The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction
Abstract In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method—the soft agar colony formation assay using polymerase chain reaction (PCR)—to evaluate tumorigenicity. MRC-5 cell...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-32442-6 |
| _version_ | 1797850137746210816 |
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| author | Daichi Nakamura |
| author_facet | Daichi Nakamura |
| author_sort | Daichi Nakamura |
| collection | DOAJ |
| description | Abstract In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method—the soft agar colony formation assay using polymerase chain reaction (PCR)—to evaluate tumorigenicity. MRC-5 cells, contaminated with HeLa cells, were cultured for up to 4 weeks in soft agar medium. Cell-proliferation-related mRNAs, Ki-67 and cyclin B, could be detected in 0.01% of HeLa cells after 5 days of culture, whereas cyclin-dependent kinase 1 (CDK1) could be detected after 2 weeks. On the other hand, CDK2, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance protein 7 (MCM7) were not useful to detect HeLa cells even after 4 weeks of culture. The cancer stem cell (CSC) markers, aldehyde dehydrogenase 1 (ALDH1) and CD133 in 0.01% of HeLa cells, could be detected 2 and 4 weeks after culture, respectively. However, another CSC marker CD44 was not useful because its expression was also detected in MRC-5 cells alone. This study suggests that the application of the PCR method to the soft agar colony formation assay could evaluate not only the tumorigenic potency in the short-term but also characterize the colonies, eventually improving the safety of CTPs. |
| first_indexed | 2024-04-09T18:56:34Z |
| format | Article |
| id | doaj.art-f2c4f6ea22834ec191058cad95fba984 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-09T18:56:34Z |
| publishDate | 2023-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-f2c4f6ea22834ec191058cad95fba9842023-04-09T11:13:06ZengNature PortfolioScientific Reports2045-23222023-04-0113111110.1038/s41598-023-32442-6The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reactionDaichi Nakamura0BoZo Research Center Inc., Tsukuba Research InstituteAbstract In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method—the soft agar colony formation assay using polymerase chain reaction (PCR)—to evaluate tumorigenicity. MRC-5 cells, contaminated with HeLa cells, were cultured for up to 4 weeks in soft agar medium. Cell-proliferation-related mRNAs, Ki-67 and cyclin B, could be detected in 0.01% of HeLa cells after 5 days of culture, whereas cyclin-dependent kinase 1 (CDK1) could be detected after 2 weeks. On the other hand, CDK2, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance protein 7 (MCM7) were not useful to detect HeLa cells even after 4 weeks of culture. The cancer stem cell (CSC) markers, aldehyde dehydrogenase 1 (ALDH1) and CD133 in 0.01% of HeLa cells, could be detected 2 and 4 weeks after culture, respectively. However, another CSC marker CD44 was not useful because its expression was also detected in MRC-5 cells alone. This study suggests that the application of the PCR method to the soft agar colony formation assay could evaluate not only the tumorigenic potency in the short-term but also characterize the colonies, eventually improving the safety of CTPs.https://doi.org/10.1038/s41598-023-32442-6 |
| spellingShingle | Daichi Nakamura The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction Scientific Reports |
| title | The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction |
| title_full | The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction |
| title_fullStr | The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction |
| title_full_unstemmed | The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction |
| title_short | The evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction |
| title_sort | evaluation of tumorigenicity and characterization of colonies in a soft agar colony formation assay using polymerase chain reaction |
| url | https://doi.org/10.1038/s41598-023-32442-6 |
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