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KIT researchers attempt to grow cardiomyocytes in Petri dishes

Regenerative medicine specialists are aiming to be able to grow complete organs from stem cells some time in the future. However, although the microscopically small cells are able to do many things, they are not able to grow organs on their own. Dr. Alexandra Rolletschek and her team at the Karlsruhe Institute of Technology (KIT) are investigating how stem cells can be grown into heart muscle cells (cardiomyocytes) in Petri dishes. The researchers’ work has shown that the cells can only differentiate in an environment that is as close as possible to the natural situation. But how can the cells be induced to spontaneously contract on glass slides? This research is not only designed to benefit humans, but also to help save the lives of animals.

Heart muscle cells grown from embryonic stem cells, shown here after immunocytochemical staining. The sarcomere protein actinin is shown in red and connexin43 (a gap junction protein that mediates cell-cell contacts) in green. The cell nuclei are stained blue. © Dr. Alexandra Rolletschek

Many heart patients who are currently waiting for tissue transplants would benefit enormously if the researchers found a way to turn embryonic stem cells into cardiomyocytes. In principle, stem cells can differentiate into any type of specialized cell. However, the molecular and cellular conditions that enable stem cells to differentiate in the natural tissue environment of an organism are highly complex; it is very difficult to mimic these conditions outside of the human body.

Doctors specializing in regenerative medicine and cell biologists who attempt to grow nerve, liver and heart tissue from stem cells face one huge obstacle. Researchers led by Dr. Alexandra Rolletschek at the Institute of Biological Interfaces I (IGB I) at the Karlsruhe Institute of Technology (KIT) are carrying out basic research in this field.

“We are mainly interested in understanding the molecular and cellular processes occurring during the development of cardiomyocyte precursors into mature cardiomyocytes,” said Rolletschek whose work has shown that placing stem cells into a Petri dish and adding a cocktail of growth factors in the hope that the cells switch on a programme that enables them to differentiate into functional cardiomyocytes is not enough.

An environment with a complex organisation

The cells of the human body do not just stick together in an unstructured manner. A complex three-dimensional network of proteins and carbohydrates (which is known as an extracellular matrix) forms a spatial scaffold into which different types of cells integrate in a precisely defined manner. Blood vessels supply the cells with nutrients and remove waste products. “Our work has shown that similar conditions also need to be present in artificial systems such as Petri dishes,” said Rolletschek.

Rolletschek and two of her colleagues use so-called co-cultures for their experiments. They place the stem cells into Petri dishes in which endothelial cells form a kind of scaffold. These cells produce growth factors that promote the growth and differentiation of the stem cells. They also form an extracellular matrix of proteins and carbohydrates into which other cells can incorporate.

Primary endothelial cells isolated from mouse hearts after 16 days of in vitro culture. Immunocytochemical staining shows the endothelial cell marker CD31 in red; collagen IV, an extracellular matrix protein that produces and releases endothelial cells is stained green. The cell nuclei are stained blue. © Dr. Andrea Rolletschek

“In our experiments involving laboratory mice we use endothelial cells that we have isolated from 17-day-old mouse embryos,” said Rolletschek. “This is a huge technical challenge as the mouse hearts are the size of a match head. However, we use these endothelial cells because they start to form blood vessels very quickly, leading to what is referred to as endocardium, a structure that corresponds to the innermost layer of tissue that lines the heart chambers.” The endocardium possesses optimal conditions for differentiation. Spontaneous contractions occur around three days after the researchers have implanted a “nest” of precursor cardiomyocytes into the mice. The cells start to contract and elongate completely on their own just like normal muscle cells. “Cells that are co-cultured with endothelial cells are able to contract for longer, i.e. approximately another week, than cells cultured without endothelial cells.

The researchers have identified one of the reasons why this happens: when they are kept in co-culture, the endothelial cells produce a molecule known as endothelin 1. This molecule diffuses into the developing cardiomyocytes where it promotes the formation of nitric oxide, a gas that increases the excitability of muscle cells.

Prospects for humans and animals

The success that regeneration medicine specialists have achieved in the growth of implantable tissue from stem cells clearly confirms the importance of endothelial cells. The methods used are constantly being improved; Rolletschek and her team make important contributions to such technological improvements. In actual fact, the researchers from Karlsruhe use far more complex systems. For example, they are currently focusing on improving what has already been achieved with cultivation by co-culturing the cells in three-dimensional peptide matrices which help make blood vessels grow even better.

One particular project that focuses more on applied aspects deals with a decisive advantage of embryonic stem cell cultures that might in the future do more than save human lives. Working in cooperation with researchers from the Institute of Human Genetics at Universität Heidelberg, Rolletschek and her team are attempting to grow cardiomyocyte precursors from embryonic stem cells with a mutation in the Shox2 (short stature homeobox 2) gene. Mice without a functional Shox2 gene die as soon as 14 days after fertilisation. These mice cannot be used for experiments even though they would be of great interest for research: Shox2 knock-out mouse embryos display severe cardiac defects and would be excellent model systems for studying the development of heart tissue. “It is actually possible to grow cardiomyocyte precursors of Shox2 mice in in vitro co-cultures and use them for research,” said Rolletschek pointing out that this would provide researchers with an experimental system that does not require experimental animals to be killed. This would then truly be “Research with heart.”

Further information:

Dr. Alexandra Rolletschek
Institute of Biological Interfaces I (IGB I)
Karlsruhe Institute of Technology (KIT)
Tel.: +49 (0) 721/ 608-28 476
E-mail: alexandra.rolletschek(at)kit.edu

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/kit-researchers-attempt-to-grow-cardiomyocytes-in-petri-dishes