Generation Of Human Induced Pluripotent Stem Cells From Severe Preeclampsia Patients And The Preliminary Study Of Differentiation Into Trophoblast-like Cells

Part I G eneration Of Human Induced Pluripotent Stem Cells From Severe Preeclampsia PatientsObjective To reprogram the cord blood CD3+ T cells of patients with severe preeclampsia into iPSC with Sendai virus,and establish IPSC lines for patients with severe PE.Methods Ficoll gradient separation method was used to separate 3 um cord blood mononuclear cells from patients with severe PE.After T cells were activated by CD3 and IL-2 for 5-7 days,they were transfected by SeV with OCT4,SOX2,KLF4 and C-MYC genes.The transfected IPSC clones were planted in MEF cultures.Alkaline phosphatase,immunocytochemical detection of pluripotency molecular markers OCT4,TRA-1-60,NANOG,SOX2,karyotype analysis were conducted after passaging in vitro.Results SeV transfected T cells showed multiple IPSC clones for 10-15 days,and the transfection efficiency was 0.02-0.04%.These IPSC alkaline phosphatase stains were positive and expressed pluripotency markers.After 40 passages,the karyotype was still normal.Conclusion A small amount of cord blood samples from patients with severe PE can be reprogrammed to IPSC by Sendai virus.IPSC has pluripotency,expands the source cell samples of IPSC,extends the clinical application of IPSC,and have a good foundation for exploring the trophoblastic dysplasia-related diseases represented by PE.Part II Specific iPSC with feeder-free differentiation into Trophoblast-like CellsObjective After iPSC was transferred to a feeder-free stable culture,the preeclampsia-specific iPSC were differentiated into Trophoblast-like cells by the small molecules(BMP4).Methods The iPSC with good condition were selected and transferred to the pre-coated Matrigel six-well plates.The iPSC were cultured with m Te SR 1 basal medium and then cultured for 5-7 days in feeder-free basal medium containing 10 ng/ml human BMP4.After cytotrophoblast(CTB)differentiation,differentiation continues to functional Trophoblast-like cells.The morphological changes of cells after differentiation were observed dynamically,and the trophoblastic markers were sequentially detected by immunological detection,and the secretion of HCG was detected.RESULTS IPSC were in good condition after multiple passages in feeder-free conditions.The differentiated cell morphology was like trophoblast,expressing CTB marker CDX2,and the positive rate was higher than 80% on the 6th day.Continued differentiation can show EVT specific Marker HLA-G and secret HCG.Conclusion The IPSC of patients with severe preeclampsia can be differentiated into CTB by BMP4 without feeder layer,and can continue to differentiate into secretory trophoblast-like cells.