Journal
JOURNAL OF CLINICAL LABORATORY ANALYSIS
Volume 36, Issue 6, Pages -Publisher
WILEY
DOI: 10.1002/jcla.24464
Keywords
electroporation; human primary chorionic villi cells; induced pluripotent stem cells; non-integration
Categories
Funding
- College Students' innovation and entrepreneurship training program of Hainan Medical University [S202111810047]
- Natural Science Foundation of Guangdong Province [2019A1515010019]
- Guizhou Provincial Health Commission Science and Technology Fund, China [gzwkj2022--067]
- Major Science and Technology Program of Hainan Province [ZDKJ202003, ZDKJ2021037]
- National Natural Science Foundation of China [81960283, 82003144, 82072880]
- Postdoctoral Science Foundation of Hainan Province
- Natural Science Foundation of Hainan Province [822MS175]
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This study successfully generated CV-iPSCs from human primary chorionic villi cells and identified the optimal electroportion conditions. These CV-iPSCs exhibited pluripotency, normal karyotype, and the ability to differentiate into three germ layers. This research provides a novel strategy for investigating the pathogenesis of diseases and gene therapy.
Background Few studies have investigated the generation of induced pluripotent stem cells (iPSCs) derived from human primary chorionic villi (CV) cells. The present study aimed to explore an optimal electroporation (EP) condition for generating non-integrated iPSCs from CV cells (CV-iPSCs). Methods The EGFP plasmid was transfected into CV cells under different EP conditions to evaluate cell adherence and the rate of EGFP positive cells. Subsequently, CV cells were transfected with the pEP4-E02S-ET2K and pCEP4-miR-302-367 plasmids under optimal EP conditions. Finally, CV-iPSC pluripotency, karyotype analysis, and differentiation ability were investigated. Results Following EP for 48 h under different conditions, different confluency, and transfection efficiency were observed in CV cells. Higher cell density was observed in CV cells exposed to 200 V for 100 s, while higher transfection efficiency was obtained in cells electroporated at a pulse of 300 V for 300 s. To generate typical primitive iPSCs, CV cells were transfected with pEP4-E02S-ET2K and pCEP4-miR-302-367 plasmids using EP and were then cultured in induction medium for 20 days under selected conditions. Subsequently, monoclonal iPSCs were isolated and were evaluated pluripotency with AP positive staining, the expression of OCT4, SOX2, and NANOG in vitro and the formation of three germ layer teratomas in vivo. Conclusion CV-iPSCs were successfully established under the conditions of 100 mu l shock cup and EP pulse of 200 V for 300 s for two times. This may provide a novel strategy for investigating the pathogenesis of several diseases and gene therapy.
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