Construction Of Human Induced Pluripotent Stem Cells And Differentiation Into Vascular Smooth Muscle Cells

Objective: Constructed induced pluripotent stem cells(iPSCs)derived from human peripheral blood mononuclear cells(PBMCs),and identified the constructed cells,which laid the foundation for the establishment of induced pluripotent stem cell bank for cardiovascular disease.basis.iPSCs were differentiated into vascular smooth cells(VSMCs)by monolayer cell differentiation,and the relevant changes during differentiation were observed.The differentiated cells were identified to ensure reliability,and sufficient research materials were provided for subsequent research.Methods: PBMCs were isolated and constructed into iPSCs by electroporation using non-integrated plasmids.The constructed iPSCs were identified by light microscopy observation of cell morphology,alkaline phosphatase staining,cellular immunofluorescence staining and in vivo three-embryonic differentiation experiments.The induced pluripotent stem cells were induced to differentiate into vascular smooth muscle cells by single-layer cell method,and the cell morphology,Calcein-AM,cellular immunofluorescence staining and Quantitative Real-time PCR(Q-PCR)were observed under light microscope.Methods The differentiated VSMCs were identified.Results: 1.PBMCs were successfully isolated and cultured,and PBMCs were induced into iPSCs by non-integrated plasmid electroporation.Under light microscopy,iPSCs exhibit a similar growth pattern to embryonic stem cells(ESCs),with large round or elliptical shapes,neat edges,and tight cell alignment.Under high magnification,the nuclei are large and round.One to many nuclei with less cytoplasm.AP staining showed that the induced pluripotent stem cells were strongly positive,showing the appearance of purple-black particles in the clones.Cellular immunofluorescence assay showed that Nanog,Sox2,Oct4 and TRA-1-60 were strongly positive in all iPSCs.In vivo three-embryonic differentiation experiments confirmed that iPSCs can differentiate into the cellular components of the three germ layers,clearly indicating that they are pluripotent.2.For the differentiated iPSCs-VSMCs,the cell morphology was spindle-shaped andpolygonal under light microscopy,and the cells were differentiated to maintain the cultured cells for Calcein-AM to confirm cell survival.The mRNA levels of ?-SMA,CNN1 and SM22? in iPSCs-VSMCs were significantly increased by iPSCs and iPSCs-VSMCs constructed by Q-PCR.Immunofluorescence staining showed that the differentiated cells expressed specific markers ?-SMA,CNN1,SM22? at the protein level.Conclusion: iPSCs were successfully constructed by PBMCs by electroporation of non-integrated plasmids,which expressed pluripotency markers and had multi-directional differentiation potential in vivo.Monolayer cell differentiation is a reliable method for the differentiation of iPSCs into VSMCs,and the differentiated cells exhibit high expression of specific markers.It laid the foundation for subsequent research.