Mini-Review: Cord Blood HSC and Physiologic Oxygen
At what oxygen level should I culture my umbilical cord-derived hematopoietic stem cells? It’s not a question you will hear on Jeopardy, but it is a critically important question for research involving cord blood stem cells.
Hematopoietic stem cells from umbilical cord blood (CB-HSC) have been shown to be beneficial for treating leukemia1,2, neuroblastoma3, and is of great interest for other disease applications. NIH-funded clinical trials listed on clinicaltrials.gov include studies of CB-HSC for immunodeficiencies, autoimmune diseases, sickle cell, cerebral palsy, complications of prematurity, and stroke. However, individual cord blood units often do not have the cell yield needed for a full transplant, requiring the pairing of HLA- partially matched cord blood units for a single transplant4. This presents obvious drawbacks in limiting the numbers of patients that can be treated.
Expanding CD34+ CB-HSC In Vitro
CD34+ cell expansion in culture holds the potential of increasing the numbers of recipients that can benefit from limited cord blood harvests. However, many risks face cells grown in vitro. Beyond the risks of contamination, exposure to room air oxygen is a major risk to CB-HSC expansion5.
CB-HSC and Oxygen In Vivo
When trying to provide the best environment for in vitro expansion, it makes sense to look to the appropriate in vivo environment for guidance. The actual oxygen levels of the bone marrow niche, a place where CB-HSC engraft and expand in vivo, have been difficult to measure. However, it is widely accepted that the hematopoietic niche is extremely low in oxygen during active CB-HSC division6. Intracellular reactive oxygen species (ROS) levels are closely associated with the ability of CB-HSC to divide and differentiate, as described in an excellent recent review7.
What Oxygen Levels Have Investigators Tried and Reported for CB-HSC Expansion?
Since the 1980s, researchers have cultured CB-HSC at a variety of different oxygen levels to enhance stem cell growth and engraftability8. Research interest has surged recently as the clinical and commercial value of stem cells has skyrocketed. A rapidly expanding body of literature provides evidence that physiologic oxygen enhances cord blood-derived CB-HSC expansion and function. Let’s look at the effects of the different oxygen levels reported.
In co-culture with MSC, one group found that 10% oxygen produced better CD34+ cell expansion from cord blood than 2%, 5%, or 21% O2 9. Without MSC, but with a combination of thrombopoietin, stem cell factor, FLt-3 ligand and IL-6, another group reported that 10% oxygen produced better CD34+ cell expansion from cord blood than room air oxygen10. This was the highest physiologic-range oxygen levels that we found in the CB-HSC literature, within the range of what might be found in arterial blood, but a higher oxygen level than would be expected in deep tissues like bone marrow. This is particularly true for the hematopoietic niche, which experiences lower blood perfusion rates. 11
Five percent oxygen is the accepted oxygen level of normal venous blood and is a very popular level chosen by researchers looking for physiologically relevant oxygen conditions.
There are some mixed findings on the in vitro expansion of cord blood HSC at 5% oxygen. One group found that cord blood progenitor cells expanded less but formed more colonies at 5% O2 than at 20% O212. Others found that cord blood hematopoietic stem and progenitor cells expanded more and repopulated better at 5%O2 than room air oxygen 13 14 15.
In co-culture, CB-HSC proliferated better at 5% oxygen than at room air. Co-cultured with adipose-derived stem cells, CB-HSC produced more CD34+ cells at 5% O2 than at room air oxygen16. Perfusion of chitosan scaffolds at 5% O2 supported the growth and repopulating function of CD34+ cells from cord blood better than 19% O217. Culturing CD34+ hematopoietic stem/precursor cells on biological scaffolds with a bone marrow stromal cell line produced up to 80-fold expansions at 5%O2, but not at 20% O2 18. Even when just storing cord blood at 4°C, cells stored under 5% O2 and 9%CO2 retained better survival and function than under room air 19.
The beneficial effect of physiologically relevant oxygen has been associated with the reduced reactive oxygen species (ROS) in culture. Like many other types of stem cells, CB-HSC were shown to experience lower levels of ROS and NADPH activity along with better expansion of CD34+ cells at 5% oxygen as compared to 21% oxygen20.
