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Cytocentric Visionaries: Roger Rönn

The Cell Environment for HSC from iPS: Why Every Hurdle Must be Overcome to Reach our Destination

This is an update on a previous interview that Dr. Alicia Henn, Chief Scientific Officer of BioSpherix, conducted with Roger Rönn, who recently finished his PhD and is now working in the laboratory of Elaine Dzierzak at the University of Edinburgh. Here we discuss his most recent paper in Stem Cells 1. This interview was edited for length.

AH: This study extends your previous findings on reactive oxygen species (ROS) in hematopoietic progenitors generated in vitro from pluripotent stem cells. Here you show that sorted low ROS vs. high ROS cells have different proliferative capacity. I thought it was interesting that even in the same culture, cells had different internal ROS levels. Why do you think that might be?

RR: We have a very artificial situation for cells outside of the body. Because the cells are exposed to higher levels of oxygen, these conditions encourage spontaneous oxidation, which significantly contribute to the build-up of ROS. When cell culture started over 50 years ago, people were first culturing cells such as fibroblasts that, while capable of only dividing a finite number of times 2 could grow in what became our standardized in vitro culture conditions. Importantly, these conditions are at atmospheric oxygen levels around 21%. Since we base our work on what has been described previously, it is easy to forget to question the more basic aspects on how we work with our cells in vitro. For instance, just because some cell types may tolerate atmospheric oxygen concentrations, assuming the same would equally apply to all various cell types may be risky. In my case, working on understanding the blood system and hematopoietic stem cells (HSCs) form during embryonic development, it is possible that HSCs may be less resilient to oxidative damage compared to other cell types. In our recent paper, we compared ROS in the HSC-like cells and endothelial cells that were generated together from human pluripotent stem cells (hPSCs) in vitro. We found that endothelial cells retained a significantly lower ROS level than the HSC-like cells, suggesting an intrinsic capacity of these cells to prevent the more extreme elevations of ROS we observed in the HSC-like population. Therefore, it is possible that the more pro-oxidative conditions associated with standard in vitro culture conditions at 21% O2 may have a more deleterious impact on HSCs compared to other cell types.


AH: In this paper you show that the cells with high ROS tend to accumulate more DNA damage as well, right?

RR: Yes, I used a method to measure the amount of DNA breaks in cells with different amounts of intracellular ROS, and high ROS cells were found to have more DNA damage. But ROS are known to also cause oxidative damage to other cellular components such as proteins and lipids so it may be that the negative effects we see is actually a sum of what would be mediated downstream through separate pathways of stress. Because it would be difficult to accurately modulate all possible pathways downstream of elevated ROS, and also since modulating singular pathways are likely to have limited efficiency, we reasoned that the most direct way to reduce the negative effects of increased ROS would be to directly try and limit the processes that form ROS in the first place, to normalize the intracellular ROS level to that of cells found in vivo.


AH: There’s an interesting new term you used in the paper, “redox window.”

RR: It’s interesting because cells do actually need ROS to some extent. At steady state, ROS do take part in how the cells convey signals internally but these levels of ROS are regulated and kept very low. So I wouldn’t think it a good idea to try and eliminate ROS all together. You just need to find a way to balance it. Usually when studying biological effects, we tend to start looking for a mechanism, one process that can be attributed as responsible for the phenotype that we observe. However, I don’t think it is that straight forward with ROS. As demonstrated in the paper many different causes, both intrinsic and extrinsic, together contribute to the total intracellular ROS level. While it is interesting to know how and through what mechanisms ROS forms, what matters to the cell is the total amount of ROS and not where and how it originates. To better understand what processes can contribute to the build-up of ROS, we used several different approaches. We inhibited the cells capacity to directly generate ROS through stress signaling or enzymatic activity, and we aimed to limit ROS that would form by spontaneous oxidation by changing the oxygen concentration from 21% to 4% inside our incubator. While inhibiting cellular process indeed helped to reduce ROS in our cells, it was changing the oxygen level down to 4%, a technical limitation at the time that was the most efficient approach in reducing the total ROS level. It is important to remember that 4% oxygen is still not as low as the cells would experience inside the body, which is somewhere around 1 to 3% depending on where you are, but it is much lower than the atmospheric 21%. Using this reduced oxygen concentration we managed to get 40% normalization towards the ROS levels found in uncultured human cord blood.


AH: Did you pre-equilibrate all your media for the 4% O2?

RR: Yes, that was necessary since directly adding media would break the hypoxia since the media, if prepared in standard in vitro conditions, would come loaded with the higher O2 concentration. I only had a hypoxic incubator to work with at the time so the only way to do this was to pre-incubate the new media in a separate dish for at least 6 hours before adding it to the cells. This certainly increased the amount of work but the results really made it worthwhile.


AH: Do you see internal ROS as a factor that might be useful clinically for assessing the ability of human HSC to engraft?

