Bernd Giebel: The current state and the therapeutic potential of MSC derived EVs.
PD Dr. rer. nat. Bernd Giebel
Bernd Giebel studied biology at the University zu Köln and received his PhD in 1996 at the Institute for Developmental Biology in Köln. In his thesis he investigated aspects of the Notch signaling pathway during early neurogenesis of Drosophila melanogaster. In 1999 he moved to the Heinrich-Heine-University of Düsseldorf and started to work with human hematopoietic stem and progenitor cells. There, he also established his research group focusing on mechanisms controlling the decision of self-renewal versus differentiation of human somatic stem cells. In 2008 he moved with his group to the Institute of Transfusion Medicine at the University Hospital Essen. Here, he continues his studies on human somatic stem cells. Via the identification of two asymmetrically segregating proteins in dividing human hematopoietic stem and progenitor cells, the tetraspanins CD53 and CD63, he became interested in extracellular vesicles (EVs).
As a second topic, in 2009, his group started to work on purification and analysis methods for EVs, setting a focus on mesenchymal stem/stromal cell-derived EVs (MSC-EVs). Together with collaboration partners the group demonstrated the therapeutic potential of prepared MSC-EVs in a human GvHD-patient and in different animal models. Accordingly, Bernd Giebel has a strong track record in the purification, characterization and analysis of the therapeutic activity of EVs and has published several articles on this topic. Due to the overall increasing interest in EV biology, the group is involved in several projects on the campus and is actively cooperating with different German and international groups.
Dr. Giebel was a member of the ME-HAD COST Action and led the working group Diagnostic & Therapeutic Potential of Microvesicles and Exosomes (ME). Since 2012 he is an active member of the International Society of Extracellular vesicles (ISEV) and coordinated an ISEV position paper regarding the therapeutic application of EVs. Furthermore, he is the president of the German Society of Extracellular Vesicles (GSEV), which he founded together with other German EV experts on March 2nd 2017. According to his expertise, Dr. Giebel is part of the scientific advisory board of two SME companies being active in the EV field.
- What pushed you towards a career in science and how did you first become interested in extracellular vesicles?
- I liked nature and animals from my earliest childhood on and it quickly became clear for me that I want to study biology.
After being educated in developmental biology, I switched to the stem cell field and searched for asymmetrically segregating proteins in dividing human hematopoietic stem/progenitor cells. Indeed, in 2005, we identified four factors, which segregated asymmetrically into the arising daughter cells. Two of them were the tetraspanins CD53 and CD63. Upon reading about tetraspanins, I realized that some groups used them as exosomal markers. Wondering what exosomes are, I started to read related to my passion in cell biological approaches quickly became fascinated by these nano-sized vesicles. Accordingly, in the resulting manuscript, which was published in 2007 in Blood, we for the first time mentioned exosomes in one of our papers.
- What was the biggest challenge you faced in your research career?
- I think the biggest challenge was to set up and to establish an own group. There are several issues that had to be solved: one has to work in a lab and educate co-workers, in parallel it is expected to write grant applications and papers and that with a position which regularly is limited with a time contract. It was a top or hop situation and a very exhaustive time, so to say, “it was quite challenging to survive”.
- What is the MSC-EVs therapeutic potential?
- I think it is quite huge if you have the right MSCs and you have the right strategy to purify them. MSC-EVs act as anti-inflammatory substances in a physiological manner and I expect that they can substitute steroids very effectively in a long-term perspective. I am convinced, they can be used for suppressing acute inflammation and promote regeneration in many degenerative diseases. To my understanding acute inflammation and regeneration is not compatible and whenever inflammations are sterile, MSC-EVs appear very promising to suppress them, which apparently promotes regeneration and recovery from disease symptoms.
- Is there any difference in the effect of application of MSCs directly and their EVs?
- Yes, I think we should consider that there are differences. There are both advantages and disadvantages in MSC-EV application compared to MSCs. First advantage is connected to the EV administration which can be sterilized by filtration.
Secondly, as EVs are once released we expect that they do not change massively in their composition. That means depending on the developmental stage of the cells or of their metabolic state they may have a certain quality, which in another setting might be different. If cells are characterized in vitro and applied in vivo, they can be reeducated by the environment and do something else than in vitro. In contrast, EVs very likely will keep the function independently of the environment.
However, reeducation of the cells might also have positive effects. If MSCs are not cultured optimally, their EVs might lack any therapeutic functions, however, due to transplantation into a certain environment, MSCs might get reeducated and start to release therapeutically active EVs in the novel environment. So, eventually the state of the MSCs and how they feel might be less critical for MSCs than for their in vitro harvested EVs. As our understanding of MSC-EVs is very preliminary, for me the testing of prepared MSC-EV fractions in functional in vitro assays is mandatory before applying these EVs into any patient.
- Do all MSCs produce therapeutic effective EVs?
- To my understanding, no. We see huge functional differences between MSC-EV fractions harvested from supernatants of MSCs of different healthy donors. As also MSCs from one given donor often appear very heterogenous, we also consider the existence of different MSCs subtypes, not all of them being therapeutically effective. That is what we addressed in our research.
- What is the current stage of your MSC-EVs investigations? What are you working at?
- We try to optimize production process, to get it licensed and to work in a scaled manner, so that it was upscaling in combination with GMP compliance and getting rid of any ultracentrifugation steps.
We believe that ultracentrifugation affects the integrity of EVs and therefore we would like to set up a UC-free EV production process.
- What in your opinion are not-to-do things when working with EVs?
- There are a number of issues. At the moment we are experiencing that NTA and other “EV-quantifying” devices are not enumerating EVs but rather nano- and microparticles which might by magnitudes outcompete the numbers of EVs being present in “EV” samples. For now, we may lack any reliable device which allows quantification of EVs in a reliable manner.
However, I think what we still should obey to the MISEV criteria and follow the guidelines provided by ISEV position papers and the EV-TRACK consortium. We should quantify particles in relation to the protein content of given samples and then check for the presence of exosomal marker proteins in Western Blots.
Furthermore, we learned that CD9, CD63 and CD81 should not be considered as pan exosome markers. Although they are helpful EV markers, they have been found as being differently expressed among different EV types.
Last but not least and as mentioned before, to my best understanding we should avoid ultracentrifugation and consider that this method affects the integrity and the morphology of many EVs. We really should be aware that UC might create a lot of artefacts.
Furthermore, we should consider that the dyes which are on the market might not specifically stain EVs. I consider that many EV-papers do investigate something else but EVs. Thus, to promote the EV field, I see it as essential that EV researchers understand their applied techniques and methods in detail and are best aware about their limitations in EV research.