Less than 3% Oxygen
Going lower than 3% oxygen is a little tricky in that with variable cell consumption rates, particularly during cell expansions, the lower the oxygen level, the higher the risk of depleting your cultures of all oxygen (unpublished observations, A. Henn). However, findings published by others indicate that human CB-HSC function, including repopulating ability of SCID mice, was enhanced at 1.5% oxygen in co-culture with MSC as compared to 5% or 21% O221. One group reported that very low oxygen (1%) maintained human cord blood CD34+ cells in a more immature, stem-like state22. Even simply storing cord blood in oxygen-impermeable bags can improve CB-HSC function. 23
Three is a magic number 24. In liquid culture, 3% O2 preserved better colony-forming activity over 28 days of culture than 20% oxygen. 25 Culture of cord blood-derived CD34+ cells on fibronectin plates and osteoblast factors (IL-6, SCF, and CXCL12) at 2-3% O2 improved cell survival 26. Cord blood CD133+ CB-HSC express genes associated with cell expansion when cultured at 3% oxygen 27.
Is 3% Oxygen the New Gold Standard? Preventing oxygen exposure of CB-HSC during isolation as well as culture
A seminal paper published in Cell last year by the Broxmeyer group provided very strong evidence that exposure of human cord blood to room air oxygen levels, even for brief periods of time (20-30 minutes) is highly detrimental to human CB-HSC yields 28. They reported that 3% oxygen was the critical threshold for this effect. Failure to pre-incubate of all materials used in their experiments, even pipette tips, in 3% oxygen overnight negated all of the benefits to CB-HSC. They saw effects in as little as 10-15 minutes of exposure to room air oxygen, they saw effects on the cells (A. Henn – personal communication with the authors).
These findings are consistent with the biology of HIF oxygen-sensing transcription factors. For more information, see this earlier post, The Biology of HIF Proteins Affects the Outcome of Your Experiments in Physiologic Oxygen: Considerations for Protocol Design.
HIF is regulated at the protein level, with a half-life on the scale of minutes. It could be that even small periods of time that the cells are in supraphysiologic oxygen, such during routine cell handling in a room-air BSC, has confounded clear results of experiments at physiologically relevant oxygen levels.
This may be particularly the case for researchers that control incubation oxygen and not cell handling oxygen. Variability in cell handling techniques may be a major cause behind mixed effects reported in the literature for all cell types at physiologic oxygen.
If you are considering expansion protocols for cord blood stem cells, you should consider controlling oxygen during all cell handling (including isolation) as well as cell incubation. Even brief periods of time in room air can affect CB-HSC growth.
Questions? Comments? Ideas? Contact us here at the Cytocentric Blog. We would love to hear your thoughts on oxygen protocols for cord blood stem cells.
1 1. Barker, J. N. et al. Results of a prospective multicentre myeloablative double-unit cord blood transplantation trial in adult patients with acute leukaemia and myelodysplasia. British journal of haematology 168, 405-412, doi:10.1111/bjh.13136 (2015).
2 2. Rio, B. et al. Decreased nonrelapse mortality after unrelated cord blood transplantation for acute myeloid leukemia using reduced-intensity conditioning: a prospective phase II multicenter trial. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 21, 445-453, doi:10.1016/j.bbmt.2014.11.009 (2015).
3 3. Jubert, C., Wall, D. A., Grimley, M., Champagne, M. A. & Duval, M. Engraftment of unrelated cord blood after reduced-intensity conditioning regimen in children with refractory neuroblastoma: a feasibility trial. Bone marrow transplantation 46, 232-237, doi:10.1038/bmt.2010.107 (2011).
4 4. Scaradavou, A. et al. Double unit grafts successfully extend the application of umbilical cord blood transplantation in adults with acute leukemia. Blood 121, 752-758, doi:10.1182/blood-2012-08-449108 (2013).
5 5. Broxmeyer, H. E., O’Leary, H. A., Huang, X. & Mantel, C. 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. Current opinion in hematology 22, 273-278, doi:10.1097/MOH.0000000000000144 (2015).
6 6. Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327-334, doi:10.1038/nature12984 (2014).
7 7. Ludin, A. et al. Reactive oxygen species regulate hematopoietic stem cell self-renewal, migration and development, as well as their bone marrow microenvironment. Antioxidants & redox signaling 21, 1605-1619, doi:10.1089/ars.2014.5941 (2014).
