September-October 2015, Volume 12, Issue 5
Hematopoietic Stem Cells Should Hold Their Breath
Published on: August 12, 2015
Mantel CR, O'Leary HA, Chitteti BR, et al. Enhancing hematopoietic stem cell transplantation efficacy by mitigating oxygen shock. Cell. 2015;161:1553-1565.
Much of what we know about hematopoietic stem cells (HSCs) revolves around our ability to take them out of a donor and transplant them into a conditioned recipient, as was discussed in greater detail in a Mini Review in The Hematologist.1 However, as is increasingly becoming clear, in many cases our experimental systems for HSCs may create observer effects, where the act of experimenting fundamentally changes the cells themselves. A recent report from Dr. Charlie Mantel and colleagues highlights just how sensitive HSCs can be to our experimental and clinical manipulations.
Several early reports have demonstrated that HSCs are present in regions of presumed hypoxia within the bone marrow. A recent direct measurement of local oxygen tension in the bone marrow of live mice showed that the bone marrow niche had considerably low oxygen (<32 mmHg) despite very high vascular density, with the lowest (~9.9 mmHg, or 1.3%) found in deeper perisinusoidal regions.2 However, when HSCs are harvested, either for subsequent clinical transplantation or for experimentation, they are harvested and processed in ambient air, which is approximately 21 percent oxygen. The authors hypothesized that this exposure to ambient air could cause a phenomenon they term “extraphysiologic oxygen shock/stress” or EPHOSS, and lead to reductions in HSC function.
They began by comparing mouse bone marrow that was harvested in ambient air, with marrow samples that were harvested in hypoxic chambers (3% oxygen content). Intriguingly, even exposures as low as 30 minutes in ambient air lead to almost 5-fold fewer phenotypically-defined HSCs than those that were maintained in 3 percent oxygen. When the bone marrow was harvested and competitively transplanted in hypoxic settings (using a custom-made mouse respirator system within the hypoxic chamber), there was a 2.5-fold increase in competitive repopulating ability, demonstrating that not only are phenotypic HSCs recovered at a higher rate in hypoxia, but they are also functional.
The increases in HSC engraftment were not due to changes in HSC homing to the marrow, nor were they due to changes in apoptosis, suggesting that alternative mechanisms were mediating the deficits in ambient air–exposed HSCs. The authors hypothesized that EPHOSS-mediated deficits in HSCs harvested in ambient air could be due to something similar to ischemia-reperfusion injury, where reactive oxygen species (ROS) from the mitochondria facilitate tissue damage. Indeed, ROS was elevated in ambient air–exposed HSCs, as was mitochondrial activity and the number of hematopoietic cells with hyperpolarized mitochondria, suggesting opening of the mitochondrial permeability transition pore (MPTP) in these air-exposed HSCs.
To test this hypothesis, the authors utilized a knockout mouse for cyclophilin D (CypD), which is a regulator of the MPTP, and previous reports have demonstrated that CypD knockout mice are protected from MPTP-mediated ischemia-reperfusion injuries. Compared with wild-type mice, bone marrow harvested in ambient air from CypD knockouts had significantly greater HSCs, with lower levels of ROS and progenitor activity, further supporting a role for EPHOSS and the MPTP in HSC regulation during harvesting in ambient air. While the authors could create a laboratory setup that allowed the harvesting of bone marrow from small mice and subsequent transplantation, all within a hypoxic chamber, the ability to replicate that method clinically would be expensive and cumbersome. Instead, the authors used the immunosuppressant cyclosporin A, which binds to CypD and prevents opening of the MPTP. When bone marrow was harvested in the continuous presence of cyclosporin A, the authors demonstrated a reversal of the deleterious effects of ambient air exposure, with significant increases in HSC recovery and competitive transplantation. These results translated to human cord blood samples harvested in cyclosporin A as well, with increases in immunophenotypically defined HSCs and repopulation into immunocompromised (SCID) mice. The authors therefore demonstrate a new pathway for regulation of HSCs and a new method of harvesting HSCs to prevent EPHOSS, either with collection in hypoxia or in the presence of cyclosporine A (Figure).
The report by Dr. Mantel et al has many important findings for the field and raises further questions. Much has been done to enumerate the total number of HSCs in organisms, including seminal work by Dr. Janis Abkowitz that utilized both experimental data and simulations to estimate the number of HSCs as being between 11,000 to 22,000 in both mice and humans.3 Given that such results were based on air-exposed cells, it will be interesting to see if the total number of HSCs within an organism is actually significantly higher. The authors used a hypoxic chamber set at 3 percent oxygen and showed that 5 percent oxygen was not able to prevent EPHOSS and increase harvested HSCs. Most of the published work in hematopoiesis, particularly with the colony-forming assay, has used “hypoxic” chambers set at 5 percent oxygen. These results indicate that studies in hematopoiesis, or for that matter broad stem cell systems in other organs as well, may need to explore lower levels, or even gradients of oxygen exposure to accurately replicate in vivo conditions. Differentiation through the “hematopoietic tree” is typically thought of as a series of divisional events, where an HSC divides to form more mature progenitors, which then divide to form lineage-committed progenitors. However, in these studies, the authors saw very significant changes in the numbers of both phenotypic and functional HSCs in as little as 30 minutes of exposure to air. This surprising finding perhaps suggests that programming of an HSC can occur rapidly and independently of cell division and warrants further exploration. Finally, the cyclosporin A protection of HSCs was also seen in human cord blood in both in vitro assays and in xenotransplantation studies and merits further clinical exploration. Several clinical trials are underway exploring methods to ex vivo manipulate cord blood units, expand cord blood units, or modulate the host to enhance engraftment. This manuscript adds another approach, and suggests that cord units should be harvested in the presence of an MPTP inhibitor.
Figure. Effects of EPHOSS on Hematopoietic Stem Cells. Hematopoietic stem cells (HSC) collected in air results in increased reactive oxygen species (ROS) production and opening of the mitochondrial permeability transition pore (MPTP) via a cyclophilin D (CypD) mediated mechanism. This oxidative stress results in lower recovery of phenotypic and functional HSCs. Conversely, the new methods of harvesting described by the authors; either in 3 percent oxygen or in the presence of the CypD inhibiting cyclosporine A, results in increased HSC recovery and transplantation.
Reprinted from Cell, Vol 161/ Issue 7, Mantel CR et al, Enhancing hematopoietic stem cell transplantation efficacy by mitigating oxygen shock, pages 1553-1565, Copyright 2015, with permission from Elsevier.
Hoggatt J. Stem cell barcoding: is it time to change our definition of a hematopoietic stem cell?. The Hematologist. 2015;12:6.
Spencer JA, Ferraro F, Roussakis E, et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature. 2014;508:269-273.
Abkowitz JL, Catlin SN, McCallie MT, et al. Evidence that the number of hematopoietic stem cells per animal is conserved in mammals. Blood. 2002;100:2665-2667.
Conflict of Interests
Dr. Hoggatt and Hannah Rasmussen indicated no relevant conflicts of interest.
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