Osteogenic Potential Reduction in Mesenchymal Stem Cells under Prolonged Simulated Microgravity

A.Y. Ratushnyy*, O.V. Grigorieva, L.B. Buravkova

Institute of Biomedical Problems, Russian Academy of Sciences, Moscow, 123007 Russia

E-mail: *Ratushkin@mail.ru

Received January 30, 2017

Full text PDF

Abstract

Increasing the duration of orbital space flights up to 6–12 months and planning interplanetary missions actualizes the need for a better understanding of the mechanisms of osteopenia caused by microgravity. Investigation of mesenchymal stem cells (MSCs) that support the tissue homeostasis under microgravity conditions allows a deeper insight into the processes underlying bone loss. The purpose of this study was to investigate the osteogenic potential of MSCs under prolonged simulated microgravity by clinorotation. Using the method of mineralized matrix detection, it has been found that MSCs osteogenic potential decreased after long-term clinorotation. The investigation of major osteogenic gene expression has showed decreased trans­criptional activity in RUNX2, ALPL-1, Col-1, but increased expression of PPARγ. One of the reasons for the decreased osteogenic potential of MSCs may be an increased level of reactive oxygen species (ROS) after 30 days of clinorotation. ROS may affect cellular signaling cascades, such as Wnt, Hedgehog and FOXO pathways, thereby leading to a shift of the differentiation potential to adipogenesis.

Keywords: mesenchymal stem cells (MSCs), simulated microgravity, osteogenic differentiation, reactive oxygen species (ROS)

Acknowledgments. The study was supported by the project no. NSh-7479.2016.4.

Figure Captions

Fig. 1. Osteogenic differentiation, alizarin red staining of mineralized matrix, light microscopy. Magnification: 100×.

Fig. 2. Quantitative evaluation of the mineralization level of extracellular MSC matrix by measuring the optical density of cohered color material (alizarin red) dissolved in DMSO.

Fig. 3. Differential expression of differentiation genes. Data are presented as mean values ? standard deviations. Asterisk indicates significant differences (p ≤ 0.05) from control.

Fig. 4. Fluorescence intensity of H2DCFDA showing the level of reactive oxygen species in cells.

