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Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue

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World Journal of Stem Cells 2017年第5期9卷 77-88页

作者： Tetsuya Ishikawa Cell Biology Core Facilities for Research and Innovative MedicineNational Cancer Center Research InstituteTokyo 104-0045Japan Central Animal Division Fundamental Innovative Oncology Core CenterNational Cancer Center Research InstituteTokyo 104-0045Japan

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 nextgeneration 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 dbS NP135, 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, TSSK1 B, 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 noncancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confir

关键词： 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

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