Isolation of mouse mesenchymal stem cells with normal ploidy from bone marrows by reducing oxidative stress in combination with extracellular matrix.

BACKGROUND

Isolation of mouse MSCs (mMSCs) with normal ploidy from bone marrow remains challenging. mMSCs isolated under 20% O(2) are frequently contaminated by overgrown hematopoietic cells, and could also be especially vulnerable to oxidative damage, resulting in chromosomal instability. Culture under low oxygen or extracellular matrix (ECM) improves proliferation of MSCs in several species. We tested the hypothesis that culture under low oxygen in combination with ECM prepared from mouse embryonic fibroblast (MEF-ECM) could be used to purify proliferative mMSCs, and to reduce oxidative damage and maintain their chromosomal stability.

RESULTS

Optimization of culture conditions under 20% O(2) resulted in immortalization of mMSCs, showing extensive chromosome abnormalities, consistent with previous studies. In contrast, culture under low oxygen (2% O(2)) improved proliferation of mMSCs and reduced oxidative damage, such that mMSCs were purified simply by plating at low density under 2% O(2). MEF-ECM reduced oxidative damage and enhanced proliferation of mMSCs. However, these isolated mMSCs still exhibited high frequency of chromosome abnormalities, suggesting that low oxygen or in combination with MEF-ECM was insufficient to fully protect mMSCs from oxidative damage. Notably, antioxidants (alpha -phenyl-t-butyl nitrone (PBN) and N-acetylcysteine (NAC)) further reduced DNA damage and chromosomal abnormalities, and increased proliferation of mMSCs. mMSCs isolated by the combination method were successfully used to generate induced pluripotent stem (iPS) cells by ectopic expression of Oct4, Sox2, Klf4 and c-Myc.

CONCLUSIONS

We have developed a technique that allows to reduce the number of karyotypic abnormalities for isolation of primary mMSCs and for limited culture period by combination of low oxygen, MEF-ECM, antioxidants and low density plating strategy. The effectiveness of the new combination method is demonstrated by successful generation of iPS cells from the isolated mMSCs. However, a culture system for mMSCs still is needed to prevent all the anomalies, especially after a long-term culture period.