Mouse clonal mesenchymal stem cells (mc-MSCs) were cultured on a Cytodex 3 microcarrier in a spinner flask for a suspension culture under hypoxia condition to increase mass productivity. The hypoxia environment was established using 4.0 mM Na2SO3 with 10 μM or 100 µM CoCl2 for 24 h in a low glucose DMEM medium. As a result, the proliferation of mc-MSCs under hypoxic conditions was 1.56 times faster than the control group over 7 days. The gene expression of HIF-1a and VEGFA increased 4.62 fold and 2.07 fold, respectively. Furthermore, the gene expression of ALP, RUNX2, COL1A, and osteocalcin increased significantly by 9.55, 1.55, 2.29, and 2.53 times, respectively. In contrast, the expression of adipogenic differentiation markers, such as PPAR-γ and FABP4, decreased. These results show that the hypoxia environment produced by these chemicals in a suspension culture increases the proliferation of mc-MSCs and promotes the osteogenic differentiation of mc-MSCs.
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Munir H, McGettrick HM (2015) Mesenchymal stem cell therapy for autoimmune disease: risks and rewards. Stem Cells Dev 24:2091–2100. https://doi.org/10.1089/scd.2015.0008
Smaldone MC, Chancellor MB (2008) Muscle derived stem cell therapy for stress urinary incontinence. World J Urol 26:327–332. https://doi.org/10.1007/s00345-008-0269-9
Fitzsimmons REB, Mazurek MS, Soos A, Simmons CA (2018) Mesenchymal stromal/stem cells in regenerative medicine and tissue engineering. Stem Cells Int. https://doi.org/10.1155/2018/8031718
Eibes G, dos Santos F, Andrade PZ et al (2010) Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system. J Biotechnol 146:194–197. https://doi.org/10.1016/j.jbiotec.2010.02.015
Zhou L, Kong J, Zhuang Y et al (2013) Ex vivo expansion of bone marrow mesenchymal stem cells using microcarrier beads in a stirred bioreactor. Biotechnol Bioprocess Eng 18:173–184. https://doi.org/10.1007/s12257-012-0512-5
Morikawa T, Takubo K (2016) Hypoxia regulates the hematopoietic stem cell niche. Pflugers Arch Eur J Physiol 468:13–22. https://doi.org/10.1007/s00424-015-1743-z
Ma T, Grayson WL, Fröhlich M, Vunjak-Novakovic G (2009) Hypoxia and stem cell-based engineering of mesenchymal tissues. Biotechnol Prog 25:32–42. https://doi.org/10.1002/btpr.128
Tsai CC, Chen YJ, Yew TL et al (2011) Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST. Blood 117:459–469. https://doi.org/10.1182/blood-2010-05-287508
Sala MA, Chen C, Zhang Q et al (2018) JNK2 up-regulates hypoxia-inducible factors and contributes to hypoxia-induced erythropoiesis and pulmonary. J Biol Chem 293:271–284. https://doi.org/10.1074/jbc.RA117.000440
Sart S, Agathos SN, Li Y (2014) Process engineering of stem cell metabolism for large scale expansion and differentiation in bioreactors. Biochem Eng J 84:74–82. https://doi.org/10.1016/j.bej.2014.01.005
Wu D, Yotnda P (2011) Induction and testing of hypoxia in cell culture. J Vis Exp. https://doi.org/10.3791/2899
Collaco CR, Hochman DJ, Goldblum RM, Brooks EG (2006) Effect of sodium sulfite on mast cell degranulation and oxidant stress. Ann Allergy, Asthma Immunol 96:550–556. https://doi.org/10.1016/S1081-1206(10)63549-1
Kaczmarek M, Cachau RE, Topol IA et al (2009) Metal ions-stimulated iron oxidation in hydroxylases facilitates stabilization of HIF-1α protein. Toxicol Sci 107:394–403. https://doi.org/10.1093/toxsci/kfn251
Jeon M-S (2011) Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method. World J Stem Cells 3:70. https://doi.org/10.4252/wjsc.v3.i8.70
Sart S, Tsai A-C, Li Y, Ma T (2014) Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev 20:365–380. https://doi.org/10.1089/ten.teb.2013.0537
Guo T, Yu L, Lim CG et al (2016) Effect of dynamic culture and periodic compression on human mesenchymal stem cell proliferation and chondrogenesis. Ann Biomed Eng 44:2103–2113. https://doi.org/10.1007/s10439-015-1510-5
Lei Y, Gojgini S, Lam J, Segura T (2011) The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels. Biomaterials 32:39–47. https://doi.org/10.1016/j.biomaterials.2010.08.103
Arora S, Srinivasan A, Leung CM, Toh Y-C (2020) Bio-mimicking shear stress environments for enhancing mesenchymal stem cell differentiation. Curr Stem Cell Res Ther 15:414–427. https://doi.org/10.2174/1574888x15666200408113630
