Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation

Journal of Biomedical Science

Ching-Feng Cheng³  Yao-Jen Chang⁸  Hwan-Wun Liu⁶  Shin-Yuan Chen⁴  Wei Wu Li⁵  Yi-Yo Chou⁵  Chao-Chuan Wu⁸  Wei-Chao Huang¹  Chia-Hsin Liao⁷  Dian-Kun Li²  Cheng-Yoong Pang⁷  Li-Yi Sun⁵ 

[1] Department of Plastic Surgery, Taipei Tzu Chi General Hospital, Taipei, Taiwan;Department of Hematology and Oncology, Taichung Tzu Chi General Hospital, Taichung, Taiwan;Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;Department of Neurosurgery, Buddhist Tzu Chi General Hospital, Hualien, Taiwan;Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, No. 707, Sec. 3, Zhongyang Rd., Hualien City 970, Hualien, Taiwan;Department of Occupational Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan;Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan;Department of Surgery, Buddhist Tzu Chi General Hospital Taipei Branch, Taipei, No. 289, Jianguo Rd., Xindian Dist., New Taipei City 231, Taiwan

关键词: Hypoxia;    Fibroblast growth factor-2;    Antioxidant;    Adipose-derived mesenchymal stem cells;   

Others  :  1137635
DOI  :  10.1186/1423-0127-20-53

 received in 2013-01-12, accepted in 2013-07-26,  发布年份 2013

【 摘 要 】

Background

Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood.

In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-L-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO₂ conditions (ImF-H).

Results

During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions.

Conclusion

Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions.

