Overcoming a major obstacle to the practical application of regenerative medicine: Paving the way for the mass production of iPS Cells

Creating iPS cells conveniently without using viruses

Professor Shinya Yamanaka of Kyoto University was awarded the 2012 Nobel Prize in Physiology or Medicine for his famous discovery that mature somatic cells such as skin or blood cells can be reprogrammed to become pluripotent iPS cells by introducing four transcriptional factors. Regenerative medicine is an innovative field of medicine which utilises iPS cells. It can potentially restore or replace damaged or lost physical functions as well as completely curing diseases or healing injuries.

A lot of clinical research has been conducted in the field of iPS cells, but there are some obstacles to its practical use, namely the difficulty in generating iPS cells that display consistent characteristics, and a very low production rate (about 1%).

Conventionally, the generation of iPS cells utilized viruses to introduce Yamanaka factors into cells, but these factors are also capable of inducing cancer in cells and creating tumors. Concerns have also been raised about the risk of parts of the viruses’ gene sequence remaining in cells. Currently, there are various methods being developed for generating iPS cells without using viruses, but all of these methods have demonstrated even poorer rates of production efficiency. In addition, the generation and culturing of iPS cells can only be done by trained experts, and these activities require specialized conditions in facilities in order to prevent contamination.

Dr. Numano explains, "We have developed a novel method of generating iPS cells by using water-in-oil droplet electroporation. Cell membranes are made of layers of oils. We have found that by momentarily applying several kilovolts to cells, with about the same voltage as static electricity, the cell membrane loosens and forms transient pores through which the Yamanaka factors can pass. This technique does not require the use of viruses.

However, commercially available electroporation equipment requires a costly pulse generator and sends out high-voltage pulses that kill more than half of the cells. Therefore, rather than implement a method using commercial electroporation equipment, we decided to develop a machine that uses direct-current electric fields to generate iPS cells in a more cell-friendly, convenient and efficient way."

Aiming for chip-based mass production of iPS cells

The procedure for this novel technique is to take an aqueous droplet several microliters in volume containing cells and four Yamanaka factors, put this droplet inside insulating oil, and then apply a direct current from a metal electrode. When the aqueous droplet is exposed to an electric field between a pair of electrodes, the droplet moves back and forth in a rapid bouncing motion between the positive and negative electrodes several hundred times in a minute.

“This technique uses the basic physical property that oil does not mix with water. When the electric field is first applied, the droplet might move to the negative electrode, for example. However, the machine switches the polarity after the droplet contacts with this electrode, and so the droplet is repelled by the negative electrode and moves toward the positive electrode. This cycle is repeated many times, making the droplet move in a bouncing motion. This creates a minute electric current that loosens the cell membranes, allowing for the Yamanaka factors to be introduced into the cells.

Although this reactor is extremely small in size — a water droplet just a few microliters in volume — the droplet contains about ten thousand cells as well as the four types of genes known as Yamanaka factors. This arrangement allows for these genes to be efficiently introduced into the cells. The droplet is insulated by the oil surrounding it, and so there is no risk of contamination in the reactor,” explains Dr. Numano.

One of the most striking points of this new method is that despite a high voltage being used to move the droplet, the electric current in the droplet is very small, and has little influence on the cells.

"We think that if we can conduct the same process in an even smaller reactor, such as picoliter-scale droplets in the micro-channels of a chip, we could further improve the diffusion efficiency of genes, allowing for better introduction of the genes into the cells.

Micro-channels are a completely closed system, with no danger of contamination. They also allow for Yamanaka factors to be introduced into the same cells, enabling a large number of iPS cells to be produced in a short period of time," she says.

This water-in-oil droplet electroporation was developed in a joint research project with Professor Takayuki Shibata and Assistant Professor Hirofumi Kurita from Toyohashi University of Technology. Professor Takayuki Shibata is a bio-MEMS (Micro Electro Mechanical System) researcher working on microneedle arrays for cells, and Assistant Professor Hirofumi Kurita is working on applications of electrostatic forces in life science. A patent has already been obtained for the device, and work is underway together with the electroporator manufacturer Nepa Gene Co., Ltd., in order to realize a practical implementation.

Dr. Numano adds, "Researchers at Juntendo University are conducting clinical research in regenerative medicine using iPS cells to treat Parkinson's Disease. We have requested Professor Wado Akamatsu, from the Center for Genomic and Regenerative Medicine in Juntendo University, to investigate the characteristics of functional differentiation in the iPS cells that we have generated.

Apart from Juntendo University, many clinical studies are currently being conducted at Kyoto University, Osaka University, Keio University, and other institutions in order to seek treatments for neurodegenerative diseases that are considered difficult to cure. We also hope to accelerate our research on mass production methods for iPS cells in order to make the fruits of our research publicly available as soon as possible."

Utilizing this technology to treat diseases by manipulating many cells at once

Before she switched to working with iPS cells, the main focus of Dr. Numano's research was on circadian rhythms in mammals.

"All animals on the earth have a circadian rhythm, which is an internal clock that works in 24-hour cycles. The circadian rhythm is regulated by some 20 types of genes known as clock genes. These clock genes work together within the brains of mammals over 24-hour cycles. This is a very robust system, and is not easily disturbed by minor genetic mutations. If we want to control clock genes, we need to manipulate multiple genes at the same time," Dr. Numano says.

For example, when flying from Japan to the US, our eyes are stimulated by light from the outside, which influences the clock genes in our brain in order to make adjustments to follow local time. However, it is difficult to shift the entire human biological clock by many hours in an instant. As a result, we become jet-lagged because it takes longer for the tissues outside of the nervous system to adjust to the local time.

"I have been working on analyzing the mechanism of the circadian rhythm. I have thought a lot about finding ways to adjust the circadian rhythm instantly. This would allow us to control physiological processes, such as to cure jet lag instantly, or to discover treatments for diseases that are related to the circadian rhythm."

In her research, she came across the electroporation method, which can manipulate many genes at once. This method may allow genes to be introduced into CAR (Chimeric antigen receptor) T cells—a type of white blood cell that specifically attacks cancer cells—to improve their abilities, meaning that this method could also be employed for cancer treatment.

"In addition to iPS cells, this electroporation method can be applied to many other types of genetic manipulation. I would like to conduct further research to refine several aspects of the method, such as identifying how many more genes we can introduce in total, and the types of cells that can be regulated with the method," said Dr. Numano about the future prospects of her research.

Reporter's Note

Dr. Numano grew up in a family with many physicians: her father was a cardiologist and her mother was a pediatrician. She recalls her father’s disappointment when he came home from his university hospital if one of his patients had died that day.

She says, "I thought of becoming a physician as well. However, through witnessing my parents’ experiences, I decided to go into basic research where I could work on ways to fundamentally treat or prevent diseases."

The Human Genome Project commenced when she was a student, and many other research efforts made progress in treating disease preventatively or at early stages. She obtained her doctorate degree while working in the laboratory of our previous Dean Yoshiyuki Sakaki, a prominent Japanese researcher who took a leading role in the Human Genome Project. After graduating, she came to Toyohashi University of Technology, and she has worked over a decade in collaboration with many researchers and companies in order to achieve her dream. Her bright, graceful smile offers the reassurance of an expert physician. We look forward to witnessing further successes in her career.