Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. May 26, 2017; 9(5): 77-88
Published online May 26, 2017. doi: 10.4252/wjsc.v9.i5.77
Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue
Tetsuya Ishikawa
Tetsuya Ishikawa, Cell Biology, Core Facilities for Research and Innovative Medicine, National Cancer Center Research Institute, Tokyo 104-0045, Japan
Tetsuya Ishikawa, Central Animal Division, Fundamental Innovative Oncology Core Center, National Cancer Center Research Institute, Tokyo 104-0045, Japan
Author contributions: Ishikawa T substantially contributed to the conception and design of the study as well as the acquisition, analysis and interpretation of the data, drafted the manuscript, made critical revisions related to the intellectual content of the manuscript, and approved the final version to be published.
Supported by the JSPS KAKENHI, No. 16K07135.
Institutional review board statement: This study was conducted with the approval of the Institutional Review Boards of the National Cancer Center of Japan and the Japanese Collection of Research Bioresources, National Institutes of Biomedical Innovation, Health and Nutrition. Written informed consent was obtained from donors for the use of their tissue in this study.
Institutional animal care and use committee statement: N/A.
Conflict-of-interest statement: The Life-Science Intellectual Property Platform Fund (LSIP) supported this work. The author confirms that the LSIP had no influence over the study design, content of the article, or selection of this journal. The author discloses patents pending (WO2013081188 A1) relevant to the work presented here.
Data sharing statement: The next-generation sequencing data in this study will be available to the public through the DDBJ Sequence Read Archive (DRA).
Open-Access: 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. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Tetsuya Ishikawa, PhD (DMSc), Laboratory Head, Central Animal Division, Fundamental Innovative Oncology Core Center, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. humanipscells@gmail.com
Telephone: +81-3-35475201 Fax: +81-3-35422548
Received: January 31, 2017
Peer-review started: February 7, 2017
First decision: March 7, 2017
Revised: April 3, 2017
Accepted: May 3, 2017
Article in press: May 5, 2017
Published online: May 26, 2017
Processing time: 107 Days and 12.7 Hours
Abstract
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.

Keywords: Colon cancer; Next-generation sequencing; Single-nucleotide variant; Genotype; Heterogeneous cancer tissue; Cancer associated fibroblast; Pre-cancer cell; Induced pluripotent stem cell; Single cell; Clonal evolution

Core tip: Ten induced pluripotent stem cell (iPSC) lines were clonally generated from heterogeneous colon cancer tissues and analyzed with next-generation sequencing. Non-synonymous single-nucleotide variants (SNVs) of the iPSC lines were not identical to the genotypes of the cancer tissues. The SNVs were de novo or pre-existing mutations that originated from a minor population within the cancer tissue. Meanwhile, the genotypes of the iPSC lines were not mutated genotypes of the cancer tissues, suggesting that the starting cells for the iPSC lines were not mature cancer cells. Thus, the genotypes of iPSC lines can be used to trace the genomic origins of single cells within heterogeneous cancer tissue.