8 8. Januszewicz, E., Cooper, I. A. & Bradley, T. R. Requirements for growth of human erythroid progenitors in nutrient agar. American journal of hematology 15, 207-217 (1983).
9 9. Andrade, P. Z. et al. Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear-cut effects on cell proliferation, differentiation and metabolism. Journal of tissue engineering and regenerative medicine, doi:10.1002/term.1731 (2013).
10 10. Tursky, M. L., Collier, F. M., Ward, A. C. & Kirkland, M. A. Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34(+) hematopoietic progenitor cells. Cytotherapy 14, 679-685, doi:10.3109/14653249.2012.666851 (2012).
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12 12. Smith, S. & Broxmeyer, H. E. The influence of oxygen tension on the long‐term growth in vitro of haematopoietic progenitor cells from human cord blood. British journal of haematology 63, 29-34 (1986).
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14 14. Luni, C. et al. Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions. Biotechnol Prog 27, 1154-1162, doi:10.1002/btpr.582 (2011).
15 15. Koller, M. R., Bender, J. G., Miller, W. M. & Papoutsakis, E. T. Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow. Experimental hematology 20, 264-270 (1992).
16 16. Andreeva, E. R. et al. Human adipose-tissue derived stromal cells in combination with hypoxia effectively support ex vivo expansion of cord blood haematopoietic progenitors. PloS one 10, e0124939, doi:10.1371/journal.pone.0124939 (2014).
17 17. Cho, C. H., Eliason, J. F. & Matthew, H. W. Application of porous glycosaminoglycan-based scaffolds for expansion of human cord blood stem cells in perfusion culture. Journal of biomedical materials research. Part A86, 98-107, doi:10.1002/jbm.a.31614 (2008).
18 18. Tiwari, A. et al. Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line. Journal of tissue engineering and regenerative medicine7, 871-883, doi:10.1002/term.1479 (2013).
19 19. Vlaski, M. et al. Hypoxia/Hypercapnia‐Induced Adaptation Maintains Functional Capacity of Cord Blood Stem and Progenitor Cells at 4° C. Journal of cellular physiology 229, 2153-2165 (2014).
20 20. Fan, J., Cai, H. & Tan, W. S. Role of the plasma membrane ROS-generating NADPH oxidase in CD34+ progenitor cells preservation by hypoxia. J Biotechnol 130, 455-462, doi:10.1016/j.jbiotec.2007.05.023 (2007).
21 21. Hammoud, M. et al. Combination of low O2 concentration and mesenchymal stromal cells during culture of cord blood CD34+ cells improves the maintenance and proliferative capacity of hematopoietic stem cells. Journal of cellular physiology 227, 2750-2758 (2012).
22 22. Cipolleschi, M. G. et al. Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro. Experimental hematology 25, 1187-1194 (1997).
23 23. Chevaleyre, J. et al. A Novel Procedure to Improve Functional Preservation of Hematopoietic Stem and Progenitor Cells in Cord Blood Stored at +4 degrees C Before Cryopreservation. Stem Cells Dev, doi:10.1089/scd.2014.0046 (2014).
24 24. Wikipedia. Three is a Magic Number. https://en.wikipedia.org/wiki/Three_Is_a_Magic_Number (2016).
25 25. Ivanovic, Z. et al. Simultaneous Maintenance of Human Cord Blood SCID‐Repopulating Cells and Expansion of Committed Progenitors at Low O2 Concentration (3%). Stem Cells 22, 716-724 (2004).
26 . 26. Dao, M. A., Creer, M. H., Nolta, J. A. & Verfaillie, C. M. Biology of umbilical cord blood progenitors in bone marrow niches. Blood 110, 74-81, doi:10.1182/blood-2006-08-034447 (2007).
27 27. Martin-Rendon, E. et al. Transcriptional profiling of human cord blood CD133+ and cultured bone marrow mesenchymal stem cells in response to hypoxia. Stem Cells 25, 1003-1012, doi:10.1634/stemcells.2006-0398 (2007).
28 28. 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).
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About The Author
Alicia D Henn, PhD, MBA
Chief Scientific Officer of BioSpherix, Ltd
Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd since 2013. 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.
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