Abstract
BackgroundEvaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. It is of importance to establish a system to accurately quantify contaminated tumorigenic cells regardless of the format of stem cell product. In the present report, we aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells. MethodsHuman mesenchymal stem cells (hMSCs) and melanoma Mewo-Luc cells constitutively expressing luciferase were used. We stained Mewo-Luc cells with a cell linker then spiked onto hMSC suspensions and hMSC sheets. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. The samples spiked with Mewo-Luc cells were subjected to qPCR and FCM analyses, respectively for the quantification of Mewo-Luc cells. ResultsTen-fold serial dilutions of Mewo-Luc cells were performed accurately with small deviation. In samples spiked with or less than 100 cells in hMSC suspensions, and samples spiked with or less than 1,000 cells in hMSC sheets showed significantly higher cell numbers in calculations by FCM, respectively (suspensions; qPCR vs FCM: 100 cells: 59 ± 25 vs 232 ± 35 cells, p = 0.022/10 cells: 21 ± 7 vs 114 ± 27 cells, p = 0.030, sheets; qPCR vs FCM: 1,000 cells: 1723 ± 258 vs 5810 ± 878 cells, p = 0.012/100 cells: 110 ± 18 vs 973 ± 232 cells, p = 0.012/10 cells: 20 ± 6 vs 141 ± 36 cells, p = 0.030). ConclusionDifferences in accuracy between quantification methods should be considered in designing a tumorigenicity study model.
Highlights
IntroductionStem cell products manufactured from various stem cell populations (e.g. bone marrow-derived hematopoietic stem or stromal cells, skeletal myoblasts, pluripotent stem cells) are being increasingly applied for clinical use worldwide [1, 2, 3, 4, 5]
Stem cell products manufactured from various stem cell populations are being increasingly applied for clinical use worldwide [1, 2, 3, 4, 5]
We aimed to examine the accuracy of the quantification of spiked cell number with commonly used 2 methods [quantitative polymerase chain reaction and flow cytometry (FCM)] in 2 formats of stem cell products [human mesenchymal stem cell-derived cell suspensions and cell sheets] spiked with genetically and fluorescently labelled positive control cells recapitulating malignant transformation [a malignant melanoma cell line constitutively expressing luciferase (Mewo-Luc) labeled with a fluorescent cell linker], respectively
Summary
Stem cell products manufactured from various stem cell populations (e.g. bone marrow-derived hematopoietic stem or stromal cells, skeletal myoblasts, pluripotent stem cells) are being increasingly applied for clinical use worldwide [1, 2, 3, 4, 5]. Technical Report Series 878 of World Health Organization entitled “Recommendation for the evaluation of animal cell cultures as substrates for the manufacture of cell banks” recommends subcutaneous transplantation of 107 of subject cells into 10 immunodeficient nude mice and a monitoring of tumor formation for more than 16 weeks [7, 8]. Evaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. We aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. Conclusion: Differences in accuracy between quantification methods should be considered in designing a tumorigenicity study model
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