Research is ongoing in Japan and overseas with the aim of realizing cell transplantation therapy using iPS cells. One safety issue of concern is the risk of tumor formation. CiRA in particular has focused its resources on this issue.

Broadly speaking, there are two main theories as to the mechanism whereby iPS cells may form tumors. One theory is that iPS cells form tumors in response either to reactivation of the reprogramming factors inserted into the cell or through damage caused to the original cell genome through the artificial insertion of the reprogramming factors. In response, a search was launched for optimal reprogramming factors which do not cause reactivation, and a method of generating iPS cells was developed in which reprogramming factors are not incorporated into the cell chromosomes and damage to the host genome is therefore avoided.

The other theory is that residues of undifferentiated cells - cells which have not successfully completed differentiation to the target cell type - or other factors lead to the formation of teratomas, a kind of benign tumor. This theory requires research on iPS cell proliferation and differentiation.

1. Search for optimal reprogramming factorsWhen Professor Shinya Yamanaka and his research team announced the successful generation of mouse iPS cells, one of the reprogramming factors they used was c-Myc, which is known to be an oncogene, that is a cancer-causing gene. There have been suggestions that this gene may be activated within the cell and cause a tumor to form. However, in 2010, CiRA Lecturer Masato Nakagawa and his team reported that L-Myc was a promising replacement factor for c-Myc. iPS cells created using L-Myc not only display almost no tumor formation, they also have a high rate of successful generation and a high degree of pluripotency.

2. Search for optimal vectorsWhen the reprogramming factors required to generate iPS cells were inserted into the cells of the skin or other body tissues, early methods employed a retrovirus or lentivirus as a "vector," or carrier. In these methods, the target genes are inserted into the viruses with the which the cells were then infected in order to deliver the target genes. When a retrovirus or lentivirus is used as a vector, however, the viruses are incorporated into the cells genomic DNA in a random fashion. This may cause some of the cells original genes to be lost, or in other cases activated, resulting in a risk of cancerous changes.

In 2008, to remedy this risk, CiRA Lecturer Keisuke Okita and his team explored the use of a circular DNA fragment known as a plasmid, which is not incorporated into the cell chromosome, as a substitute to the retrovirus or lentivirus methods. In this way, they developed a method of generating iPS cells in which the reprogramming factors are not incorporated into the cell chromosome. In 2011, Okita and his team further improved the efficiency generation by introducing into a self-replicating episomal plasmid six factors - OCT3/4, SOX2, KLF4, LIN28, L-MYC, and p53shRNA.

3. Establishing a method for generating and screening safe cellsOnce iPS cells have been induced to differentiate into the target somatic cells using the appropriate genes and gene insertion methods as explained above, the differentiated cells can be relied upon not to revert to the undifferentiated state. However, there may sometimes be a residue of undifferentiated cells which have not completed the process of differentiation into the target cells, and it is possible that these cells, however few, may form a tumor. Scientists had already established that different iPS cell lines, even if generated from the same individual using the same method, might nevertheless display differences in proliferation and differentiation potentials. This meant that, if iPS cells with low differentiation potential were used, there was a risk that a residue of cells in the cell group might fail to fully differentiate and result in the formation of a teratoma. In 2013, a team led by CiRA Lecturer Kazutoshi Takahashi and Dr. Michiyo Aoi, now an assistant professor at Kobe University, developed a simple method to screen for iPS cell lines that have high potential to differentiate into nerve cells. There is also a risk of tumorigenesis from genomic or other damage arising at the iPS cell generation stage or at the subsequent culture stage. CiRA Assistant Professor Akira Watanabe and his team have developed a sensitive method to detect genomic and other damage in iPS cells using the latest equipment.

4. Developing a reliable method of differentiation into the target cell typeIn cell transplantation therapy, iPS cells are not transplanted directly into the human body. Instead, cells are transplanted after first being differentiated into the target cell type. It is therefore important to develop a reliable method of inducing iPS cells to differentiate into the target cell type. CiRA is currently working to develop technology for differentiation into a range of different cell types from iPS cells. CiRA Professor Jun Takahashi and his team have developed a highly efficient method of inducing iPS cells to differentiate into dopamine-producing nerve cells. In 2014, CiRA Professor Koji Eto and his team reported a method of producing platelets from iPS cells that is both reliable and can yield high volumes. These findings represent a major step toward iPS cell-based regenerative medicine for nerve diseases such as Parkinsons disease and blood diseases such as aplastic anemia.

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