Building embryos only from cells

Working alongside the team of Charité’s Transgenics Core Facility Transgene Technologies, Berlin-based researchers Ellen Na and Dr. Geert Michel have succeeded in reconstructing mouse embryos using three different cell lines. This first-ever successful reconstruction could herald the start of something much bigger. If everything continues to go as well as it has until now, this could lead to a drastic reduction in the number of animals used in research. What Ellen Na and Dr. Geert Michel succeeded in developing during those long days and nights spent in Charité's transgenic technologies laboratory is so tiny that it cannot be seen with the naked eye. And yet, their work has made it onto the cover of the renowned journal Nature Cell Biology.

The cover image of the journal's August 2018 edition depicts high magnification microscope images of mouse embryos. What is spectacular about these spherical objects – structures comprising yellow, purple and turquoise cells – is that they did not develop naturally. Instead, the two Charité researchers 'assembled’ them in the laboratory, using three different mouse cell lines: embryonic stem cells (ESCs), which go on to form the embryo; trophoblast stem cells (TSCs), which go on to form the placenta; and extraembryonic endoderm (XEN), which delimit the embryo and go on to form the yolk sac.

No other researchers had ever succeeded in creating an ETX embryo* which so closely resembles a fully-formed natural embryo.  “We were the first,” says Ellen Na proudly. A medical technologist by training, Ellen Na has worked at Charité for more than 30 years, conducting research on both human and animal cells. Successfully creating something that has never been done before and which could potentially result in thousands of animals being spared, is “a damned good feeling,” she says. The researchers initially embarked on this work following a request from a Charité-based research group, who needed TSCs derived from blastocysts. Until that moment, ESCs had been the only cell types routinely produced by the Core Facility. While researching TSCs, Ellen Na quickly realized that XEN cells would need to be added to any cell culture intended to represent the blastocyst stage of embryonic development. “Articles on the reconstruction of hair follicles from individual component cell types gave me the idea that we should try this using the three types of stem cell present in blastocysts,” says Ellen Na. It did not take much to stir the enthusiasm of her colleague, Dr. Geert Michel.

As the Head of Transgenic Technologies at Charité’s Research Facility for Experimental Medicine (FEM), Dr. Michel oversees a highly dedicated team committed to producing genetically modified animal models for use in research. Since shortly after its foundation, the main goals of the FEM service unit have been to reduce the number of animals used during production and develop new alternatives to animal research. Since its inception, it has benefited greatly from Charité’s continued assistance and support. Given the many methodological improvements within the areas of animal model production, cryopreservation and reconstitution, the ETX embryo idea seemed to hold considerable promise.

Creating identical baseline conditions for different cell lines
A previous article had reported on in vitro aggregates of embryonic and trophoblast stem cells. However, these co-cultures had limited potential for further development or did not develop at all. Ellen Na came up with the idea of expanding the work previously done with ESCs and TSCs by adding a third stem cell type, XEN cells. “That was of immense importance,” says biologist Dr. Michel. He adds: “Another distinct advantage was the fact that we found one culture medium for all three of the cell lines, because these three cell line types can only interact naturally if grown under identical conditions from the outset.”
This was precisely where other researchers had failed. Once again, it was Ellen Na who then decided to spend considerable time and effort researching growth media which would sustain these cell cultures. After extensive research and practical experiments, she finally found a growth medium whose composition was suitable for all three cell types. Using this culture medium, the ESCs, TSCs and XENs were initially grown and expanded in separate cultures. The researchers then prepared a cell suspension containing specific amounts of each of these cell types. A major milestone had been reached.

Yet there was one more challenge left to conquer. Cells have the tendency to sink and attach themselves to the bottom of the flask, where they then continue growth as a 2D cell culture. To prevent this, and to enable 3D spheroid formation, Ellen Na experimented with ‘ultra-low attachment plates’ containing 384 tiny round-bottom wells. These plates prevent the cells from settling, apparently causing them to do precisely what they are supposed to do: “Once the cell suspension is added to the wells, the different cell types will self-assemble and form aggregates,” explains Ellen Na. “After several days, we saw embryo-like structures, which is precisely what we had wanted to achieve.” The researchers had completed a further crucial step towards producing an ETX embryo. However, were these structures truly identical to ‘real’ embryos, or were their similarities purely external?