Berry JD, Liovic P, Šutalo ID et al (2016) Characterisation of stresses on microcarriers in a stirred bioreactor. Appl Math Model 40:6787–6804. https://doi.org/10.1016/j.apm.2016.02.025
Berra E, Benizri E, Ginouvès A et al (2003) HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1α in normoxia. EMBO J 22:4082–4090. https://doi.org/10.1093/emboj/cdg392
Cash TP, Pan Y, Simon MC (2007) Reactive oxygen species and cellular oxygen sensing. Free Radic Biol Med 43:1219–1225. https://doi.org/10.1016/j.freeradbiomed.2007.07.001
Lewis AC, Roberts DJ (2005) New techniques for following the oxidation of sodium sulfite in mass-transfer studies. Ind Eng Chem Res 44:183–185. https://doi.org/10.1021/ie049412x
Zhao B, Li Y, Tong H et al (2005) Study on the reaction rate of sulfite oxidation with cobalt ion catalyst. Chem Eng Sci 60:863–868. https://doi.org/10.1016/j.ces.2004.09.064
Teti G, Focaroli S, Salvatore V et al (2018) The hypoxia-mimetic agent cobalt chloride differently affects human mesenchymal stem cells in their chondrogenic potential. Stem Cells Int. https://doi.org/10.1155/2018/3237253
Wenger RH, Kvietikova I, Rolfs A et al (1997) Hypoxia-inducible factor-1α is regulated at the post-mRNA level. Kidney Int 51:560–563. https://doi.org/10.1038/ki.1997.79
Vincent AS, Lim BG, Tan J et al (2004) Sulfite-mediated oxidative stress in kidney cells. Kidney Int 65:393–402. https://doi.org/10.1111/j.1523-1755.2004.00391.x
Salakou S, Kardamakis D, Tsamandas AC et al (2007) Increased bax/bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with Myasthenia gravis. Vivo (Brooklyn) 21:123–132
Frith JE, Thomson B, Genever PG (2010) Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods 16:735–749. https://doi.org/10.1089/ten.tec.2009.0432
Whitman NA, Lin ZW, Kenney RM et al (2019) Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats. Arch Biochem Biophys 671:8–17. https://doi.org/10.1016/j.abb.2019.05.025
Kannan S, Ghosh J, Dhara S (2020) Osteogenic differentiation potential and marker gene expression of different porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media. bioRxiv 2020.04.27.063230. https://doi.org/10.1101/2020.04.27.063230
Mylotte LA, Duffy AM, Murphy M et al (2008) Metabolic flexibility permits mesenchymal stem cell survival in an ischemic environment. Stem Cells 26:1325–1336. https://doi.org/10.1634/stemcells.2007-1072
Lo T, Ho JH, Yang MH, Lee OK (2011) Glucose reduction prevents replicative senescence and increases mitochondrial respiration in human mesenchymal stem cells. Cell Transplant 20:813–825. https://doi.org/10.3727/096368910X539100
Pattappa G, Heywood HK, de Bruijn JD, Lee DA (2011) The metabolism of human mesenchymal stem cells during proliferation and differentiation. J Cell Physiol 226:2562–2570. https://doi.org/10.1002/jcp.22605
Razban V, Lotfi AS, Soleimani M et al (2012) HIF-1α overexpression induces angiogenesis in mesenchymal stem cells. Biores Open Access 1:174–183. https://doi.org/10.1089/biores.2012.9905
Van Pham P, Vu NB, Phan NK (2016) Hypoxia promotes adipose-derived stem cell proliferation via VEGF. Biomed Res Ther 3:476–482. https://doi.org/10.7603/s40730-016-0004-x
Mayer H, Bertram H, Lindenmaier W et al (2005) Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation. J Cell Biochem 95:827–839. https://doi.org/10.1002/jcb.20462
Wagegg M, Gaber T, Lohanatha FL et al (2012) Hypoxia promotes osteogenesis but suppresses adipogenesis of human mesenchymal stromal cells in a hypoxia-inducible factor-1 dependent manner. PLoS ONE 7:1–11. https://doi.org/10.1371/journal.pone.0046483
Il YH, Moon YH, Kim MS (2016) Effects of CoCl2 on multi-lineage differentiation of C3h/10T1/2 mesenchymal stem cells. Korean J Physiol Pharmacol 20:53–62. https://doi.org/10.4196/kjpp.2016.20.1.53
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1A4A1016793), the National Research Foundation of Korea (NRF-2020R1F1A1048494), and Inha University Research Grant, Korea.
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Kim, H., Kwon, S. Dual effects of hypoxia on proliferation and osteogenic differentiation of mouse clonal mesenchymal stem cells. Bioprocess Biosyst Eng (2021). https://doi.org/10.1007/s00449-021-02563-1
- Mesenchymal stem cells
- Microcarrier culture