technology

rooted in cutting-edge science

Our approach and difference with preexisting methods

We selectively produce a large amount of ventricular type cardiomyocytes from iPS cells. Our original purification method (patented in major countries), which eliminates undifferentiated cells and non-cardiomyocytes, minimizes the risk of teratoma formation and arrhythmia.

The transplanted differentiated cardiomyocytes are efficiently engrafted in the host myocardium for a long period of time, and is expected to contribute to the long-term improvement of cardiac function of patients with heart failure.

Among the many technologies related to cardiac regenerative medicine, the transplant method of differentiated cardiomyocytes is an extremely important factor that determines the therapeutic effect. Methods of transplantation of differentiated cardiomyocytes derived from human iPS cells are roughly classified into two methods, cell sheet transplantation and direct administration to the heart.

Cell sheet transplantation is considered to be the easiest method of transplantation with less burden on patients at the time of transplantation, but in cell sheet transplantation, the epicardium and fat layer are between the left ventricular myocardium and the transplanted cell sheet. Therefore, it has been reported that the electrical connection with the recipient's heart is not made, there is a possibility that contraction of the host's cardiac muscle and the transplanted cardiomyocytes cannot be synchronized, and that long-term survival may be difficult. These may affect the effectiveness etc. On the other hand, direct injection method of differentiated cardiomyocytes has been reported that the differentiated cardiomyocytes transplanted directly into the myocardium can electrically couple with the recipient's cardiomyocytes, and good results have been demonstrated in various animal experiments.

Original technologies in each step with a view of industrialization

iPSC Production

In the conventional iPS cell production method, skin tissue of the patient is collected and reprogramming genes are directly inserted into the genome. However, the possibility of tumorigenicity could not be denied with this method because the genome itself could be damaged. In our method, we can generate iPSCs without direct insertion of reprogramming genes into the genome by using a special vector, and we have succeeded in significantly reducing the possibility of tumorigenicity. In addition, since iPS cells can be generated from only one drop of blood, the burden on patients can be reduced.

iPS cells have self-renewal ability and pluripotency similar to ES cells, but there is a difference in differentiation ability and quality depending on the cell line compared with ES cells, so it is important to secure the quality of iPS cells. We focused on H1 foo, a linker histone protein specifically expressed in oocytes, and we have succeeded in generating iPSCs with enhanced efficiency of embryoid bodies and chimera formation by adding this gene during iPS cell reprogramming.

Related patents
Title of invention Patent number Date of application Status
Method for producing induced pluripotent stem cells JP5856949 US9447432B2 EP2559757B1 2011/4/15 JPN, US, EU etc
METHOD FOR PRODUCING HIGH-QUALITY iPS CELLS PCT/JP2016/003282 2016/7/11 PCT application
Differentiation
Differentiation

immunofluorescence staining for myosin light chain 2a (MLC2a) and myosin light chain 2v (MLC2v) was performed. Most purified CMs were MLC2v-positive ventricular CM(Tohyama S, et al. Cell Metabolism, 2016)

A large number of cardiomyocytes are required to cardiac regenerative medicine, and several steps are required to differentiate undifferentiated iPS cells into cardiomyocytes. From the many research results of the developmental study of the heart including ours, with related to the induction and differentiation of cardiomyocyte from pluripotent stem cells, it has been found that combining proteins such as BMP/Activin signal and Wnt signal that are involved in early cardiac development can significantly increase the efficiency of differentiation. In addition, research on replacing recombinant proteins with chemical compounds has progressed, and it has become possible to produce cardiomyocytes more inexpensively and efficiently.

On the other hand, not only the signal but also the culture environment is important for efficient cardiomyocyte differentiation. We have already succeeded in developing a clinically applicable level of cardiomyocyte differentiation culture solution through collaboration with companies, and high cardiomyocyte differentiation efficiency can be achieved by combining with a low molecular compounds-based differentiation induction method. In addition, the cardiomyocytes that we produce are ventricular specific ones that work for the pumping function of the heart.

