope,the nuclei of the clone cells were large and the cytoplasm was small,and the cell spacing was very close,morphologically similar to that of ESCs.The selected iPSCs like colonies were reddish brown with AKP staining,Oct-4 and SSEA-3immunocytochemical staining.After 5 days of suspension culture,the isolated iPSCs-like cells formed a simple EB,and formed a significant lumen EB after 7 days,the resulting embryoid bodies were moved into a new 24-well plate,and the cells spontaneously differentiated into neuroepithelium cells after 7 days of adherent growth,EB adherent differentiated cells expressed the marker genes of three germ layeres including AFP(Alpha-fetoprotein,endoderm),Brachyury(mesoderm)and AAT(Alpha-1-antitrypsin,ectoderm).The results indicated that the iPSCs-like cell colonies showed the characteristics of pluripotent stem cells.Therefore,HEK-293T cells could be reprogrammed to iPSCs by SPION-PEI mediated the overexpression of single transcription factor Oct 4 and combined treatment with CHIR99021,PD0325901and A-83-01.Conclusions:The concentration of SPION-PEI belows 50 ng/?l has no obvious toxic effect on HEK-293T cells.The transfection effect is optimal under the condition that the mass ratio of SPION-PEI to Oct4 plasmid DNA is 1:1,the concentration of SPION-PEI/Oct4 complex is 1.5 ng/?l,the external magnetic field is applied for 20 minutes,and the 100nM PMA is added to pretreat 30min.SPION-PEI mediated the overexpression of a single transcription factor Oct4 while simultaneously using the small molecules including CHIR9921,PD0325901 and A-83-01,which could convert HEK-293T cells into iPSCs.The application of magnetic nanotechnology to over-expression of single transcription factor Oct 4in HEK-293T cells combined with small molecules to generate iPSCs will lay a foundation for further improving its safety and developing clinical disease diagnosis and treatment as well as regenerative medicine.