Complex analyses were required in order to determine the truth, but the two researchers did not have the necessary funding. “At that point, we had hit the end of the road, as we were unable to analyze these structures further,” explains Geert Michel. In addition to giving presentations, the two researchers repeatedly sought contact with a range of different experts until, approximately six months later, Berlin’s excellent research network and a lucky coincidence came to their aid. A researcher from the Max Planck Institute for Molecular Genetics provided a crucial piece of information: the renowned developmental biologist and expert in mammalian embryonic development, Prof. Magdalena Zernicka-Goetz, would be in Berlin the following day. Perhaps there might be an opportunity to find the appropriate partner for the analysis of the ETX embryos.

The two researchers didn’t need any further encouragement. Overnight, they put together a presentation that would present the salient details of their project in a matter of 10 seconds. The following day, armed with their ‘pitch for the busy researcher’, Ellen Na and Geert Michel went to meet the leading expert in their field. Despite her extremely tight schedule, Prof. Zernicka-Goetz spent an entire hour with her Berlin-based colleagues. “She was hugely interested and would have loved to have taken me back with her on the plane,” reminisces Ellen Na.

Testing confirms: these are embryonic structures
The flight to Cambridge came three weeks later, carrying the researchers and her precious cargo: three cell lines and several AggreWellTM plates. With a total of 1,200 ‘microwells’ per well, these 24-well plates represented another step up from those previously used, enabling the researchers to produce more than 1,000 embryos simultaneously. Despite the fact that Na and Michel had not previously worked with these plates, the experiments conducted in Cambridge ran like clockwork. “My colleague instructed the most highly renowned experts in embryonic development research in the world in how to use our methodology and techniques, and was celebrated like a star,” says a proud Geert Michel. “It’s the sort of thing that only happens once in a lifetime." Another unique experience was the outcome of subsequent analyses conducted by Magdalena Zernicka-Goetz, which confirmed that the structures produced were in fact embryos. Ample material for a cover story in Nature Cell Biology.

Work on the next installment is ongoing. After all, the question that remains is whether these embryos are capable of developing into mice. With work on the transfer of embryos into live surrogates already underway in Berlin, and subsequent analyses once again scheduled to be performed in Cambridge, researchers should have an answer to this question by mid-2019. Describing the current state of the research, Dr. Michel says: “We are already seeing successful implantation, but we don’t know yet how these cell structures will continue to develop inside the uterus.”

Thousands of mice would be spared every year
If successful, and results prove reliably reproducible, this project will deliver a whole range of potential uses. One of these is to achieve a reduction in research using animals. It is conceivable that future drug development research could test potential new drugs on embryos produced in a culture dish; similarly, one might be able to study the implantation of embryos in vitro, without sacrificing a single animal’s life. The manufacture of cell lines from transgenic mice holds at least as much promise in this regard. ‘Basic’ (laboratory-based) researchers, for instance, use genetically modified mice to help them understand disease mechanisms and find new treatments – and they need these ‘knockout mice’ in huge numbers. The embryos used to produce these mice are taken from donor animals, which are killed in the process. Artificially produced embryos would put an end to this practice. “We are full of hope that our approach will one day enable us to eliminate the need for embryo donors,” says Geert Michel. He adds: “This would result in an enormous reduction in the number of research animals used.”

Ellen Na has since moved to a different research group. However, it is her sincere wish that this project should be continued in some form. A second published article would be nice. It would of course be even better if her method were to become firmly established as a humane alternative to animal research. “That,” says the Berlin-based researcher who once studied Chinese and Korean Studies and Linguistics, “would constitute a truly happy ending.”

*ETX is a new term created from the initial letters of the three cell lines (ESC, TSC, and XEN) used.

Links

Transgene Technologien

Contact

Dr. rer. nat. Geert Michel
Leitung Serviceeinrichtung Transgene Technologien
Charité - Universitätsmedizin Berlin
Campus Benjamin Franklin
Krahmerstraße 6
12207 Berlin
t: +49 30 8445 3809