Cytocentric Visionaries: Donald Phinney
Part 3: The Cell Cycle Is the Redox Cycle, and All Clinical MSC are Stressed
In Part Two, Chief Scientific Officer Alicia Henn talked with Dr. Phinney about MSC, the exosomes that they produce and therapeutics. Today we talk about MSC and oxidative stress in vitro. This conversation on the importance of studying the cellular redox balance in MSC cells was edited for length.
You set up MSC and macrophage to interact on a stage to study them. Any good play begins with a stressor and in this case, it was oxidative stress.
DP: This is something that has evolved over a number of years now. It is quite clear that bone marrow is a low oxygen environment. Oxygen saturation varies across the bone marrow and sinusoids, but it is certainly low. For many years, because they had little choice in the early days, people grew cells in atmospheric oxygen.
There are papers going back 20 years or longer showing effects of oxygen, particularly upon hematopoiesis, and then upon marrow stromal cells. One of the biggest stresses on cells is changes in oxygen saturation.
Cells have very complicated mechanisms to regulate that phenomenon. Many of the antioxidant proteins and transcription factors that regulate stress responses are intimately interacting with cell cycle regulators, with NF-kappaB and p53. People are actually calling the Cell Cycle the Redox Cycle now.
Mouse MSC are super-sensitive to oxidative stress and we are just seeing all kinds of fascinating things. An important point is that oxidative stress or reactive oxygen species (ROS) play critical fundamental roles in cell biology and disease pathophysiology as well. We have an interest in Fanconi Anemia, bone marrow failure syndromes, and how changes in p53 and ROS affect all these processes. Some cellular differentiation events are promoted by ROS and some are inhibited. It is a very exciting area to be studying right now because it is all so interactive.
Why weren’t ROS and physiologic oxygen so important before now?
DP: It’s evolving. In the old days, just being able to establish cultures was, in some cases, a herculean effort. Developing proper media formulations took precedent. In the back of people’s heads, they knew oxygen was important, but you needed specialized equipment. Looking at some of the key factors driving events for cell physiology took precedence. In the MSC field for example, now we know most of the master regulators that drive bone and cartilage differentiation and the signaling pathways that promote growth and self-renewal.
So as these things get teased out, you start to realize that redox balance affects all of these processes. So it becomes very interesting to study how oxygen and oxidative stress responses impact these known pathways.
As to chambers and oxygen-regulated incubators, they are much more common now. The cost isn’t any more expensive than setting up a standard tissue culture lab. We are at the point now where we grow all of our primary mouse cells in closed, low oxygen and we are beginning to think that we should grow all of our human cells under those conditions as well.
Obviously the work we described in that nature communications paper [Phinney et al., 2015] suggests that even though human MSC seem to grow well in atmospheric oxygen, they are clearly under stress and that is affecting their biology.
I envision producing MSC in stressful physiologic conditions to get them to export exosomes so we can use those for treatments.
DP: I would argue that probably all MSCs that are used clinically are stressed. I don’t know of a facility that grows cells in low oxygen. Most facilities are using additives to drive expansion to high levels and that’s a stressor for sure.
So if you’re culturing MSC at physiologic oxygen and you take them out in the air for just a little while to handle them and put them back, what does that do to the their stress level as compared with having unbroken physiologic conditions.
DP: We actually did that in the early days. It’s much more severe for mouse MSCs. We originally got an oxygen controlled incubator, but we would change the cultures in the hood before we got the glove box. It turns out if you bring cells in and out of different oxygen levels, it affects them much more severely. You actually upregulate massive amounts of p53 and the cells do much worse.
That makes sense right? If you shift them back and forth it’s like reperfusion injury, where it’s not the ischemia but the reperfusion that does them in. We’ve definitely seen that in the mouse MSCs. It’s much better not to shift them back and forth.
Thank you, Dr. Phinney for sharing your findings and your enthusiasm with us. Is there anything else you’d like to tell readers?
DP: The value of studying the cellular redox balance is critical. Obviously, trying to replicate the physiological conditions that your cells are normally in is critically important.
This concludes our conversation with Dr. Phinney. If you would like to be featured in our Cytocentric Visionary Series, contact us. We would love to hear about your work.
Phinney, D. G., et al. (2015), Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs, Nat Commun, 6, 8472.
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.