Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow‐Derived Mouse Mesenchymal Stem Cells via a p53‐Dependent Mechanism: Implications for Long‐Term Culture Expansion
Open Access
- 9 April 2012
- journal article
- research article
- Published by Oxford University Press (OUP) in The International Journal of Cell Cloning
- Vol. 30 (5), 975-987
- https://doi.org/10.1002/stem.1069
Abstract
Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long‐term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2‐associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300‐fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen‐induced stress response was dependent upon p53 since siRNA‐mediated knockdown of p53 in wild‐type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long‐term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen‐induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long‐term expansion in vitro. Disclosure of potential conflicts of interest is found at the end of this article.Keywords
Funding Information
- National Institute of Health (R01 NS052301-01A2)
This publication has 55 references indexed in Scilit:
- Selective Inhibition of Mitochondrial JNK Signaling Achieved Using Peptide Mimicry of the Sab Kinase Interacting Motif-1 (KIM1)ACS Chemical Biology, 2011
- p85α Regulates Osteoblast Differentiation by Cross-talking with the MAPK PathwayOnline Journal of Public Health Informatics, 2011
- Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent statusBMC Cell Biology, 2011
- O2 regulates stem cells through Wnt/β-catenin signallingNature, 2010
- The Role of Hypoxia in Bone Marrow–Derived Mesenchymal Stem Cells: Considerations for Regenerative Medicine ApproachesTissue Engineering, Part B: Reviews, 2010
- Loss of p53 causes mitochondrial DNA depletion and altered mitochondrial reactive oxygen species homeostasisBiochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
- Comparative study of mesenchymal stem cells from C57BL/10 and mdx miceBMC Cell Biology, 2008
- Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cellsBiochemical and Biophysical Research Communications, 2006
- Direct Activation of Bax by p53 Mediates Mitochondrial Membrane Permeabilization and ApoptosisScience, 2004
- Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell deathCell, 1993