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Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue
AIM: To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. METHODS: Human iPSC lines were clonally established by selecting independent single colonies expanded from he...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Baishideng Publishing Group Inc
2017
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440771/ https://www.ncbi.nlm.nih.gov/pubmed/28596815 http://dx.doi.org/10.4252/wjsc.v9.i5.77 |
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author | Ishikawa, Tetsuya |
author_facet | Ishikawa, Tetsuya |
author_sort | Ishikawa, Tetsuya |
collection | PubMed |
description | AIM: To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. METHODS: Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer (OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. RESULTS: In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells. CONCLUSION: The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from. |
format | Online Article Text |
id | pubmed-5440771 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Baishideng Publishing Group Inc |
record_format | MEDLINE/PubMed |
spelling | pubmed-54407712017-06-09 Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue Ishikawa, Tetsuya World J Stem Cells Basic Study AIM: To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. METHODS: Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer (OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. RESULTS: In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells. CONCLUSION: The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from. Baishideng Publishing Group Inc 2017-05-26 2017-05-26 /pmc/articles/PMC5440771/ /pubmed/28596815 http://dx.doi.org/10.4252/wjsc.v9.i5.77 Text en ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved. http://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. |
spellingShingle | Basic Study Ishikawa, Tetsuya Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
title | Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
title_full | Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
title_fullStr | Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
title_full_unstemmed | Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
title_short | Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
title_sort | next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue |
topic | Basic Study |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440771/ https://www.ncbi.nlm.nih.gov/pubmed/28596815 http://dx.doi.org/10.4252/wjsc.v9.i5.77 |
work_keys_str_mv | AT ishikawatetsuya nextgenerationsequencingtraceshumaninducedpluripotentstemcelllinesclonallygeneratedfromheterogeneouscancertissue |