Hajime Ohgushi (Principal Research Scientist), the Research Institute for Cell Engineering (Director: Jun Miyake) of the National Institute of Advanced Industrial Science and Technology (AIST) (President: Hiroyuki Yoshikawa), in a joint research with Masahiro Go (Chief Research Scientist) of Stem Cell Sciences Ltd., transduced a single gene into human mesenchymal stem cells and successfully restored their proliferation and differentiation abilities (osteogenic differentiation)

With the help of an AIST-developed technology, culture-expanded mesenchymal stem cells from the marrow of a patient are being used for regeneration medical therapy to his/her damaged bone or heart. However, the clinical application of this technology is limited because the proliferation and differentiation abilities of these cells decline drastically within several weeks after the culture. In the joint research, the researchers transduced a gene, Nanog or Sox2, which is expressed by embryonic stem cells, with the aid of a retrovirus into stem cells with reduced proliferation and differentiation abilities.

The proliferation and differentiation abilities of the cells into which the Nanog gene was transduced were either restored to the normal levels or increased in comparison to their initial (right after the primary culture) levels. The proliferation and differentiation abilities were not restored in the cells into which only the Sox2 gene was transduced; however, they were restored when these cells were cultured with a protein named basic fibroblast growth factor (b-FGF).

Because the cells used in this research were cryo-preserved cells that had already been used for clinical purposes, the research results will soon be applicable to clinical use, and the application of regeneration medicine are expected to expand considerably.

The research results will be published shortly in the electronic version of a scientific journal of the U.S.A. named “Experimental Cell Research.”

Although organ transplantation is widely used to treat damaged organ, there is a serious shortage of organ donors. For example, an increasing number of Japanese patients go abroad to undergo heart transplantation because of the shortage in Japan. This trend has grown to become a social issue. As a possible solution to this problem, application of regenerative medicine using mesenchymal stem cells from the marrow of patients has already been started.

Professor Shinya Yamanaka of Kyoto University successfully generated induced pluripotent stem cells (iPS cells) by transducing 4 genes (Oct4, Sox2, Klf4, and Myc) into skin cells. A research group at the University of Wisconsin also succeeded in generating iPS cells by transducing 4 genes (Oct4, Sox2, Nanog, and Lin28). These research results are epoch making in the sense that they showed the possibility of alteration of cell function by multiple gene transduction.

However, it is extremely difficult to control expression of multiple transduced genes for a prolonged period. In addition, it is highly probable that iPS cells will form a teratoma after their transplantation. There are many issues to be resolved before iPS cells are ready for clinical application.

Since 2001, AIST has been working on culture, proliferation, and differentiation of pluripotent mesenchymal stem cells reside in the marrow of patients; the culture-expanded mesenchymal stem cells have been transplanted to patients suffering from bone and joint diseases or heart disease in university hospitals with which the AIST jointly conducts research. However, we had to discontinue the treatment of some patients halfway because their numbers did not increase as expected due to the lack of their proliferation ability.

Under these circumstances, we started this joint research with Stem Cell Sciences Ltd. about 3 years ago on how to develop the culture system of mesenchymal stem cells while maintaining their differentiation ability for the prolonged periods. As the aim of this study, we have been attempting to determine whether the proliferation ability of mesenchymal stem cells can be maintained by transduction of a gene actively expressed in embryonic stem cells into these mesenchymal stem cells.

The approach we adopted in the research showed that single gene transduction restores cell function. In this sense, the research results can soon be applied practically (clinically) because we already have used cryo-preserved mesenchymal stem cells for the patients and present study utilized the mesenchymal stem cell.

AIST cryopreserves mesenchymal stem cells cultured from the marrow of humans under informed consents of the donors. We thawed these mesenchymal stem cells and transduced them with an activated retrovirus into which the Nanog or Sox2 gene had been cloned. We selected only the gene-transduced cells and cultured them for several weeks.

The proliferation ability of the control cells into which the gene had not been transduced declined and showed only 10 times proliferation by culture for 3 to 6 weeks. On the other hand, in the same condition of the culture, the cells into which the Nanog gene had been transduced showed 1,000 times proliferation, i.e., we succeeded to generate the proliferation ability with several hundred fold higher only by transduction with the Nanog gene compared with non-transduced cells. (Figure 1)

These Nanog-transduced cells were cultured with a differentiation-inducing factor, and they differentiated into bone cells. Differentiation was scarcely evident in the control cells. (Figure 2: The red-colored cell represents the differentiated bone cell). It has an approximately 100-fold higher differentiation ability (osteogenic differentiation) as compared to the control cells.

We confirmed from these results that transduction with the Nanog gene enhances the proliferation and differentiation abilities of human mesenchymal stem cells by approximately 100 times.

At the same time, transduction with the Sox2 gene also restores these abilities if the stem cells are cultured with a protein named basic fibroblast growth factor (b-FGF), although transduction with only the Sox2 gene fails to show any effect.

Fig1: Proliferation curve of mesenchymal stem cells

Fig2: Osteogenic differentiation of mesenchymal stem cells (red stained areas indicate the differentiation)

We aim to improve the method of gene transduction, and it will be ready for treating patients suffering from intractable diseases.