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Method for identifying low-quality pluripotent stem cells developed using large-scale analysis

In a large-scale analysis of human iPS/ES cells, a CiRA research team has discovered the existence of low-quality iPS cell lines which, when induced to differentiate into neurons, leave a residue of undifferentiated cells that cause the formation of teratomas after transplantation into mouse brains. The researchers further established that a characteristic gene was active in these cell lines. Among the team members were Drs. Michiyo Aoi, an assistant professor at Kobe University Graduate School of Medicine and a former CiRA researcher, Mari Ohnuki, a researcher at CiRA, Kazutoshi Takahashi, a lecturer at CiRA, and Professor Shinya Yamanaka, director of CiRA.

In the last few years, there have been a wide variety of studies on the differences between iPS cells and ES cells. The problem with these studies, however, is that they have so far used a relatively small number of cell lines for comparison and that culture conditions have not been uniform. In the present research, 49 human iPS cells lines and 10 human ES cell lines were cultured under the same conditions and their properties compared. 

The 49 human iPS cell lines were established from four different types of somatic cell (dermal fibroblasts, dental pulp stem cells, umbilical cord blood cells, and peripheral blood monocytes) using three different gene transduction methods (retrovirus, episomal plasmid, and Sendai virus). The additional 10 human ES cell lines were cultured using the same methods. The various gene expression patterns and DNA methylation patterns of the cell lines were then compared. Under the system used in the study, no gene was found which could serve independently as an indicator to distinguish human iPS cells from ES cells. 

To test the differentiation potential of iPS cells and ES cells, 40 iPS cell lines and 10 ES cell lines were differentiated into neurons in vitro. It was confirmed that all cell lines differentiated  into neurons with a success rate of 80% or higher, but in some of the iPS cell lines, a residue of 10% or higher of cells was found to remain undifferentiated (Fig. 1). Moreover, when the induced cells were transplanted into mouse brain, these low-quality cell lines were found to form teratomas. To identify these low-quality cell lines in advance, gene expression patterns were compared between the "defective" cell lines and the "good" cell lines. This resulted in the discovery of a potential indicator gene group with consistent strong expression in the low-quality cell lines. The findings also suggested that the expression of this gene group may be connected with the insertion into the human genome of an endogenous retrovirus in prehistoric times. 

The present research studied a large number of iPS and ES cell lines and examined the differences between them, but no gene was identified which could serve independently as an indicator to distinguish between the two cell types. It was also established that, after iPS cells are differentiated into neurons, there are some cell lines in which a residue of low-quality cells remains undifferentiated, and that there is a certain gene group with a higher level of expression in these than in good-quality cell lines. It is hoped that the application of these findings will make it possible to screen out these low-quality iPS cells before use, for instance in regenerative medicine. 

The findings of the research were published in the week of November 18, 2013, in the online version of the U.S. scientific journal Proceedings of the National Academy of Sciences

PNAS_koyanagi_Fig2A.png

Fig.1 Discovery of gene group to identify low-quality iPS cells
Forty human iPS cell lines and ten human ES cell lines were differentiated into neurons and the proportion of cells remaining undifferentiated was measured using the undifferentiated cell marker OCT3/4 as indicator. Low-quality cell lines were labeled "defective" and successfully differentiated cell lines "good". 

Title of the paper:
"Differentiation defective phenotypes revealed by large scale analysis of human pluripotent stem cells"

Authors: 
Michiyo Koyanagi-Aoi, Mari Ohnuki, Kazutoshi Takahashi, Keisuke Okita, Hisashi Noma, Yuka Sawamura, Ito Teramoto, Megumi Narita, Yoshiko Sato, Tomoko Ichisaka, Naoki Amano, Akira Watanabe, Asuka Morizane, Yasuhiro Yamada, Tosiya Sato, Jun Takahashi and Shinya Yamanaka 

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