Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Long-Duration Three-Dimensional Spheroid Culture Promotes Angiogenic Activities of Adipose-Derived Mesenchymal Stem Cells

  • Lee, Jun Hee (Laboratory for Vascular Medicine & Stem Cell Biology, Medical Research Institute, Department of Physiology, School of Medicine, Pusan National University) ;
  • Han, Yong-Seok (Medical Science Research Institute, Soonchunhyang University Seoul Hospital) ;
  • Lee, Sang Hun (Medical Science Research Institute, Soonchunhyang University Seoul Hospital)
  • Received : 2015.09.08
  • Accepted : 2015.11.11
  • Published : 2016.05.01
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Abstract

Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes increased in a time-dependent manner. After re-attachment of MSC spheroids to the plastic dish, their adhesion capacity and morphology were not altered. MSC spheroids showed enhanced production of hypoxia-induced angiogenic cytokines such as vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF), and hepatocyte growth factor (HGF). In addition, spheroid culture promoted the preservation of extracellular matrix (ECM) components, such as laminin and fibronectin, in a culture time- and spheroid size-dependent manner. Furthermore, phosphorylation of AKT, a cell survival signal, was significantly higher and the expression of pro-apoptotic molecules, poly (ADP ribose) polymerase-1 (PARP-1) and cleaved caspase-3, was markedly lower in the spheroids than in MSCs in monolayers. In the murine hindlimb ischemia model, transplanted MSC spheroids showed better proliferation than MSCs in monolayer. These findings suggest that MSC spheroids promote MSC bioactivities via secretion of angiogenic cytokines, preservation of ECM components, and regulation of apoptotic signals. Therefore, MSC spheroid-based cell therapy may serve as a simple and effective strategy for regenerative medicine.

