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Effects of Long-Term In Vitro Expansion on Genetic Stability and Tumor Formation Capacity of Stem Cells

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Abstract

Stem cell therapeutics are emerging as novel alternative treatments for various neurodegenerative diseases based on their regenerative potentials. However, stem cell transplantation might have side effects such as tumor formation that limit their clinical applications. Especially, in vitro expansion of stem cells might provoke genetic instability and tumorigenic potential. To address this issue, we analyzed genomic alterations of adult human multipotent neural cells (ahMNCs), a type of human adult neural stem cells, after a long-term in vitro culture process (passage 15) using sensitive analysis techniques including karyotyping, array comparative genomic hybridization (aCGH), and whole exome sequencing (WES). Although karyotyping did not find any major abnormalities in chromosomal number or structure, diverse copy number variations (CNVs) and genetic mutations were detected by aCGH and WES in all five independent ahMNCs. However, the number of CNVs and genetic mutations did not increase and many of them did not persist as in vitro culture progressed. Although most observed CNVs and genetic mutations were not shared by all five ahMNCs, nonsynonymous missense mutations at MUC4 were found in three out of five long-term cultured ahMNC lines. The genetic instability did not confer in vivo tumorigenic potential to ahMNCs. Collectively, these results indicate that, although genetic instability can be induced by long-term in vitro expansion of stem cells, it is not sufficient to fully exert tumor formation capacity of stem cells. Other functional effects of such genetic instability need to be further elucidated.

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Funding

This research was supported by grants from Ministry of Food and Drug Safety (14172MFDS974 and 18172MFDS182), National Research Foundation (2016R1A5A2945889 and 2019R1F1A1060243), Korea Basic Science Institute (2020R1A6C101A191), and the Samsung Biomedical Research Institute grant (SMX1161411).

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Author notes

  1. Hyun Nam, In-Hee Lee, Jason K. Sa and Sung Soo Kim contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy & Cell Biology, Sungkyunkwan University School of Medicine, 2066 Seobu-ro, Suwon, Gyeonggi-do, 16419, South Korea

    Hyun Nam, Sung Soo Kim, Hee-Jang Pyeon, Kee Hang Lee, Kyunghoon Lee & Kyeung Min Joo

  2. Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea

    Hyun Nam, Sung Soo Kim, Hee-Jang Pyeon, Kee Hang Lee, Kyunghoon Lee, Sun-Ho Lee & Kyeung Min Joo

  3. Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul, 06351, South Korea

    Hyun Nam, Sung Soo Kim, Hee-Jang Pyeon, Kee Hang Lee, Sun-Ho Lee & Kyeung Min Joo

  4. Computational Health Informatics Program, Boston Children’s Hospital, Boston, MA, 02115, USA

    In-Hee Lee

  5. Department of Biomedical Sciences, Korea University College of Medicine, Seoul, South Korea

    Jason K. Sa

  6. Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, 06351, South Korea

    Sung Soo Kim, Sun-Ho Lee & Kyeung Min Joo

  7. Biomedical Institute for Convergence at Sungkyunkwan University (BICS), Sungkyunkwan University, Suwon, 16419, South Korea

    Kyunghoon Lee & Kyeung Min Joo

  8. Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 06351, South Korea

    Sun-Ho Lee

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  1. Hyun Nam
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Contributions

Hyun Nam, In-Hee Lee, and Jason K Sa designed and performed all the experiments. They analyzed the data of those experiments and wrote the manuscript. Hee-Jang Pyeon was involved in the characterization of ahMNCs. Hee-Jang Pyeon participated in the visualization of data generated from aCGH and WES. Kee Hang Lee was involved in the tumorigenicity of ahMNCs. Kyeunghoon Lee advised the whole research procedures. Sun-Ho Lee and Kyeung Min Joo designed the research concept, guided the experiments and corrected the manuscript. Especially, Sung Soo Kim made a meaningful contribution to the revision. All authors read and approved the final manuscript.

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Correspondence to Sun-Ho Lee or Kyeung Min Joo.

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Supplementary Information

Supplementary Figure 1.

Analysis of CNVs of ahMNCs. Total number (A) and accumulated sizes (B) of gain or loss of CNVs were presented at each chromosome. In this analysis, data of Table 1 were utilized. (PPTX 52 kb)

Supplementary Table 1.

Detected CNVs in aCGH analysis (XLSX 19 kb)

Supplementary Table 2.

Detected genetic mutations in WES (XLSX 141 kb)

Supplementary Table 3.

qPCR data with human-specific Alu primers (XLSX 13 kb)

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Nam, H., Lee, IH., Sa, J.K. et al. Effects of Long-Term In Vitro Expansion on Genetic Stability and Tumor Formation Capacity of Stem Cells. Stem Cell Rev and Rep 18, 241–257 (2022). https://doi.org/10.1007/s12015-021-10290-z

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