Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification.
Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification.
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DOI：
10.1007/s12015-022-10402-3
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发表时间：
2022-12
影响因子：
4.8
通讯作者：
Taniguchi, Hideki
中科院分区：
文献类型：
--
作者：
Yasui, Ryota;Matsui, Atsuka;Sekine, Keisuke;Okamoto, Satoshi;Taniguchi, Hideki
关键词：
对于安全的再生药物，细胞衍生产品中受污染或残留的致瘤性未分化细胞必须通过敏感的检测进行严格评估。尽管体外核酸测试提供了特别敏感的致瘤性相关测定，但人多能干细胞（hPSC）的可检测性部分地受到每次测试的少量RNA输入量的限制。为了克服这一限制，我们开发了逆转录环介导等温扩增（RT-LAMP）检测，是高度基因特异性和强大的干扰物质。LAMP可以容易地测定每次测试的微克级输入样品，并在简单的一锅反应中检测到0.00002% hiPSC污染的等效模型。为了评价细胞来源的总RNA，RT-LAMP利用多种多能性RNA检测hPSC来源的三系细胞中掺入的hPSC。我们还开发了多重RT-LAMP检测，并进一步应用于原位细胞成像，实现了多能性蛋白和RNA的特异性共染色。我们的尝试揭示了RT-LAMP方法在致瘤性相关检测中的实用性，支持再生医学的实际应用。在线版本包含补充材料，可通过10.1007/s12015-022-10402-3获取。
For safe regenerative medicines, contaminated or remaining tumorigenic undifferentiated cells in cell-derived products must be rigorously assessed through sensitive assays. Although in vitro nucleic acid tests offer particularly sensitive tumorigenicity-associated assays, the human pluripotent stem cell (hPSC) detectability is partly constrained by the small input amount of RNA per test. To overcome this limitation, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays that are highly gene specific and robust against interfering materials. LAMP could readily assay microgram order of input sample per test and detected an equivalent model of 0.00002% hiPSC contamination in a simple one-pot reaction. For the evaluation of cell-derived total RNA, RT-LAMP detected spiked-in hPSCs among hPSC-derived trilineage cells utilizing multiple pluripotency RNAs. We also developed multiplex RT-LAMP assays and further applied for in situ cell imaging, achieving specific co-staining of pluripotency proteins and RNAs. Our attempts uncovered the utility of RT-LAMP approaches for tumorigenicity-associated assays, supporting practical applications of regenerative medicine. The online version contains supplementary material available at 10.1007/s12015-022-10402-3.
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影响因子：
5.6
作者：
Avendaño C;Patarroyo MA
通讯作者：
Patarroyo MA
影响因子：
3.7
作者：
Gropp M;Shilo V;Vainer G;Gov M;Gil Y;Khaner H;Matzrafi L;Idelson M;Kopolovic J;Zak NB;Reubinoff BE
通讯作者：
Reubinoff BE
影响因子：
2.7
作者：
Basu, Amar S.
通讯作者：
Basu, Amar S.
影响因子：
64.8
作者：
La Manno G;Soldatov R;Zeisel A;Braun E;Hochgerner H;Petukhov V;Lidschreiber K;Kastriti ME;Lönnerberg P;Furlan A;Fan J;Borm LE;Liu Z;van Bruggen D;Guo J;He X;Barker R;Sundström E;Castelo-Branco G;Cramer P;Adameyko I;Linnarsson S;Kharchenko PV
通讯作者：
Kharchenko PV
影响因子：
3.1
作者：
Jones M;Williams J;Gärtner K;Phillips R;Hurst J;Frater J
通讯作者：
Frater J