Cytocentric Visionaries: Hal Broxmeyer, Indiana University
How the Shock of Room Air Oxygen Reduces Stem Cell Yields
Alicia Henn, PhD MBA, Chief Scientific Officer, BioSpherix
Dr. Hal Broxmeyer is a Distinguished Professor, Professor of Microbiology/Immunology and Co-Leader of the Program on Hematopoiesis, Malignant Hematology, and Immunology at Indiana University Simon Cancer Center. He has chalked up well over 1,000 scientific publications and his work has been seminal to the field of hematopoietic stem cell transplantation. His group’s 2015 paper in Cell and the subsequent review article are landmark papers that demonstrate how room air oxygen exposure during cell isolation inflicts damage on freshly isolated stem cells.
Here, we talk with Dr. Broxmeyer about his work. This interview was conducted by email and edited for length.
AH: We see you as a Cytocentric Visionary because of your decades of work elucidating the biology of hematopoietic progenitor cells in physiologically relevant oxygen conditions. Why were you interested in investigating the effects of room air exposure on HPC?
HB:The bone marrow microenvironment where hematopoietic stem (HSC) and progenitor (HPC) cells mainly reside in the adult is hypoxic (~1-5% oxygen), compared to that of ambient air (~20% oxygen; normoxia). Even cord blood is lower in oxygen than ambient air.
We have been interested in the comparative effects of hypoxia and normoxia on the growth of HPCs and HSCs since the early 1980s. That’s when Randy Yerden first loaned us his “oxyreducer” to do some preliminary studies on colony formation by HPCs prior to us purchasing it. We clearly showed that colony formation was much improved when the cells were grown in hypoxic compared to normoxic cultures. We also showed that the cells grown in hypoxia were more sensitive to growth regulating cytokines, which made sense since lowered oxygen is a more physiological environment for these cells. Yet, all studies performed prior to our recent paper , including our own, processed the cells in ambient air prior to culture.
Charlie Mantel, a Research Associate, questioned whether or not the exposure of mouse bone marrow cells to ambient air, even for very short periods of time, were stressing these cells such that they were differentiating. After years of work by Charlie, Heather O’Leary, a post-doc, Scott Cooper, a Research Associate, myself and others in the lab, we became convinced that even a very short term exposure to air caused the rapid differentiation of HSCs to HPCs through a phenomenon that Charlie termed Extra Physiological Shock/Stress (EPHOSS).
Over five years of work by many different people, we were able to gain some mechanistic insight into EPHOSS, which acted through a Mitochondrial Permeability Transition Pore (MPTP)-Cyclophilin D- p53 axis, and involved the oxygen-induced release of reactive oxygen species (ROS). We also implicated HIF-1alpha and the hypoxamir, miR210 in this phenomenon.
AH: Do you think that the peripheral blood samples or cells from solid tissues types might undergo the same type of shock?
HB: We have not yet tested peripheral blood for effects on HSCs and HPCs, but my guess is that the same phenomenon of EPHOSS will apply. In fact as I added to the Cell paper with the permission of the Editor, I believe that this phenomenon will be broadly applicable to all cells in the body (stem, progenitor, precursor, and more mature), including tumor cells, that reside in an in vivo hypoxic environment. To get more insight into these thoughts see a subsequent review we wrote .
I do believe that EPHOSS will have broad impact on the metabolism of cells that normally reside in an hypoxic environment and are then collected and studied in ambient air.
If you think about personalized medicine approaches to cancer, and the need to evaluate gene or protein expression patterns and the response of these tumors and their cells to different modulating agents, it may be that the study of these cells in ambient air, even if they are subsequently placed into hypoxia, may not reflect the true state and responsiveness of these cells as they would be when residing in vivo. Thus, one might get a false or not accurate understanding of how best to treat the individual. This area clearly needs investigation.
AH: Do we need to go back and put a footnote on all previous studies that says “Performed in non-physiologic conditions,” or some other cautionary note?
HB: Wouldn’t this be an interesting thought? I seriously doubt that it will ever happen. However, it would be nice if others were able to follow up in this area for different types of cells.
Most important is the rigor needed in these experiments.
For example, we had to be very careful about air contamination in our experiments using the hypoxic chamber. Everything in the hypoxic chamber had to be pre-equilibrated to the lowered oxygen tension of 3% for at least 18 hours prior to processing the cells. This included glassware, plasticware, solutions, growth factors, etc. See the Cell paper for details.
The first experiments that were done were in a “RUBE GOLDBERG” type hypoxic chamber apparatus that Charlie put together himself that used duct tape to keep the oxygen levels in the chamber low, which gave us enough confidence in the results. But, it was not until we had a better more professional chamber that was more quality controlled that we got the reproducible effects we reported.
AH: There are costs to a researcher for abandoning their own methods after publication. Have you seen your recent findings inspire major changes by others?
HB: I have not yet seen follow-up on the hypoxia studies. I have heard from others that they are working on the use of cyclosporine A, as we noted in the Cell paper, to compensate the opening of the MPTP, so that the studies can be done in air. However, cyclosporine A is not an easy compound to work with as it is not easy to get into solution, and if cells are exposed to it too long in culture it is toxic.
We spent a long time finding the right concentrations of cyclosporine A to use for our studies and each investigator will have to work this out for themselves.
So in the meantime we are studying other compounds and means, without need for hypoxic collections and processing of cells to mitigate the EPHOSS effect which occur when these cells are exposed to ambient air.
AH: Why do you think, after a flurry of interest in oxygen in vitro in the 1950’s, 60’s, and 70's, that interest in oxygen had fallen off?
HB: This is like all science. It keeps moving forward which is what makes scientific investigation so exciting. Science knowledge moves on, as it should, and investigators like to be part of what is considered the next “hot” area.
Sometimes this is necessary in order to be competitive for publishing your work and attaining external peer-reviewed funding. However, sometimes, old areas reappear when a new advancement is made. I never left working in the area of hypoxia but now we are on to another aspect of this area, one that I feel is very important.
AH: How are your findings going to change the future of clinical use of hematopoietic stem cells and progenitors?
HB: This remains to be seen. I have been interested in cord blood transplantation since the early 1980s when I helped to initiate this clinical field. My lab continues to focus on basic science cell and intracellular mechanistic studies and pre-clinical animal models to better understand the regulation of HSCs, HPCs, and hematopoiesis in order to improve Hematopoietic Cell Transplantation in collaboration with our clinical colleagues.
We are working on different aspects of this, but one aspect is to improve the quality and quantity of collected cord blood, so that the cord blood banks have more potent cells to provide. Thus, we are currently following up on the results of our paper in Cell to find an easier, more simple and inexpensive means to counter the EPHOSS effect.
In addition to finding other means to collect a more potent stem cell population, we continue to study how to better engraft these cells. We continue to study the effects of dipeptidylpeptidase 4/CD26 and its inhibition, as well as a number of new small molecules to enhance homing and engraftment.
AH: Thank you for taking time to answer our questions here at the Cytocentric Blog. We look forward to seeing your work spread across the community.
HB: Thank you giving me the opportunity to respond to your questions.
1. Mantel, C.R., et al., Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock. Cell, 2015. 161(7): p. 1553-65.
2. Broxmeyer, H.E., et al., The importance of hypoxia and extra physiologic oxygen shock/stress for collection and processing of stem and progenitor cells to understand true physiology/pathology of these cells ex vivo. Curr Opin Hematol, 2015. 22(4): p. 273-8.