Comparison and optimization of nonviral transfection methods for human pluripotent stem cell line (H1) under xeno-free culture system

doi:10.3877/cma.j.issn.2095-1221.2021.05.008

Abstract

Abstract:

Objective

Human pluripotent stem cells (hPSCs) emerge as an increasingly important role in stem cell therapy. Systematic and comparative study of nonviral transfections under xeno-free culture could contribute to the clinical research.

Methods

Used chemically defined E8 medium on Vitronectin to culture hPSCs line H1, and compared the transfection efficiency, cell viability and differentiation markers among blank control group and nonviral transfection groups (including electroporation, lipofection, and calcium phosphate transfection) . One-way ANOVA with Tukey post test was used for multiple comparisons.

Results

Cells in all groups exhibited normal hPSCs morphology and expressed pluripotency markers SSEA1^-TRA-1-60⁺ (> 99.4﹪) . Stem cells were severely damaged by calcium phosphate transfection. Electroporation and lipofection did less damage to stem cells and had efficient stem cells transfection under xeno-free culture. As to transfection efficiency, lipofection group had the highest GFP transfection efficiency [ (7.99±0.47) ﹪, P < 0.01], compared to control group, electroporation group and calcium phosphate transfection group [ (0.46±0.10) ﹪、(0.06±0.10) ﹪、(0.14±0.24) ﹪] , respectively. After optimizing quantity and program, transfection efficiency of electroporation reached (65.47±3.07) ﹪, while electroporation and calcium phosphate transfection only reached (14.7 ± 0.26) ﹪ and (4.84±0.25) ﹪, and the difference was statistically significant (P < 0.01) .

Conclusion

Electroporation and lipofection enabled robust generation of GFP-positive hPSCs in xeno-free culture. After optimization, electroporation (program A13) had the highest transfection efficiency.

Key words: Xeno-free, Human pluripotent stem cells, Nonviral transfection

Kai Liu. Comparison and optimization of nonviral transfection methods for human pluripotent stem cell line (H1) under xeno-free culture system[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(05): 305-310.

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Figures/Tables 10

References 17

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