无异源培养条件下人多能干细胞系H1非病毒转染方法的比较和优化

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

中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (05) : 305 -310. doi: 10.3877/cma.j.issn.2095-1221.2021.05.008

论著

无异源培养条件下人多能干细胞系H1非病毒转染方法的比较和优化

刘锴¹^,^†(

)

^1. 100050　北京，首都医科大学附属北京友谊医院科研实验中心

收稿日期:2020-09-17 出版日期:2021-10-01
通信作者: 刘锴
基金资助:
北京市自然科学基金(7192048); 北京市李桓英医学基金会人才培养基金(lhyjjhrcpy2018-06)

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

Kai Liu¹^,^†()

^1. Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

Received:2020-09-17 Published:2021-10-01
Corresponding author: Kai Liu

全文 ( ) 下载PDF ( ) 导出引用

目的

将人类多能干细胞（hPSCs）进行基因编辑和分化是干细胞治疗的发展趋势。所以在无异源（Xeno-free）的培养条件下，系统地比较研究非病毒的干细胞转染方法有助于临床科研的发展。

方法

采用E8和Vitronectin人类干细胞Xeno-free培养系统培养人类H1干细胞系，从细胞活力、干细胞分化标记和转染效率三个方面比较并优化了3种主流的非病毒转染方法，分为空白对照组（正常培养的H1，未做任何处理）、转染GFP荧光质粒的电转组、脂质体组和磷酸钙组。多组间比较采用单因素方差分析和Tukey多重比较。

结果

3种转染方法对H1细胞的分化都没有显著的影响，转染后各组99.4﹪以上的细胞分型为SSEA1^-TRA-1-60⁺。从形态上观察，磷酸钙转染对干细胞损伤严重；电转和脂质体转染对细胞的损伤较小，且可以在Xeno-free培养系统下对干细胞形成有效转染。转染效率方面：优化前，与对照组、电转组、磷酸钙组的GFP阳性细胞率[（0.46±0.10）﹪、（0.06±0.10）﹪、（0.14±0.24）﹪]相比，脂质体组的转染效率（7.99±0.47）﹪最高（P均< 0.01）。优化试剂浓度和程序后，电转组的转染效率升高到（65.47±3.07）﹪，比优化后的脂质体组和磷酸钙组的效率都要高[（14.7±0.26）﹪和（4.84±0.25）﹪]，差异有统计学意义（P < 0.01）。

结论

电转和脂质体转染适用于Xeno-free培养系统下人类多能干细胞系H1的转染，优化后利用电转程序A13转染H1细胞可以获得本实验中最高的转染效率。

关键词: 无异源, 人多能干细胞, 非病毒转染

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

图1 荧光显微镜观察1 μg荧光质粒转染后48 h细胞转染效率比较（×40）

图2 流式细胞仪检测活力细胞和GFP阳性细胞百分比的圈门策略

图3 1 μg荧光质粒转染后统计GFP阳性细胞百分比

图4 流式细胞仪检测1 μg荧光质粒转染后Ghost表达

图5 1 μg荧光质粒转染后细胞绝对数量和活力

图6 流式细胞仪检测1 μg荧光质粒转染后SSEA1^-TRA-1-60⁺细胞的百分比

图7 优化后磷酸钙各组和脂质体各组的GFP阳性细胞百分比

图8 优化后磷酸钙各组和脂质体各组的GFP阳性细胞百分比

图9 荧光显微镜观察优化后不同电转程序A12、A13、A23、A27的转染效率（×20）

图10 优化后不同电转组的GFP阳性细胞百分比

1	Vo LT, Daley GQ. De novo generation of hscs from somatic and pluripotent stem cell sources[J]. Blood, 2015, 125(17): 2641-2648.
2	Dao Thi VL, Wu X, Belote RL, et al. Stem cell-derived polarized hepatocytes[J]. Nat Commun, 2020, 11(1): 1677. doi: 10.1038/s41467-020-15337-2.
3	Karagiannis P, Takahashi K, Saito M, et al. Induced pluripotent stem cells and their use in human models of disease and development[J]. Physiol Rev, 2019, 99(1):79-114.
4	Sachamitr P, Hackett S, Fairchild PJ. Induced pluripotent stem cells: Challenges and opportunities for cancer immunotherapy[J]. Front Immunol, 2014, 5:176.
5	Hamieh M, Dobrin A, Cabriolu A, et al. Car t cell trogocytosis and cooperative killing regulate tumour antigen escape[J]. Nature, 2019, 568(7750):112-116.
6	Themeli M, Kloss CC, Ciriello G, et al. Generation of tumor-targeted human t lymphocytes from induced pluripotent stem cells for cancer therapy[J]. Nat Biotechnol, 2013, 31(10):928-933.
7	Maxwell KG, Augsornworawat P, Velazco-Cruz L, et al. Gene-edited human stem cell-derived beta cells from a patient with monogenic diabetes reverse preexisting diabetes in mice[J]. Sci Transl Med, 2020, 12(540):eaax9106.
8	Uenishi G, Theisen D, Lee JH, et al. Tenascin c promotes hematoendothelial development and t lymphoid commitment from human pluripotent stem cells in chemically defined conditions[J]. Stem Cell Reports, 2014, 3(6):1073-1084.
9	Park MA, Jung HS, Slukvin I. Genetic engineering of human pluripotent stem cells using piggybac transposon system[J]. Curr Protoc Stem Cell Biol, 2018, 47(1):e63.
10	Hu K, Yu J, Suknuntha K, et al. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells[J]. Blood, 2011, 117(14):e109-119.
11	Chen G, Gulbranson DR, Hou Z, et al. Chemically defined conditions for human ipsc derivation and culture[J]. Nat Methods, 2011, 8(5):424-429.
12	Soh CL, Huangfu D. Crispr/cas9-mediated mutagenesis of human pluripotent stem cells in defined xeno-free e8 medium[J]. Methods Mol Biol, 2017, 1498: 57-78.
13	Trusler O, Huang Z, Goodwin J, et al. Cell surface markers for the identification and study of human naive pluripotent stem cells[J]. Stem Cell Res, 2018, 26:36-43.
14	Shubhra Q TH, Oyane A, Araki H, et al. Calcium phosphate nanoparticles prepared from infusion fluids for stem cell transfection: Process optimization and cytotoxicity analysis[J]. Biomater Sci, 2017, 5(5): 972-981.
15	Stewart CC, Steinkamp JA. Quantitation of cell concentration using the flow cytometer[J]. Cytometry, 1982, 2(4): 238-243.
16	Sinenko SA, Ponomartsev SV, Tomilin AN. Human artificial chromosomes for pluripotent stem cell-based tissue replacement therapy[J]. Exp Cell Res, 2020, 389(1): 111882.
17	Yu J, Hu K, Smuga-Otto K, et al. Human induced pluripotent stem cells free of vector and transgene sequences[J]. Science, 2009, 324(5928): 797-801.

[1]	周逸凡, 金颖. ERK信号通路在人多能干细胞的多能性状态调控中的作用[J/OL]. 中华细胞与干细胞杂志(电子版), 2023, 13(01): 27-35.
[2]	吴倩, 张梅. 胰岛类器官研究进展[J/OL]. 中华细胞与干细胞杂志(电子版), 2021, 11(02): 120-124.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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