References

  1. Zayzafoon M., Meyers V.E., McDonald J.M. Microgravity: The immune response and bone. Immunol. Rev., 2005, vol. 208, no. 1, pp. 267–280. doi: 10.1111/j.0105-2896.2005.00330.x.
  2. Berg-Johansen B., Liebenberg E.C., Li A., Macias B.R., Hargens A.R., Lotz J.C. Spaceflight-induced bone loss alters failure mode and reduces bending strength in murine spinal segments. J. Orthop. Res., 2016, vol. 34, no. 1, pp. 48–57. doi: 10.1002/jor.23029.
  3. Shi D., Meng R., Deng W., Ding W., Zheng Q., Yuan W., Liu L., Zong C., Shang P., Wang J. Effects of microgravity modeled by large gradient high magnetic field on the osteogenic initiation of human mesenchymal stem cells. Stem Cell Rev., 2010, vol. 6, no. 4, pp. 567–578. doi: 10.1007/s12015-010-9182-x.
  4. Oganov V. S. Bone System, Weightlessness, and Osteoporosis. Voronezh, Nauchn. Kn., 2014. 291 p. (In Russian)
  5. Jha R., Wu Q., Singh M., Preininger M.K., Han P., Ding G., Cho H.C., Jo H., Maher K.O., Wagner M.B., Xu C. Simulated microgravity and 3D culture enhance induction, viability, proliferation and differentiation of cardiac progenitors from human pluripotent stem cells. Sci. Rep., 2016, vol. 6, art. 30956, pp. 1–14. doi: 10.1038/srep30956.
  6. Lin S.C., Gou G.H., Hsia C.W., Ho C.W., Huang K.L., Wu Y.F., Lee S.Y., Chen Y.H. Simulated microgravity disrupts cytoskeleton organization and increases apoptosis of rat neural crest stem cells via upregulating CXCR4 expression and RhoA-ROCK1-p38 MAPK-p53 signaling. Stem Cells Dev., 2016, vol. 25. no. 15, pp. 1172–1193. doi: 10.1089/scd.2016.0040.
  7. Wehland M., Aleshcheva G., Schulz H., Saar K., Hübner N., Hemmersbach R., Braun M., Ma X., Frett T., Warnke E., Riwaldt S., Pietsch J., Corydon T.J., Infanger M., Grimm D. Differential gene expression of human chondrocytes cultured under short-term altered gravity conditions during  parabolic flight maneuvers. Cell Commun. Signaling, 2015, vol. 13, no. 18, pp. 1–13. doi: 10.1186/s12964-015-0095-9.
  8. He L., Pan S., Li Y., Zhang L., Zhang W., Yi H., Song C., Niu Y. Increased proliferation and adhesion properties of human dental pulp stem cells in PLGA scaffolds via simulated microgravity. Int. Endod. J., 2016, vol. 49, no. 2, pp. 161–173. doi: 10.1111/iej.12441.
  9. Buravkova L.B., Romanov Yu.A., Konstantinova N.A. Buravkov S.V., Gershovich Yu.G., Grivennikov I.A. Cultured stem cells are sensitive to gravity changes. Acta Astronaut., 2008, vol. 63, nos. 5–6, pp. 603–608. doi: 10.1016/j.actaastro.2008.04.012.
  10. Gershovich Yu.G., Buravkova L.B. Effects of microgravity simulation on the production of interleukins in culture of human mesenchymal stromal cells. Hum. Physiol., 2011, vol. 37, no. 7, pp. 860–865. doi: 10.1134/S0362119711070139.
  11. Friedenstein A.J., Gorskaja J.F., Kulagina N.N. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp. Hematol., 1976, vol. 4, no. 5, pp. 267–274.
  12. Dominici M., Le Blanc K., Mueller I., Slaper-Cortenbach I., Marini F., Krause D., Deans R., Keating A., Prockop D., Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, vol. 8, no. 4, pp. 315–317. doi: 10.1080/14653240600855905.
  13. Caplan A.I. Mesenchymal stem cells. J. Orthop. Res., 1991, vol. 9, no. 5, pp. 641–650. doi: 10.1002/jor.1100090504.
  14. Richardson S.M., Kalamegam G., Pushparaj P.N., Matta C., Memic A., Khademhosseini A., Mobasheri R., Poletti F.L., Hoyland J.A., Mobasheri A. Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration. Methods, 2016, vol. 99, pp. 69–80. doi: 10.1016/j.ymeth.2015.09.015.
  15. Caplan A.I., Dennis J.E. Mesenchymal stem cells as trophic mediators. J. Cell Biochem., 2006, vol. 98, no. 5, pp. 1076–1084. doi: 10.1002/jcb.20886.
  16. Gershovich P.M., Gershovich Iu.G., Buravkova L.B. Cytoskeleton structures and adhesion properties of human stromal precursors under conditions of simulated microgravity. Tsitologiia, 2009, vol. 51, no. 11, pp. 896–904. (In Russian)
  17. Gershovich Yu.G., Buravkova L.B. Morphofunctional status and osteogenic differentiation potential of human mesenchymal stromal precursor cells during in vitro modeling of microgravity effects. Kletochnye Tekhnol. Biol. Med., 2007, no. 4, pp. 196–202. (In Russian)
