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

Fan G, Wen L, Li M, Li C, Luo B, Wang F, Zhou L, Liu L - BMC Cell Biol. (2011)

Bottom Line: 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.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.The effectiveness of the new combination method is demonstrated by successful generation of iPS cells from the isolated mMSCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Science, Sun Yat-Sen University, Guangzhou, China.

ABSTRACT

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.

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Purification of mMSCs by culture under 2% O2 condition. (A) Flow cytometry analysis of epitopes on primary mMSCs. Few cells show CD45 and CD11b antigens by culture under 2% O2 compared to under 20% O2. (B) Flow cytometry analysis of epitopes on mMSCs at passage one. CmMSCs show epitopes similar to IDmMSCs. (C) Differentiation of CmMSCs. Adipogenesis, osteogenesis, and chondrogenesis were indicated by Oil Red, Alizarin Red (pH 4.1) and Alcian blue, respectively. Scale bar = 100 μm.
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Figure 3: Purification of mMSCs by culture under 2% O2 condition. (A) Flow cytometry analysis of epitopes on primary mMSCs. Few cells show CD45 and CD11b antigens by culture under 2% O2 compared to under 20% O2. (B) Flow cytometry analysis of epitopes on mMSCs at passage one. CmMSCs show epitopes similar to IDmMSCs. (C) Differentiation of CmMSCs. Adipogenesis, osteogenesis, and chondrogenesis were indicated by Oil Red, Alizarin Red (pH 4.1) and Alcian blue, respectively. Scale bar = 100 μm.

Mentions: More homogeneous cell population of fibroblast-like cells were found in mMSCs cultured under 2% O2, compared to those under 20% O2 (Figure 2A). Flow cytometry analysis showed that contamination by hematopoietic cells was greatly reduced by hypoxic culture. Under low oxygen, cell populations showed less expression of CD45 and CD11b antigens while maintaining high expression of Sca-1 and CD44 (Figure 3A), indicative of MSCs in most species. These results indicate that low oxygen improves purification of mMSCs, consistent with observations on rat MSCs [38].


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

Fan G, Wen L, Li M, Li C, Luo B, Wang F, Zhou L, Liu L - BMC Cell Biol. (2011)

Purification of mMSCs by culture under 2% O2 condition. (A) Flow cytometry analysis of epitopes on primary mMSCs. Few cells show CD45 and CD11b antigens by culture under 2% O2 compared to under 20% O2. (B) Flow cytometry analysis of epitopes on mMSCs at passage one. CmMSCs show epitopes similar to IDmMSCs. (C) Differentiation of CmMSCs. Adipogenesis, osteogenesis, and chondrogenesis were indicated by Oil Red, Alizarin Red (pH 4.1) and Alcian blue, respectively. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141734&req=5

Figure 3: Purification of mMSCs by culture under 2% O2 condition. (A) Flow cytometry analysis of epitopes on primary mMSCs. Few cells show CD45 and CD11b antigens by culture under 2% O2 compared to under 20% O2. (B) Flow cytometry analysis of epitopes on mMSCs at passage one. CmMSCs show epitopes similar to IDmMSCs. (C) Differentiation of CmMSCs. Adipogenesis, osteogenesis, and chondrogenesis were indicated by Oil Red, Alizarin Red (pH 4.1) and Alcian blue, respectively. Scale bar = 100 μm.
Mentions: More homogeneous cell population of fibroblast-like cells were found in mMSCs cultured under 2% O2, compared to those under 20% O2 (Figure 2A). Flow cytometry analysis showed that contamination by hematopoietic cells was greatly reduced by hypoxic culture. Under low oxygen, cell populations showed less expression of CD45 and CD11b antigens while maintaining high expression of Sca-1 and CD44 (Figure 3A), indicative of MSCs in most species. These results indicate that low oxygen improves purification of mMSCs, consistent with observations on rat MSCs [38].

Bottom Line: 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.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.The effectiveness of the new combination method is demonstrated by successful generation of iPS cells from the isolated mMSCs.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Science, Sun Yat-Sen University, Guangzhou, China.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus