成果報告書詳細
管理番号20130000000277
タイトル*平成24年度中間年報 「ヒト幹細胞産業応用促進基盤技術開発 ヒト幹細胞実用化に向けた評価基盤技術の開発」 (タカラバイオ株式会社)
公開日2014/8/20
報告書年度2012 - 2012
委託先名タカラバイオ株式会社
プロジェクト番号P10027
部署名バイオテクノロジー・医療技術部
和文要約
英文要約Title: Beyond the Fusion of Academia and Industry in Japan: an Integrated System for High Quality and Large Scale Production of hPSCs and Derivative Cells

The TAKARA BIO Inc. plays the following roles:
2. Development of quality evaluation and control for hPSC lines
(1) Development of profiling/evaluation hardware and reagents for quality control of hPSCs
1. Construction of the PrimerArray kit
Based on the literature information on ISCI, the list of genes specifically revealed by the human embryonic stem cell, Takara Bio has developed a set of real-time RT-PCR primers for the analysis of the expression of genes associated with ES cell pluripotency. This set can be used to analyze the gene expression in differentiated and undifferentiated ES cells. Each array contains 96 primer pairs representing 88 pluripotency-related genes and 8 housekeeping genes. When comparing an unknown sample to a control sample (or a control data), gene expression differences can be expressed using the relative quantification method. In addition, expression levels of multiple genes can be screened simultaneously.

2. Development of the effective index by the Next-generation sequencer
It remained 500, the oncogene and the functional gene of embryonic stem cell undifferentiated maintenance were selected, the probe (bait) for condensing those gene regions was designed (bait number: 22,336 / bait cover length : 2.69Mb), and the capturing kit was prepared.
By using this kit, some ES cells genomes were sequenced by HiSEQ2000, then the 9400 mutations were detected. Although these will be the calculation mutation of one base per about 500bp per sample, the mapping is determined that false positives are often detected. So it is necessary to find a method of data analysis for higher accuracy. Now, we have redesigned the probes to capturing the genomes evenly, and added some genes involved in DNA repair to this capturing kit to enhance the completeness and accuracy.

3. Verification of the known index candidate by microarrays
The place which conducted revelation analysis by Agilent SurePrint G3 Human GE 8x60K about three steps of cultivated samples of KhES-1, -3 and H1 (At Early and Late passage), while most change of a gene expression pattern was not accepted, many revelation change was checked by Differentiated.
However, between the embryonic stem cell lines, some difference was observed in revelation.
In addition to the expression analysis of gene expression analysis, we performed on culture samples described above, the analysis of SNP / LOH / CNV due to the bead array Illumina BeadChip HumanOmni2.5-Quad, the standard confirmed the findings of the stability of the genome expression profile and by culture methods such. The detected copy number variation and genotyping by Bead Array for each cell line before and after differentiation and culture period were different from that have been detected in the region CNVs known. Among them, it is a significant copy number variation region exists in the H1 strain chromosome 7 of differentiated culture sample was suggested. Therefore, we tried to detect copy number variation using the data in the target Exome resequencing analysis was carried out by described above, and the results of detection by Exome data were almost identical by the bead array detection. So chromosome 7 of the strain H1 was seemed to get some CNV in differentiated culture. It was suggested that there was a significant risk for occurring the mutation of ES cells genome not only in expansion culture but also in differentiated culture.
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