RR: People in my field have long sought ways to maintain and expand functional human HSCs in the lab as a way to improve therapy. One donated unit of cord blood is usually not enough for treating a fully grown adult, but if you could expand the HSCs contained, even if only by two-fold, it could dramatically improve clinical treatment of blood disease. Unfortunately, efforts aimed at expanding HSCs have had limited success, and the HSCs quickly lose their long-term functionality when they are cultured outside the body. I think one key factor involved in this loss of functionality is because the HSCs become damaged from oxidation. Therefore, I was very excited when a paper from the Broxmeyer laboratory 3 recently demonstrated that protecting cord blood cells from exposure to atmospheric oxygen significantly improved the number of functional HSCs that could engraft.


AH: What are different ways that researchers could reduce ROS for cells?

RR: We used small molecules to either inhibit or block processes where the cell itself increases the intracellular ROS level. While these strategies helped to reduce the total level of ROS and provided evidence that ROS originates from multiple processes, the main factor that helped to decrease ROS was reducing the cultures oxygen concentration to limit spontaneous ROS formation.


AH: So do you see research heading toward physiologically relevant oxygen in the future?

RR: It’s probably heading in that direction, but it’s not going very fast. I think people in the field might get somewhat uncomfortable considering the possibility that something as fundamental as our current in vitro working methods, something we take for granted and is dogma, might actually be a major reason why HSC generation so far has been unsuccessful. That’s a big deal. Very many people believe that we just need to understand hematopoietic development a little better, perhaps find some missing cytokine to add, or over express a key gene, but what I’m talking about goes outside that concept. I am suggesting that the reason why we have been failing might not be the developmental process, but rather that the cells we have been trying to make never really had a chance from the beginning due to detrimental oxidative damage occurring from our in vitro culture methods. Conditions that, when you think about it, are far from embryonically relevant. Of course, this does not exclude the possibility that there could be a need for additional factors, such as more accurate cytokine stimulation, for successful HSC generation, but I am certain that as long as the cells we generate have elevated ROS, which has been proven to be incompatible with function for true adult HSCs 4, we will not be able to successfully generate HSCs. As I demonstrated, reducing the oxygen concentration had a dramatic impact on the function of hematopoietic progenitors generated in vitro, but it is clear that more work is needed before we successfully can generate cells that have ROS levels similar to their in vivo counterparts.

In general, I think it makes sense to culture our cells with parameters that most accurately recapitulate the natural conditions for the cell in vivo. While HSCs may be extraordinarily sensitive to oxidative damage and therefore depend most on such conditions for retaining their functionality, it might be that other cell types also could benefit from reduced oxidative stress. Even if the cells may be resilient to oxidative damage such as DNA breaks, when you expose them to non-physiological oxygen levels they consequently will be more likely to accumulate damage, increasing the risk of transformation and other abnormalities. Increased oxidation could also be a problem in terms of the stability of several proteins and small molecules added to the culture. And another situation where this unwanted oxidation might be critical is when doing screenings for drugs and molecules.


AH: So you think that the physiologically relevant oxygen is important for its affect on the actual drug substance?

RR: Rapid oxidation is a very common cause for instability and short half-life of many small molecules. Drugs that may have great in vivo potential may be tested with pro-oxidative in vitro conditions. Because of this some drugs might not get detected due to their rapid spontaneous oxidation. Evaluating the function of drugs in culture conditions that more accurately reflect the in vivo environment in which the drugs are ultimately intended to be used would make sense. I think this could be the difference between a drug being considered interesting or just being thrown to the side.


AH: Is there anything else that you would like people to know about your work right now?

RR: I cannot get into too much detail on exactly what I am working on but creating cells that have normalized ROS levels, potentially with engraftment capacity, continues to be a major goal. Reaching this goal certainly won’t be easy. But what really drives me is that, after spending years thinking about what could be the reason behind the difficulty to generate functional HSCs in the lab, I finally have an alternative theory to explain it. I can’t give the full details of this Model of Functional Impairment by External Oxidative Insult in this interview, but if any reader would be interested it can be found in my PhD-thesis, which is available online as open access here.

To summarize, while it is possible that reducing oxidative damage alone won’t be sufficient to enable successful generation of functional HSCs, I am fully convinced that preventing an increase in damaging ROS will, like for adult HSCs, be a critical feature. In other words, if we are to reach our destination, sooner or later we must overcome every hurdle that blocks our path.


AH: We will be watching your work as you progress, Dr. Rönn. Thank you for your time, your enthusiasm, and your insights.

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1. Ronn, R. E., Guibentif, C., Saxena, S. & Woods, N. B. Reactive Oxygen Species Impair the Function of CD90+ Hematopoietic Progenitors Generated from Human Pluripotent Stem Cells. Stem cells (Dayton, Ohio) 35, 197-206, doi:10.1002/stem.2503 (2017).
2. Hayflick, L. & Moorhead, P. S. The serial cultivation of human diploid cell strains. Experimental cell research 25, 585-621 (1961).
3. Mantel, C. R. et al. Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock. Cell 161, 1553-1565, doi:10.1016/j.cell.2015.04.054 (2015).
4. Yahata, T. et al. Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells. Blood 118, 2941-2950, doi:10.1182/blood-2011-01-330050 (2011).