Cytocentric Visionaries: Dr. Adrien Moya, University of Miami
Part 2: Focusing on Cell Fate
In Part One of this two-part interview, we talk with Dr. Moya about his recent paper on the importance of proper glucose and oxygen levels for MSC in regenerative medicine constructs. Here we talk about the implications of this work.
So you found that Multipotent Stromal Cells (MSC) die deep inside hydrogel constructs not because of lack of oxygen, but because of lack of glucose. What impact could this have on future work on bone tissue regeneration?
I think that one of the key issues overlooked in that regard is cell density in cell constructs. If you put too many cells, no matter the amount of glucose you’re adding, the cells will not survive. This is because the microenvironment of the implanted cells will become deleterious faster than the cells can adapt. As a result, there will be massive cell death no matter how much glucose you use.
If you extend this to in vivo applications, many studies use a huge amount of cells. It can be more than a hundred million in some clinical trials. If a hundred million cells are implanted, how many of these cells are still alive after one week post implantation? If the answer is only ten thousand then maybe with only a hundred thousand cells, the same amount of surviving cells could have been achieved after one week post implantation.
Implanting a hundred thousand cells instead of a hundred million could have saved a lot of cell expansion time. This cell expansion time is precious as amplifying MSC (and stem cells in general) from a few cells to one hundred thousand instead of one hundred million, would have saved a lot of passages and better preserved MSC stemness and regeneration potential. This may ultimately improve their clinical outcomes.
I believe it is an important argument to make for tissue engineering applications.
You have to get those cells through that first initial implantation time without burning through all the local glucose?
Yes, the two first weeks post-implantation are the most important ones because that’s when most of the cells die. Massive cell death always occurs at the center where it’s a more hypoxic and ischemic environment because neither nutrients nor oxygen can get there quickly enough. As a result, cell survival is always better at the surrounding part of the tissue engineered construct. Once the construct is vascularized then the environment present at the core of the cell construct is no longer ischemic.
In this paper you looked at the in vivo oxygen levels of these implants, at the edges and at the core. That is often a point that is missed.
We also looked at the oxygen levels within the implants both at the core and at on the surface before implantation. When the external oxygen tension is at 21% O2 (air), the oxygen tension present at the border of the cell constructs was roughly around 16% O2 whereas the one at the center was 5% O2. So, in fact, there is a huge oxygen gradient in this type of tissue engineering constructs.
What kind of technical problems did you encounter in your work?
The BioSpherix system we used at the lab is really good. However, we always have to be careful that the hypoxic chamber is well calibrated, mainly because that 1% oxygen tension is really different from 0.1% oxygen tension. Therefore, any calibration problem could create an artefact in the data collected from these experiments.
The 3D culture itself was not very hard. MSC were loaded in fibrin hydrogels which is fairly easy to achieve. However, what can be hard is to perform tests from these 3D cultures. For example, we had to come up with ways to degrade the hydrogel without destroying our cells, for staining live and dead cells and flow cytometry analyses. In that regard, one should be careful that the degradation of the hydrogel does not harm the cells. So that part was a bit tricky.
Also, we have tried to come up with a system that would allow us to study the metabolism of MSC in vivowithout being invaded by host cells. For these experiments, we used diffusion chambers to keep the cells from the host out, but they were very time consuming to make. We were then able extract the cells from the cell-constructs which were imbedded in the diffusion chamber. Apart from these hurdles, we performed simple, yet robust experiments, and I think that they addressed accurately some of the critical questions.
MSC are being targeted for tissue engineering to so many different types of tissues, and those different places all have different physiologic oxygen levels. Do you think we should treat MSC differently for different injuries?
Yes, I think we should. First of all, I think we should be careful of using the term MSC. Indeed, you are surely aware there is not only MSC isolated from bone marrow, but it also derived from adipose tissue. Although they share the same name, and some key features, they also possess very different attributes.
For example, we have found that for bone tissue engineering applications, MSC derived from adipose tissues do not work very well in comparison to bone marrow isolated MSC. So certainly, there is the issue of which type of MSC we use for which tissue engineering application.
Moreover, one should be careful when using the acronym MSC as it refers to too many definitions: Mesenchymal Stem Cells, Multipotent Stromal Cells, Medicinal Signaling Cells, Marrow Stromal Cells, and even Multi-factor Secretory Cells, etc…
In my opinion, the best course of action for the tissue engineering applications, would be to develop protocols that best preserve (least disturb) the MSC from extraction, through expansion and up to implantation. We have to prepare these cells for either the environment or their intended task once implanted. If these aspects are not taken into account when developing protocols for cell expansion, I believe we are not to get the best physiological responses and ultimately clinical outcomes from these cells.
Is there anything else that you want our readers to know about your work?
As you may have noticed during this interview, I feel quite strongly about the cell fate after implantation. And I believe too few people are focused on that aspect.
Many research groups often look at the chemical or the biological outcomes of the implanted tissue constructs, however cell survival does not interest a lot of people. From my perspective, it’s important to know how many implanted cells survive, what are the underlying mechanism of their survival and how they contribute to the biological response.
Moreover, thinking about the role of MSC in regenerative medicine, I would argue that it is likely that a cell struggling to survive will not going to be a cell that will efficiently repair tissues. They will redirect their energy toward survival instead of fueling the regenerative response.
Also, I would also thank all of the people in my lab for the support, especially Nathanaël, but also the director Hervé and my former PhD PI, Delphine. I would also like to thank again the CNRS (centre national de la recherche scientifique) and the DGA (direction generale de l'armement) for funding this research. Finally, thank you BioSpherix for the good equipment you have provided us. It gave us the necessary tools to study MSC in their adequate environments (Normoxia, Hypoxia and Anoxia). Well done!
Thank you for your time and your kind words, Dr. Moya. We will be looking for your future work in Miami, and with MIAMI cells!
This concludes our interview series with Dr. Adrien Moya.
If you would like to be featured in our Cytocentric Visionary Series, contact us. We would love to hear about your work.
- Moya A, Paquet J, Deschepper M, Larochette N, Oudina K, Denoeud C, Bensidhoum M, Logeart‐Avramoglou D, Petite H: Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly use Intracellular Energy Reserves through Glycolysis Explains Poor Cell Survival After Implantation. STEM CELLS 2017.
About the Author
Alicia D Henn, PhD, MBA
Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.