【 授权许可】

   
2013 Sun et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Yao Y, Zhang F, Wang L, Zhang G, Wang Z, Chen J, Gao X: Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction. J Biomed Sci 2009, 16:74. BioMed Central Full Text
• [2]Liu AM, Lu G, Tsang KS, Li G, Wu Y, Huang ZS, Ng HK, Kung HF, Poon WS: Umbilical cord-derived mesenchymal stem cells with forced expression of hepatocyte growth factor enhance remyelination and functional recovery in a rat intracerebral hemorrhage model. Neurosurgery 2010, 67:357-365. discussion 365-356
• [3]Wong VW, Rustad KC, Glotzbach JP, Sorkin M, Inayathullah M, Major MR, Longaker MT, Rajadas J, Gurtner GC: Pullulan hydrogels improve mesenchymal stem cell delivery into high-oxidative-stress wounds. Macromol Biosci 2011, 11:1458-1466.
• [4]Ulloa-Montoya F, Verfaillie CM, Hu WS: Culture systems for pluripotent stem cells. J Biosci Bioeng 2005, 100:12-27.
• [5]King JA, Miller WM: Bioreactor development for stem cell expansion and controlled differentiation. Curr Opin Chem Biol 2007, 11:394-398.
• [6]Sun LY, Hsieh DK, Yu TC, Chiu HT, Lu SF, Luo GH, Kuo TK, Lee OK, Chiou TW: Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells. Bioelectromagnetics 2009, 30:251-260.
• [7]Sun LY, Hsieh DK, Syu WS, Li YS, Chiu HT, Chiou TW: Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential. Cell Prolif 2010, 43:445-456.
• [8]Meuleman N, Tondreau T, Delforge A, Dejeneffe M, Massy M, Libertalis M, Bron D, Lagneaux L: Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-MEM medium. Eur J Haematol 2006, 76:309-316.
• [9]Pochampally RR, Smith JR, Ylostalo J, Prockop DJ: Serum deprivation of human marrow stromal cells (hMSCs) selects for a subpopulation of early progenitor cells with enhanced expression of OCT-4 and other embryonic genes. Blood 2004, 103:1647-1652.
• [10]Muller AM, Davenport M, Verrier S, Droeser R, Alini M, Bocelli-Tyndall C, Schaefer DJ, Martin I, Scherberich A: Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue. Tissue Eng Part A 2009, 15:869-875.
• [11]Martin I, Muraglia A, Campanile G, Cancedda R, Quarto R: Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology 1997, 138:4456-4462.
• [12]Sheehy EJ, Buckley CT, Kelly DJ: Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells. Biochem Biophys Res Commun 2012, 417:305-310.
• [13]Hung SP, Ho JH, Shih YR, Lo T, Lee OK: Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells. J Orthop Res 2012, 30:260-266.
• [14]Hung SC, Pochampally RR, Hsu SC, Sanchez C, Chen SC, Spees J, Prockop DJ: Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo. PLoS One 2007, 2:e416.
• [15]Liu H, Xue W, Ge G, Luo X, Li Y, Xiang H, Ding X, Tian P, Tian X: Hypoxic preconditioning advances CXCR4 and CXCR7 expression by activating HIF-1alpha in MSCs. Biochem Biophys Res Commun 2010, 401:509-515.
• [16]Lin TM, Tsai JL, Lin SD, Lai CS, Chang CC: Accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants. Stem Cells Dev 2005, 14:92-102.
• [17]Wang KH, Kao AP, Wangchen H, Wang FY, Chang CH, Chang CC, Lin SD: Optimizing proliferation and characterization of multipotent stem cells from porcine adipose tissue. Biotechnol Appl Biochem 2008, 51:159-166.
• [18]Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS: Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004, 14:311-324.
• [19]Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I: Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007, 327:449-462.
• [20]Park BS, Kim WS, Choi JS, Kim HK, Won JH, Ohkubo F, Fukuoka H: Hair growth stimulated by conditioned medium of adipose-derived stem cells is enhanced by hypoxia: evidence of increased growth factor secretion. Biomed Res 2010, 31:27-34.
• [21]Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, Pell CL, Johnstone BH, Considine RV, March KL: Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004, 109:1292-1298.
• [22]Zhu Y, Liu T, Song K, Fan X, Ma X, Cui Z: Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct 2008, 26:664-675.
• [23]Ikegame Y, Yamashita K, Hayashi S, Mizuno H, Tawada M, You F, Yamada K, Tanaka Y, Egashira Y, Nakashima S, et al.: Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy. Cytotherapy 2011, 13:675-685.
• [24]Yang XF, He X, He J, Zhang LH, Su XJ, Dong ZY, Xu YJ, Li Y, Li YL: High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells. J Biomed Sci 2011, 18:59. BioMed Central Full Text
• [25]Bianchi G, Banfi A, Mastrogiacomo M, Notaro R, Luzzatto L, Cancedda R, Quarto R: Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 2003, 287:98-105.
• [26]Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF: FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol 2005, 203:398-409.
• [27]Griesche N, Luttmann W, Luttmann A, Stammermann T, Geiger H, Baer PC: A simple modification of the separation method reduces heterogeneity of adipose-derived stem cells. Cells Tissues Organs 2010, 192:106-115.
• [28]Lin PC, Chen YL, Chiu SC, Yu YL, Chen SP, Chien MH, Chen KY, Chang WL, Lin SZ, Chiou TW, Harn HJ: Orphan nuclear receptor, Nurr-77 was a possible target gene of butylidenephthalide chemotherapy on glioblastoma multiform brain tumor. J Neurochem 2008, 106:1017-1026.
• [29]Cawthon RM: Telomere measurement by quantitative PCR. Nucleic Acids Res 2002, 30:e47.
• [30]Chiu SC, Chen SP, Huang SY, Wang MJ, Lin SZ, Harn HJ, Pang CY: Induction of apoptosis coupled to endoplasmic reticulum stress in human prostate cancer cells by n-butylidenephthalide. PLoS One 2012, 7:e33742.
• [31]Potdar PD, D’Souza SB: Ascorbic acid induces in vitro proliferation of human subcutaneous adipose tissue derived mesenchymal stem cells with upregulation of embryonic stem cell pluripotency markers Oct4 and SOX 2. Hum Cell 2010, 23:152-155.
• [32]Kanda Y, Hinata T, Kang SW, Watanabe Y: Reactive oxygen species mediate adipocyte differentiation in mesenchymal stem cells. Life Sci 2011, 89:250-258.
• [33]Tamama K, Kawasaki H, Kerpedjieva SS, Guan J, Ganju RK, Sen CK: Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition. J Cell Biochem 2011, 112:804-817.
• [34]Song H, Cha MJ, Song BW, Kim IK, Chang W, Lim S, Choi EJ, Ham O, Lee SY, Chung N, et al.: Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex. Stem Cells 2010, 28:555-563.
• [35]Robson CN, Gnanapragasam V, Byrne RL, Collins AT, Neal DE: Transforming growth factor-beta1 up-regulates p15, p21 and p27 and blocks cell cycling in G1 in human prostate epithelium. J Endocrinol 1999, 160:257-266.
• [36]Tsai CC, Chen YJ, Yew TL, Chen LL, Wang JY, Chiu CH, Hung SC: Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST. Blood 2011, 117:459-469.
• [37]Adesida AB, Mulet-Sierra A, Jomha NM: Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells. Stem Cell Res Ther 2012, 3:9. BioMed Central Full Text
• [38]Nekanti U, Dastidar S, Venugopal P, Totey S, Ta M: Increased proliferation and analysis of differential gene expression in human Wharton’s jelly-derived mesenchymal stromal cells under hypoxia. Int J Biol Sci 2010, 6:499-512.
• [39]Rosova I, Dao M, Capoccia B, Link D, Nolta JA: Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 2008, 26:2173-2182.
• [40]Rochefort GY, Delorme B, Lopez A, Herault O, Bonnet P, Charbord P, Eder V, Domenech J: Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia. Stem Cells 2006, 24:2202-2208.
• [41]Neubauer M, Fischbach C, Bauer-Kreisel P, Lieb E, Hacker M, Tessmar J, Schulz MB, Goepferich A, Blunk T: Basic fibroblast growth factor enhances PPARgamma ligand-induced adipogenesis of mesenchymal stem cells. FEBS Lett 2004, 577:277-283.