Dual effects of hypoxia on proliferation and osteogenic differentiation of mouse clonal mesenchymal stem cells

Abstract

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    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

    Article  PubMed  Google Scholar 

  2. 2.

    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

    Article  PubMed  Google Scholar 

  3. 3.

    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

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    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

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    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

    CAS  Article  Google Scholar 

  6. 6.

    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

    CAS  Article  Google Scholar 

  7. 7.

    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

  8. 8.

    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

  9. 9.

    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

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    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

    CAS  Article  Google Scholar 

  11. 11.

    Wu D, Yotnda P (2011) Induction and testing of hypoxia in cell culture. J Vis Exp. https://doi.org/10.3791/2899

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    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

    CAS  Article  Google Scholar 

  13. 13.

    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

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    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

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    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

    Article  PubMed  Google Scholar 

  16. 16.

    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

    Article  PubMed  Google Scholar 

  17. 17.

    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

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    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

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    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

    Article  Google Scholar 

  20. 20.

    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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    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

    CAS  Article  Google Scholar 

  23. 23.

    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

    CAS  Article  Google Scholar 

  24. 24.

    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

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    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

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    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

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    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

    CAS  Google Scholar 

  28. 28.

    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

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    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

  31. 31.

    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

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    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

    Article  PubMed  Google Scholar 

  33. 33.

    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

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    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

    Article  Google Scholar 

  36. 36.

    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

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    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

    CAS  Article  Google Scholar 

  38. 38.

    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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Soonjo Kwon.

Ethics declarations

Conflict of interest

The authors declare that there are no conflict of interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Keywords

  • Hypoxia
  • Mesenchymal stem cells
  • Proliferation
  • Differentiation
  • Microcarrier culture