Large-scale cell culture

Although a method has been reported for producing a large number of differentiated cells in a mass by three-dimensional culture, this method has several problems. First, it is difficult to completely remove undifferentiated stem cells that cause tumorigenesis when the cell mass increases. In addition, there are other problems such as the reduction of cell proliferation efficiency, the inability to control the size of cell mass uniformly, and the instability of cardiomyocyte differentiation efficiency.

We have established a two-dimensional mass culture system using multi-layered culture plates. In the case of using a multi-layered culture plate, it was an issue that the proliferation of human iPS cells becomes unstable because the layers cannot be cultured uniformly. By using the forced ventilation system, it has become possible to stabilize the proliferation of human iPS cells and to obtain a sufficiently large number of differentiated cardiomyocytes for transplantation into patients.

Related patents
Title of invention Patent number Date of application Status
Cell culture device and its use JP2019062840A 2017/10/3  
Purification

It has been revealed that undifferentiated iPS cells remain after induction of differentiation into cardiomyocytes, and when these undifferentiated cells are mixed in when transplanted into a patient's body, they form teratoma, which is a major problem. Therefore, before transplanting cardiomyocytes, it is necessary to remove undifferentiated cells that cause tumorigenesis and purify only cardiomyocytes.

In the past, the mainstream method was to separate cells one by one using FACS cell sorter and collect only cardiomyocytes. By analyzing energy metabolism of iPS cells and cardiomyocytes in detail, we have succeeded in developing culture solutions, removing undifferentiated cells inexpensively and easily, and recovering a large number of cardiomyocytes. This technological breakthrough has dramatically increased the possibility of iPS cell-derived cardiomyocyte transplantation.

Related patents
Title of invention Patent number Date of application Status
Method for purifying cardiac myocyte and presumptive cardiac myocyte derived from stem cell and fetus JP5312804, JP5756145 US9115342 EP1983042 2007/1/31 JPN, US, EU, etc
Methods for inducing cell death in pluripotent stem cells and differentiated cells other than cardiac myocytes JP5620905 US8735152 EP2415862 2010/3/29 JPN, US, EU, etc
New undifferentiated stem cell removal and myocardial purification and refinement culture medium PCT/JP2015/071048 JP6333378 2015/7/16 JPN,PCT application
Undifferentiated stem cell removal agent and method for removing undifferentiated stem cells PCT/JP2017/044937 2016/12/15 PCT application
Spheroid

When transplanting purified cardiomyocytes to heart tissue, it has been confirmed that only a small number of cells can be finally engrafted when transplanting disjointed cardiomyocytes one by one. We have been searching for alternative method that can achieve high engraftment rate.

As a result, we have developed a method for efficient transplantation by creating "cardiomyocyte spheroid", in which about 1,000 cardiomyocytes are aggregated, and transplanting them into the heart tissue. Using this method, it has been confirmed that cardiomyocytes made from iPS cells engraft in heart tissue with a high rate, and cardiomyocytes grow further after engraftment and significantly improve actual cardiac function. Our company name, Heartseed, is named after transplanting this "cardiomyocyte spheroid", which will be the heart's seed (Heartseed), to a site that has become necrotic due to myocardial infarction and the like.

In addition, we are developing special transplantation devices for transplantation of cardiomyocyte spheroid and establishing a cell transplantation system that does not rely on the skill of the operator. Development of such a system to reduce the technical burden of such a transplant operator and to transplant cardiomyocyte spheroid in an efficient manner is expected to play an important role in the stability of the therapeutic effect after cell transplantation and the spread of this therapy.

Related patents
Title of invention Patent number Date of application Status
Method for constructing mass of myocardial cells and use of the myocardial cell mass JP5523830 US9109205 EP2172542 2008/7/31 JP, US, EU, etc