Turning back the clock

Ethically superior

A little like cooking

Comprehensive screenings

Necessary risks

Transplants with iPS cells

Contractions in the Petri dish

People

Experience and intuition

The moment Matthias Mueller learned about the discovery of induced pluripotent stem cells (iPS), it was immediately apparent to him that this was something he wanted to explore in more detail. Now – about a decade later – the biochemist has played a decisive role in developing the method and establishing it within NIBR. Numerous research groups already use the enormous potential of iPS cells to study the mechanisms of diseases and to test substances.

Text by Annette Ryser

Table of contents

Turning back the clock

Ethically superior

A little like cooking

Comprehensive screenings

Necessary risks

Transplants with iPS cells

Contractions in the Petri dish

This article was originally published in August 2015.
Published on 30/06/2020

It is truly a miracle of nature: a tiny, fertilized egg has the potential to become a complete person – something which scientists call totipotency. However, after just three cell divisions, the daughter cells become too differentiated and lose this ability.

And yet these embryonic stem cells can still do a great deal – they can develop into any cell type in the human body, which is why they are also called “pluripotent.” However, when they have morphed into muscle, nerve or blood cells they have become too specialized and lose their pluripotent quality.

The development of our cells is thus a one-way street – at least in a living organism. However, when it comes to cells in the laboratory this principle does not apply. This breakthrough by Nobel Prize winners John B. Gurdon and Shinya Yamanaka has opened the door to countless therapies.

Turning back the clock

Back in the 1950s and 1960s, the English developmental biologist Sir John B. Gurdon succeeded in cloning a frog of the genus Xenopus at Oxford University.

To do that, he transplanted a nucleus from a fully grown intestinal cell into a frog egg. This then developed into an embryo that was genetically identical to the donor of the nucleus. Gurdon was thus able to demonstrate that it was possible to start over again from the beginning and reset the biological clock.

Forty years later, Japanese researcher Shinya Yamanaka discovered that virtually every adult cell can be transformed back into a stem cell – and all without the need for a clone. He called these artificially reprogrammed cells induced pluripotent stem cells (or iPS).

The results achieved by Gurdon and Yamanaka turned the knowledge of cell development completely on its head. For their work, the two scientists were awarded the Nobel Prize in physiology or medicine in 2012.

“When Yamanaka published his findings in 2006, there were huge reverberations in science and the media,” recalls Matthias Mueller, iPS Specialist in the Developmental and Molecular Pathways (DMP) department at the Novartis Institutes for BioMedical Research (NIBR). “It was immediately clear to us just what a fascinating discovery this was,” continues the biochemist. He first heard about the method by e-mail from a colleague: “My lab assistant and I then checked out the article in Cell magazine. Immediately, we both said to each other that we had to get on board here too.”

Ethically superior

Back in 2006, Mueller was already Lab Head at NIBR and specialized in the creation of transgenic mice. However, as with the majority of new technologies, the creation of iPS cells first had to establish itself. When NIBR Basel jumped on the bandwagon four years ago, Matthias Mueller was the obvious choice and was entrusted with the job. Since then, the scientist has significantly refined the method, which has become an integral part of research within NIBR.

“iPS cells are such an attractive prospect for researchers, as they usually dispense with controversial embryonic stem cells,” explains Mueller.

Up to now, cell development studies have had to rely on stem cells from human embryos left over following in vitro fertilization. This was constantly called into question from an ethical point of view and such research is also strictly regulated. With this in mind, iPS cells have become established in recent years as an alternative to embryonic stem cells. Above all, researchers at NIBR currently use them to grow cell cultures that originate genetically from individual patients and carry their pathogenic genes.

“In theory, all possible cell types can be generated from the iPS cells, many aspects of the disease can be recreated and these can then be examined on a cellular level,” explains Mueller. New substances can then be tested with the help of these adapted cells.

In future, it is also planned to use iPS cells in gene and cell therapies. It is conceivable that genetically adjusted transplants can be generated from patient cells using iPS cells that should not be rejected by the patient.

A little like cooking

“Interestingly, the method for producing iPS cells has remained practically unchanged since Yamanaka’s studies,” explains Matthias Mueller.

Back then, the Japanese researchers identified four transcription factors which – when expressed in any cell – can transform it back into a stem cell. While the procedure was further perfected in the following years, it remained fundamentally unchanged.

“It is not actually a difficult procedure, which is why it has already been outsourced in some cases by research companies,” says Mueller. However, if time is of the essence or the process involves reprogramming unusual cells then Mueller and his team create the cells themselves.

The actual challenge is the subsequent differentiation of the iPS cell to the desired cell type. As Mueller explains: “The process is a little like cooking – you need a certain amount of experience, talent and the right instincts for the recipe to be a success.”

