We are developing pre-disease models for clinical studies (PreDMoC) by gene modification and regenerative cell technology. Using these models,we are engaged in research subjects focused on how to prevent the progress of diseases (Fig. 1), e.g.from pre-diabetes to diabetes, immunodeficiency, inflammation, and cancer metastasis.

In the pre-disease stage, healthy cells are contacting a few disease cells. In the early stage of cancer metastasis, for instance, a few cancer cells contact cells of healthy tissues. In order to analyze the intercellular molecular communication at the frontier of such heterogeneous cell contacts, a unique single-cell analysis method has been developed.

Dynamic expression of whole connexin family was analyzed comprehensively and Cx30.3 was found to be a unique isoform with specific novel functions such as a undifferentiated state marker and a cell-cell contact-activated expression. We also focus the potential novel functions of exosomes relevant to intercellular communication between remote cells.

Disease progression is not necessarily a gradual and slow worthening process. It occasionally is a rapid and stepwise worthening one. A sign of such a rapid worthening will be found in the pre-disease stage. During the pre-disease stage, healthy cells and disease cells are competing. When the disease cell mass and malignancy surpass the preventive potential by healthy cells, a rapid and stepwise worthening occurs.

The purpose of “Cell Science for Disease Prevention” is to detect and analyze the disease-healthy competing state and to find an efficient way to be a healthy state. Research subjects are the survey of molecular markers for quantitative expression of disease-healthy competing state, the threshold analysis of healthy to disease conversion, and the development of methods to strengthen healthy cells.

Eight genes were selected as diabetes-related genes: (Pdx-1, Irs-1, Kir6.2, Irs-2, Gk, Shp, Hnf-1α, Hnf-1β). One or two of them in ES cells were knocked down by RNAi method or knocked out by gene targeting method. In total 38 knocked-down or overexpressing model cell lines were developed.

Moreover, Gk knocked-out mouse was developed and registered as B6;129-Gck^tm1Tms. The casual blood glucose level and glucose tolerance in this mouse showed a middle level between those of healthy and diabetic mice. Bred with high fat diet, this mouse became diabetes. Therefore, this Gk(+/-) line was defined as a pre-diabetes model mouse (Fig. 3).

【Analyses using pre-diabetes model】
◎Effects of transplantation of insulin generating cells (Fig. 4).

◎Effects of hyperglycemia on immune functions: Decrease in NKT cells in adipose tissue、Effects of NKT cell transplantation on the improvement of glucose tolerance (Fig. 5).

◎Influence of hyperglycemia on the metastasis of melanoma cells

◎Effects of anti-inflammation nucleic acid reagents on the improvement of glucose tolerance

Insulin generating cells for diabetes patients, and NKT cells and lymphocytes for immune therapy may be called as cell medicine if they are effective for the therapy.

In contrast, if healthy cells are strengthened and prevent the invasion of disease cells, the healthy cells are effective for the therapy. Not only the transplantation of these strengthened cells but also the injection of factors that may strengthen healthy cells in vivo is promising.

Mouse ES cells were cultured by hanging drop method to obtain embryoid bodies. When spermine was added to embryoid bodies, peripheral and frontier cells were killed. To our surprise, skeletal muscle cells showed 3-dimensional growth (Fig. 6). Spermine was originally recognized as a growth inhibitor of muscle cells. In fact, frontier cells were killed. However, a marked growth of muscle cells occurred successively and there appeared a large number of spherical cells that were thought to be stem cells of muscle tissue. In this case, spermine may be regarded as a cell activator. Skeletal muscle chipshowed an insulin responsive glucose uptake.

Regulatory Science is
(1)Toxicological science for the decision of regulation
(2)Scientific management to fulfill regulation values
(3)Analytical science to confirm the fulfillment of regulation values

　 We are engaged in regulatory science for the food safety and the validation of microbiological tests. One of the research subjects is stanard material of viable microbial cells.Preparation of single-cell with colony forming potency was demonstrated using a cell sorter (Fig. 8).

Rapid decision is important in (3). 2NBDG was developed for the photometric rapid detection of glucose uptake by viable cells. Density slicer was developed for the efficient separation of viable cells from food matrices, which was essential for non-culture rapid methods. Automatic system was developed for the simultaneous measurement of 3D-images of every colony in multiple plates.

The selection of indicators for toxicity evaluation is important in (1). From the viewpoint of bioethics, the pain suffered by animals during toxicity tests should be as small as possible. The toxicity may be evaluated from the slight effects by small quantity of materials on the pre-disease state. In this sense, pre-diabetes model mice are more sensitive than diabetes model mice in the evaluation of active factors that may improve or worthen blood glucose tolerance.