  18. Dedolph R.R., Dipert M.H. The physical basis of gravity stimulus nullification by clinostat rotation. Plant Physiol., 1971, vol. 47, no. 6, pp. 756–764. doi: 10.1104/pp.47.6.756.
  19. Yan M., Wang Y., Yang M., Liu Y., Qu B., Ye Z., Liang W., Sun X., Luo Z. The effects and mechanisms of clinorotation on proliferation and differentiation in bone marrow mesenchymal stem cells. Biochem. Biophys. Res. Commun., 2015, vol. 460, no. 2, pp. 327–332. doi: 10.1016/j.bbrc.2015.03.034.
  20. Chen Z., Luo Q., Lin C., Song G. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells through down regulating the trans­criptional co-activator TAZ. Biochem. Biophys. Res. Commun., 2015, vol. 468, nos. 1–2, pp. 21–26. doi: 10.1016/j.bbrc.2015.11.006.
  21. Zuk P.A., Zhu M., Mizuno H., Huang J., Futrell J.W., Katz A.J., Benhaim P., Lorenz H.P., Hedrick M.H. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng., 2001, vol. 7, no. 2, pp. 211–228. doi: 10.1089/107632701300062859.
  22. Buravkova L.B., Grinakovskaia O.S., Andreeva E.P., Zhambalova A.P., Kozionova M.P. Characteristics of human lipoaspirate-isolated mesenchymal stromal cells cultivated under a lower oxygen tension. Tsitologiia, 2009, vol. 51, no. 1, pp. 5–11. (In Russian)
  23. Corydon T.J., Kopp S., Wehland M., Braun M., Schütte A., Mayer T., Hülsing T., Oltmann H., Schmitz B., Hemmersbach R., Grimm D. Alterations of the cytoskeleton in human cells in space proved by life-cell imaging. Sci. Rep., 2016, vol. 6, art. 20043, pp. 1–14. doi: 10.1038/srep20043.
  24. Meyers V.E., Zayzafoon M., Douglas J.T., McDonald J.M. RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity. J. Bone Miner. Res., 2005, vol. 20, no. 10, pp. 1858–1866. doi: 10.1359/JBMR.050611.
  25. Chen Z., Luo Q., Lin C., Kuang D., Song G. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation. Sci. Rep., 2016, vol. 6, art. 30322, pp. 1–11. doi: 10.1038/srep30322.
  26. Buravkova L.B., Gershovich P.M., Gershovich J.G., Grigor'ev A.I. Mechanisms of gravitational sensitivity of osteogenic precursor cells. Acta Nat., 2010, vol. 2, no. 1, pp. 28–36.
  27. Jeon M.J., Kim J.A., Kwon S.H., Kim S.W., Park K.S., Park S.W., Kim S.Y., Shin C.S. Activation of peroxisome proliferator-activated receptor-gamma inhibits the Runx2-mediated trans­cription of osteocalcin in osteoblasts. J. Biol. Chem., 2003, vol. 278, no. 26, pp. 23270–23277. doi: 10.1074/jbc.M211610200.
  28. Denu R.A., Hematti P. Effects of oxidative stress on mesenchymal stem cell biology. Oxid. Med. Cell. Longevity, 2016, vol. 2016, art. 2989076, pp. 1–9. doi: 10.1155/2016/2989076.
  29. Atashi F., Modarressi A., Pepper M.S. The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: A review. Stem Cells Dev., 2015, vol 24, no. 10, pp. 1150–1163. doi: 10.1089/scd.2014.0484.
  30. Tormos K.V., Anso E., Hamanaka R.B., Eisenbart J., Joseph J., Kalyanaraman B., Chandel N.S. Mitochondrial complex III ROS regulate adipocyte differentiation. Cell Metab., 2011, vol. 14, no. 4, pp. 537–544. doi: 10.1016/j.cmet.2011.08.007.
  31. Almeida M., Han L., Martin-Millan M., O'Brien C.A., Manolagas S.C. Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factor- to forkhead box O-mediated trans­cription. J. Biol. Chem., 2007, vol. 282, no. 37, pp. 27298–27305. doi: 10.1074/jbc.M702811200.
  32. Iyer S., Ambrogini E., Bartell S.M., Han L., Roberson P.K., de Cabo R., Jilka R.L., Weinstein R.S., O'Brien C.A., Manolagas S.C., Almeida M. FOXOs attenuate bone formation by suppressing Wnt signaling. J. Clin. Invest., 2013, vol. 123, no. 8, pp. 3409–3419. doi: 10.1172/JCI68049.
  33. Kim W.K., Meliton V., Bourquard N., Hahn T.J., Parhami F. Hedgehog signaling and osteogenic differentiation in multipotent bone marrow stromal cells are inhibited by oxidative stress. J. Cell. Biochem., 2010, vol. 111, no. 5, pp. 1199–1209. doi: 10.1002/jcb.22846.

For citation: Ratushnyy A.Y., Grigorieva O.V., Buravkova L.B. Osteogenic potential reduction in mesenchymal stem cells under prolonged simulated microgravity. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 2, pp. 206–216. (In Russian)


The content is available under the license Creative Commons Attribution 4.0 License.