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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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Antioxidants increase proliferation of mMSCs. (A) Comparison of spontaneous formation of micronucleus in primary mMSCs supplemented with or without antioxidants (800 μM PBN+5 mM NAC). (B) Effects of antioxidants on number of mMSCs. Bone marrow cells were plated at a density of 2 × 105 cells/cm2 on 6-well and cultured under 2% O2 with or without addition of antioxidants for 5 days, and cells were counted by hemocytometer. (C) Effects of H2O2 on the proliferation of mMSCs. Primary mMSCs were replated at 1000 cells/cm2 and cultured for two days, then H2O2 were added and cell number counted after 24 h. (D) Micronucleus analysis on mMSCs. (E) Karyotype analysis of mMSCs on plastic or ECM treated with H2O2. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01; n = 3. (F) A representative proliferation curve of mMSCs from three independent experiments. Data are expressed as the mean ± SD. *p < 0.05; n = 3. Abbreviations: Con., control; PN, PBN+NAC; CmMSCs, mMSCs on plastic as controls; EmMSCs, mMSCs on MEF-ECM; PCmMSCs, mMSCs on plastic in the presence of PBN and NAC; PEmMSCs, mMSCs on MEF-ECM in the presence of PBN and NAC.
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Figure 5: Antioxidants increase proliferation of mMSCs. (A) Comparison of spontaneous formation of micronucleus in primary mMSCs supplemented with or without antioxidants (800 μM PBN+5 mM NAC). (B) Effects of antioxidants on number of mMSCs. Bone marrow cells were plated at a density of 2 × 105 cells/cm2 on 6-well and cultured under 2% O2 with or without addition of antioxidants for 5 days, and cells were counted by hemocytometer. (C) Effects of H2O2 on the proliferation of mMSCs. Primary mMSCs were replated at 1000 cells/cm2 and cultured for two days, then H2O2 were added and cell number counted after 24 h. (D) Micronucleus analysis on mMSCs. (E) Karyotype analysis of mMSCs on plastic or ECM treated with H2O2. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01; n = 3. (F) A representative proliferation curve of mMSCs from three independent experiments. Data are expressed as the mean ± SD. *p < 0.05; n = 3. Abbreviations: Con., control; PN, PBN+NAC; CmMSCs, mMSCs on plastic as controls; EmMSCs, mMSCs on MEF-ECM; PCmMSCs, mMSCs on plastic in the presence of PBN and NAC; PEmMSCs, mMSCs on MEF-ECM in the presence of PBN and NAC.

Mentions: The chromosome abnormalities in early passage mMSCs indicate that culture under low oxygen is not sufficient to protect mMSCs from oxidative damage. To further decrease possible accumulation of oxidative damage and therefore improve long-term viability of mMSCs, a combination of two antioxidants (800 μM PBN and 5 mM NAC) was used for initial isolation of mMSCs. PBN is a free radical spin trapping agent and has been shown to reduce oxidative damage and extend lifespan of human fibroblasts and peritoneal mesothelial cells [45,46]. NAC is an antioxidant that acts as the precursor of glutathione and as a scavenger of free radicals [47]. Antioxidants reduced number of spontaneous micronucleus (Figure 5A) and significantly improved proliferation (Figure 5B), and increased normal karyotype of primary mMSCs (Table 1), suggesting that antioxidants could effectively reduce oxidative damage to mMSCs.


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)

Antioxidants increase proliferation of mMSCs. (A) Comparison of spontaneous formation of micronucleus in primary mMSCs supplemented with or without antioxidants (800 μM PBN+5 mM NAC). (B) Effects of antioxidants on number of mMSCs. Bone marrow cells were plated at a density of 2 × 105 cells/cm2 on 6-well and cultured under 2% O2 with or without addition of antioxidants for 5 days, and cells were counted by hemocytometer. (C) Effects of H2O2 on the proliferation of mMSCs. Primary mMSCs were replated at 1000 cells/cm2 and cultured for two days, then H2O2 were added and cell number counted after 24 h. (D) Micronucleus analysis on mMSCs. (E) Karyotype analysis of mMSCs on plastic or ECM treated with H2O2. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01; n = 3. (F) A representative proliferation curve of mMSCs from three independent experiments. Data are expressed as the mean ± SD. *p < 0.05; n = 3. Abbreviations: Con., control; PN, PBN+NAC; CmMSCs, mMSCs on plastic as controls; EmMSCs, mMSCs on MEF-ECM; PCmMSCs, mMSCs on plastic in the presence of PBN and NAC; PEmMSCs, mMSCs on MEF-ECM in the presence of PBN and NAC.
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Figure 5: Antioxidants increase proliferation of mMSCs. (A) Comparison of spontaneous formation of micronucleus in primary mMSCs supplemented with or without antioxidants (800 μM PBN+5 mM NAC). (B) Effects of antioxidants on number of mMSCs. Bone marrow cells were plated at a density of 2 × 105 cells/cm2 on 6-well and cultured under 2% O2 with or without addition of antioxidants for 5 days, and cells were counted by hemocytometer. (C) Effects of H2O2 on the proliferation of mMSCs. Primary mMSCs were replated at 1000 cells/cm2 and cultured for two days, then H2O2 were added and cell number counted after 24 h. (D) Micronucleus analysis on mMSCs. (E) Karyotype analysis of mMSCs on plastic or ECM treated with H2O2. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01; n = 3. (F) A representative proliferation curve of mMSCs from three independent experiments. Data are expressed as the mean ± SD. *p < 0.05; n = 3. Abbreviations: Con., control; PN, PBN+NAC; CmMSCs, mMSCs on plastic as controls; EmMSCs, mMSCs on MEF-ECM; PCmMSCs, mMSCs on plastic in the presence of PBN and NAC; PEmMSCs, mMSCs on MEF-ECM in the presence of PBN and NAC.
Mentions: The chromosome abnormalities in early passage mMSCs indicate that culture under low oxygen is not sufficient to protect mMSCs from oxidative damage. To further decrease possible accumulation of oxidative damage and therefore improve long-term viability of mMSCs, a combination of two antioxidants (800 μM PBN and 5 mM NAC) was used for initial isolation of mMSCs. PBN is a free radical spin trapping agent and has been shown to reduce oxidative damage and extend lifespan of human fibroblasts and peritoneal mesothelial cells [45,46]. NAC is an antioxidant that acts as the precursor of glutathione and as a scavenger of free radicals [47]. Antioxidants reduced number of spontaneous micronucleus (Figure 5A) and significantly improved proliferation (Figure 5B), and increased normal karyotype of primary mMSCs (Table 1), suggesting that antioxidants could effectively reduce oxidative damage to mMSCs.

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