Mesenchymal Stem Cells for Cellular Therapies and Regenerative Medicine: 

Cytocentric oxygen control makes sense from the molecular level through to the bottom line.

Mesenchymal stem cells (MSC) have shown tremendous promise as a cellular therapy for indications as varied as arthritis [1], diabetes [2], cardiac disease [3], wound repair [4], graft-versus-host disease [5], ALS [6], spinal cord injury [7], even infectious disease [89]. MSC also have the potential to transdifferentiate into multiple cell lineages for regenerative medicine applications [10]. But for MSC to live up to that promise, the best conditions for expanding and studying these critical cells in the laboratory must be established.

MSC originate in tissues that normally experience low oxygen levels

There are very few live cell types that normally experience room air oxygen (21%) in vivo, as inspired oxygen mixes with expired carbon dioxide in the body. In the lungs, oxygen is only at 10-13%. Venous blood contains only 5% oxygen. See Carreau et al. for oxygen levels of other tissues [11]. Many types of mesenchymal stem cells currently in study for their therapeutic effects originate from tissues that are normally even lower in oxygen, such as bone marrow [12], and umbilical cord [13].

These facts hold an important implication that is often overlooked: MSC culture or handling without controlling oxygen below room air levels results in a supraphysiologic oxygen state.

Supraphysiologic oxygen culture damages mitochondria in mesenchymal stem cells ex vivo.

A recent Nature Communications paper by Phinney et al. has reported on the response of mesenchymal stem cells (MSC) cultured in standard room-air oxygen to oxidative stress [14]. They found that the supraphysiologic oxygen levels experienced by the cells in this suboptimal condition were associated with the exportation of partially depolarized mitochondria to macrophage.

The Phinney publication builds upon findings reported in an earlier mitophagy paper from the same group showing that MSC suffer mitochondrial damage in room air oxygen cell culture [15]. They also showed that the effect of atmospheric oxygen on mesenchymal stem cells may be mediated by p53-dependent mechanisms.

Supraphysiologic oxygen affects MSC function.

As the Phinney paper describes, MSC can have potent anti-inflammatory effects on other cell types such as local macrophage. The authors show that MSC release miRNA-containing exosomes that reduce inflammatory activation of macrophage. This could help explain the paracrine mechanism behind how MSC reduce inflammation as a cellular therapy. However, as other groups have shown, the human MSC secretome changes when they are cultured in supraphysiologic oxygen[16] [17].

Supraphysiologic oxygen profoundly affects mesenchymal stem cell proliferation and differentiation.

There is increasing consensus that supraphysiologic oxygen exposure reduces proliferation and clonogenic capacity of MSC from many different tissues. Just in the last year alone, there have been findings published on the detrimental effects of oxygen on the proliferation of MSC from: lacrimal gland[18], fat [19], bone marrow [20], and umbilical cord [21] [13].

Oxidative stress has also been found to affect differentiation of MSC into tissues as diverse as adipose tissue [22], neural cell types [23], and osteoblasts [24].

Regulated at the protein level upon minutes of oxygen exposure [25], HIF proteins may mediate the loss of MSC proliferative and differentiation capacity [26] [27].

This means that even brief handling of cells in supraphysiologic oxygen of room air biological safety cabinets may affect MSC batch yields, as is the case with hematopoietic stem cells[28].

The in vitro yield and batch-to-batch consistency of MSC are cellular therapy business problems.

Any nascent cellular therapy company will succeed or fail based not only upon the clinical efficacy of the cells they produce, but also the effectiveness and reliability of cell production processes in their laboratories. Profit-draining COGS can be dramatically affected by the materials needed for cell growth in vitro and the yields of cells produced from batch to batch [29].

This means that to protect the bottom line of MSC-based cellular therapy businesses, mesenchymal stem cells must have full-time protection from exposure to oxygen.

Cytocentric cell production is good for MSC cellular therapy business

From the level of regulation of individual proteins through to the economics of the whole cellular therapy enterprise, the scientific literature holds evidence that oxygen may dictate the success of an MSC cellular therapeutic. These findings highlight the critical importance of maintaining unbroken physiologically relevant oxygen levels for MSC during cell handling as well as incubation because Cells Need Physiologic Simulationfor the MSC industry to succeed.

1.         1. Tanaka, Y., Human mesenchymal stem cells as a tool for joint repair in rheumatoid arthritis. Clinical and experimental rheumatology, 2015. 33(4 Suppl 92): p. 58.

2.         2. Holditch, S.J., A. Terzic, and Y. Ikeda, Concise review: pluripotent stem cell-based regenerative applications for failing β-cell function. Stem cells translational medicine, 2014: p. sctm. 2013-0184.

3.         3. Kim, J., L. Shapiro, and A. Flynn, The clinical application of mesenchymal stem cells and cardiac stem cells as a therapy for cardiovascular disease. Pharmacology & therapeutics, 2015.

4.         4. Isakson, M., et al., Mesenchymal Stem Cells and Cutaneous Wound Healing: Current Evidence and Future Potential. Stem Cells International, 2015. 2015.

5.         5. Chen, X., et al., Efficacy of Mesenchymal Stem Cell Therapy for Steroid-Refractory Acute Graft-Versus-Host Disease following Allogeneic Hematopoietic Stem Cell Transplantation: A Systematic Review and Meta-Analysis.PloS one, 2015. 10(8): p. e0136991.

6.         6. Hajivalili, M., et al., Mesenchymal Stem Cells in the Treatment of Amyotrophic Lateral Sclerosis. Current stem cell research & therapy, 2015.

7.         7. Watanabe, S., et al., Early Transplantation of Mesenchymal Stem Cells After Spinal Cord Injury Relieves Pain Hypersensitivity Through Suppression of PainRelated Signaling Cascades and Reduced Inflammatory Cell Recruitment. Stem Cells, 2015. 33(6): p. 1902-1914.

8.         8. Hao, Q., et al., Study of Bone Marrow and Embryonic Stem Cell-Derived Human Mesenchymal Stem Cells for Treatment of Escherichia coli Endotoxin-Induced Acute Lung Injury in Mice. Stem cells translational medicine, 2015: p. sctm. 2015-0006.

9.         9. Simonson, O.E., et al., In vivo effects of mesenchymal stromal cells in two patients with severe acute respiratory distress syndrome. Stem cells translational medicine, 2015: p. sctm. 2015-0021.

10.       10. Atashi, F., A. Modarressi, and M.S. Pepper, The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review. Stem Cells Dev, 2015. 24(10): p. 1150-63.

11.       11. Carreau, A., et al., Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. Journal of cellular and molecular medicine, 2011. 15(6): p. 1239-1253.

12.       12. Spencer, J.A., et al., Direct measurement of local oxygen concentration in the bone marrow of live animals.Nature, 2014. 508(7495): p. 269-73.

13.       13. Devito, L., et al., Wharton's jelly mesenchymal stromal/stem cells derived under chemically defined animal product-free low oxygen conditions are rich in MSCA-1(+) subpopulation. Regen Med, 2014. 9(6): p. 723-32.

14.       14. Phinney, D.G., et al., Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nat Commun, 2015. 6: p. 8472.

15.       15. Boregowda, S.V., et al., Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion. Stem Cells, 2012. 30(5): p. 975-87.

16.       16. Paquet, J., et al., Oxygen Tension Regulates Human Mesenchymal Stem Cell Paracrine Functions. Stem Cells Transl Med, 2015. 4(7): p. 809-21.

17.       17. Teixeira, F.G., et al., Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome? Stem Cell Res Ther, 2015. 6(1): p. 133.

18.       18. Roth, M., et al., The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland-Derived Mesenchymal Stem Cells. Invest Ophthalmol Vis Sci, 2015. 56(8): p. 4741-52.

19.       19. Salgado, A.J., et al., Mesenchymal stem cells secretome as a modulator of the neurogenic niche: basic insights and therapeutic opportunities. Front Cell Neurosci, 2015. 9: p. 249.

20.       20. Fan, L., et al., Low oxygen tension enhances osteogenic potential of bone marrow-derived mesenchymal stem cells with osteonecrosis-related functional impairment. Stem Cells Int, 2015. 2015: p. 950312.

21.       21. Reppel, L., et al., Hypoxic Culture Conditions for Mesenchymal Stromal/Stem Cells from Wharton’s Jelly: A Critical Parameter to Consider in a Therapeutic Context. Current stem cell research & therapy, 2014. 9(4): p. 306-318.

22.       22. Higuchi, M., et al., Differentiation of human adipose-derived stem cells into fat involves reactive oxygen species and Forkhead box O1 mediated upregulation of antioxidant enzymes. Stem cells and development, 2012. 22(6): p. 878-888.

23.       23. Drela, K., et al., Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner. Cytotherapy, 2014.

24.       24. Bai, X.-c., et al., Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-κB.Biochemical and biophysical research communications, 2004. 314(1): p. 197-207.

25.       25. Jewell, U.R., et al., Induction of HIF-1alpha in response to hypoxia is instantaneous. FASEB J, 2001. 15(7): p. 1312-4.

26.       26. Tsai, C.-C., et al., Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST. Blood, 2011. 117(2): p. 459-469.

27.       27. Guarnerio, J., et al., Bone marrow endosteal mesenchymal progenitors depend on HIF factors for maintenance and regulation of hematopoiesis. Stem cell reports, 2014. 2(6): p. 794-809.

28.       28. Mantel, C.R., et al., Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock.Cell, 2015. 161(7): p. 1553-65.

29.       29. Simaria, A.S., et al., Allogeneic cell therapy bioprocess economics and optimization: single-use cell expansion technologies. Biotechnol Bioeng, 2014. 111(1): p. 69-83.

 


alicia author iconAbout 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.