It sometimes takes months to create the desired cell type, during which time the cells are stored in incubators and their quality is checked on a regular basis. The researchers have to replace their growth medium and add certain substances each day according to a strict protocol. “This is in addition to the variants that can be traced to genetic differences in the patient material or the reprogramming,” adds Mueller.

Nonetheless, in the four years since Mueller’s team has been responsible for iPS cells, the method has been constantly improved. It is now firmly established within NIBR thanks to his dedication. In the DMP department in particular, he has already worked closely with many research groups that now prefer using cell cultures created from iPS cells instead of classic cell lines or primary cells. “These interactions are also encouraged thanks to the open laboratory structure in the Chipperfield Building – the distances between us are short.”

Comprehensive screenings

One example of this interaction came in 2014, when Mueller collaborated with researchers from Disease Area Musculoskeletal (MSD) and DMP in creating motor neurons from a skin cell of a patient with ALS (amyotrophic lateral sclerosis). Some ALS patients have a genetic defect that results in the formation of RNA aggregates in the motor neurons. As these may play a role in the development of the disease, researchers are looking for an active substance that can dissolve these aggregates. Thanks to these cells, it has been possible to examine several substances.

Some promising candidates were found and are currently undergo-ing further analysis. “Our screening record in terms of scope is almost 100 000 substances,” Mueller says proudly. Such numbers are only possible if the cells he uses are of outstanding quality and the interaction with the screening specialists from the Center for Proteomic Chemistry (CPC) runs smoothly.

The success of the method has proven that Mueller was right. iPS cell technology is very important at NIBR nowadays and is used in the further development of CRISPR technology, among others. The Neuroscience group was set up in a completely new way in 2013, with the iPS cell method as one of the key technologies.

Despite this, iPS was a controversial issue for quite some time. “We had to deal with several challenges,” recalls Mueller. The method remains expensive, which is mainly due to the time and effort that are required in the differentiation phase. There are also differences between the cell types: Neurons and heart muscle cells are easier to create as a lot is already known about their development. “When working with other cells, such as liver cells, we sometimes have to improvise and develop the method further in small steps,” explains Mueller.

Necessary risks

Problems can also occur because the differentiated cells are too young. After three months in a Petri dish, the development status of a human cell corresponds to that of a fetus. “This is a problem with some cell types. For example, the liver cells in a fetus react differently in many ways compared to those in a grown adult,” explains Mueller.

This is particularly relevant when testing the toxic effects of a drug. As part of the “liver project” at NIBR, Mueller and his team intend to research methods for artificially accelerating the aging process.

The group’s innovation drive is high. As Matthias Mueller comments: “The existing protocols are sometimes too complicated and too difficult to follow in practice. We are always trying to find simpler and shorter processes.”

Finally, there are still many uncertain aspects to the process, mean-ing there is a certain risk that the final cell types will not meet the necessary requirements. “This risk is something we simply have to take – there’s no getting away from it. You have to be ready to invest a great deal of time and to live with a certain degree of uncertainty – only then can you take advantage of the enormous potential of iPS cells.”

Matthias Mueller in his laboratory on the Novartis Campus in Basel where he differentiates induced pluripotent stem cells (iPS cells) to achieve the desired cell type. The cells are given a nutrient solution, stored in incubators and their quality and characteristics are checked on a regular basis.

Transplants with iPS cells

In contrast to pharmaceutical research, the use of iPS cells in stem cell transplants is still a dream for the future. However, hopes are high. The first clinical trials of an implant of this type were started last fall at the RIKEN Institute in Kobe, Japan. A patient suffering from agerelated macular degeneration (AMD) received a transplant of retinal pigment epithelium that had been created from her own skin cells. Novartis is also involved in the ophthalmology field as it is here that the chances of success are currently greatest.

“We are simply not as far down the line in other fields,” admits Matthias Mueller. Safety is the greatest problem here. The method for creating iPS cells in particular poses a certain risk that cancer will develop sooner or later in those cells.

“In the womb, the development of the embryo is subject to a strict selection process,” says Mueller. The result of these processes is that the embryo develops into a viable organism. Up to now, it has hardly been possible to imitate these selection processes in the laboratory. “However, in order to use such cells as part of a therapy their genome has to be absolutely flawless. We cannot transplant cells or organs into patients if we are not absolutely sure that they are safe.”

Myotubes (green) cocultured with iPS-derived neurons (red).

Contractions in the Petri dish

Given the high potential and the fast development of the field, Mueller still considers his job to be both a challenge and a privilege. “My work is always exciting – there is no drudgery. It is fascinating to see just how our cells develop.”

In the future, he intends to work with his team in refining existing processes and developing more complex systems, which consist of different cell types in three dimensions. “We have already been able to create mixed cultures from motor neurons and muscle cells. When the cell types interact, we see the contractions in the Petri dish – just like in a living skeletal muscle. It really is a sight to behold.”

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