Keywords

References

  1. Assmus, B., Honold, J., Schächinger, V., Britten, M. B., Fischer-Rasokat, U., Lehmann, R., Teupe, C., Pistorius, K., Martin, H., Abolmaali, N. D., Tonn, T., Dimmeler, S. and Zeiher, A. M. (2006) Transcoronary transplantation of progenitor cells after myocardial infarction. N. Engl. J. Med. 355, 1222-1232. https://doi.org/10.1056/NEJMoa051779
  2. Baer, P. C., Griesche, N., Luttmann, W., Schubert, R., Luttmann, A. and Geiger, H. (2010) Human adipose-derived mesenchymal stem cells in vitro: evaluation of an optimal expansion medium preserving stemness. Cytotherapy 12, 96-106. https://doi.org/10.3109/14653240903377045
  3. Bartosh, T. J., Ylöstalo, J. H., Mohammadipoor, A., Bazhanov, N., Coble, K., Claypool, K., Lee, R. H., Choi, H. and Prockop, D. J. (2010) Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc. Natl. Acad. Sci. U.S.A. 107, 13724-13729. https://doi.org/10.1073/pnas.1008117107
  4. Bhang, S. H., Cho, S. W., La, W. G., Lee, T. J., Yang, H. S., Sun, A. Y., Baek, S. H., Rhie, J. W. and Kim, B. S. (2011) Angiogenesis in ischemic tissue produced by spheroid grafting of human adiposederived stromal cells. Biomaterials 32, 2734-2747. https://doi.org/10.1016/j.biomaterials.2010.12.035
  5. Bruder, S. P., Jaiswal, N. and Haynesworth, S. E. (1997) Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J. Cell. Biochem. 64, 278-294. https://doi.org/10.1002/(SICI)1097-4644(199702)64:2<278::AID-JCB11>3.0.CO;2-F
  6. Burdick, J. A. and Vunjak-Novakovic, G. (2009) Engineered microenvironments for controlled stem cell differentiation. Tissue Eng. Part A 15, 205-219. https://doi.org/10.1089/ten.tea.2008.0131
  7. Chacko, S. M., Ahmed, S., Selvendiran, K., Kuppusamy, M. L., Khan, M. and Kuppusamy, P. (2010) Hypoxic preconditioning induces the expression of prosurvival and proangiogenic markers in mesenchymal stem cells. Am. J. Physiol. Cell Physiol. 299, C1562-C1570. https://doi.org/10.1152/ajpcell.00221.2010
  8. Cheng, N. C., Wang, S. and Young, T. H. (2012) The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials 33, 1748-1758. https://doi.org/10.1016/j.biomaterials.2011.11.049
  9. Cochrane, D. J., Stannard, S. R., Firth, E. C. and Rittweger, J. (2010) Comparing muscle temperature during static and dynamic squatting with and without whole-body vibration. Clin. Physiol. Funct. Imaging 30, 223-229. https://doi.org/10.1111/j.1475-097X.2010.00931.x
  10. Colter, D. C., Class, R., DiGirolamo, C. M. and Prockop, D. J. (2000) Rapid expansion of recycling stem cells in cultures of plasticadherent cells from human bone marrow. Proc. Natl. Acad. Sci. U.S.A. 97, 3213-3218. https://doi.org/10.1073/pnas.97.7.3213
  11. da Silva Meirelles, L., Chagastelles, P. C. and Nardi, N. B. (2006) Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J. Cell Sci. 119, 2204-2213. https://doi.org/10.1242/jcs.02932
  12. English, K., French, A. and Wood, K. J. (2010) Mesenchymal stromal cells: facilitators of successful transplantation? Cell Stem Cell 7, 431-442. https://doi.org/10.1016/j.stem.2010.09.009
  13. Erices, A., Conget, P. and Minguell, J. J. (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br. J. Haematol. 109, 235-242. https://doi.org/10.1046/j.1365-2141.2000.01986.x
  14. Fiorina, P., Jurewicz, M., Augello, A., Vergani, A., Dada, S., La Rosa, S., Selig, M., Godwin, J., Law, K., Placidi, C., Smith, R. N., Capella, C., Rodig, S., Adra, C. N., Atkinson, M., Sayegh, M. H. and Abdi, R. (2009) Immunomodulatory function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes. J. Immunol. 183, 993-1004. https://doi.org/10.4049/jimmunol.0900803
  15. Frisch, S. M. and Screaton, R. A. (2001) Anoikis mechanisms. Curr. Opin. Cell Biol. 13, 555-562. https://doi.org/10.1016/S0955-0674(00)00251-9
  16. Gonzalez, M. A., Gonzalez-Rey, E., Rico, L., Büscher, D. and Delgado, M. (2009) Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology 136, 978-989. https://doi.org/10.1053/j.gastro.2008.11.041
  17. Grossmann, J. (2002) Molecular mechanisms of "detachment-induced apoptosis--Anoikis". Apoptosis 7, 247-260. https://doi.org/10.1023/A:1015312119693
  18. Hahn, J. Y., Cho, H. J., Kang, H. J., Kim, T. S., Kim, M. H., Chung, J. H., Bae, J. W., Oh, B. H., Park, Y. B. and Kim, H. S. (2008) Pretreatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction. J. Am. Coll. Cardiol. 51, 933-943. https://doi.org/10.1016/j.jacc.2007.11.040
  19. Han, Y. S., Lee, J. H., Jung, J. S., Noh, H., Baek, M. J., Ryu, J. M., Yoon, Y. M., Han, H. J. and Lee, S. H. (2015) Fucoidan protects mesenchymal stem cells against oxidative stress and enhances vascular regeneration in a murine hindlimb ischemia model. Int. J. Cardiol. 198, 187-195. https://doi.org/10.1016/j.ijcard.2015.06.070
  20. Haycock, J. W. (2011) 3D cell culture: a review of current approaches and techniques. Methods Mol. Biol. 695, 1-15. https://doi.org/10.1007/978-1-60761-984-0_1
  21. Hill, E., Boontheekul, T. and Mooney, D. J. (2006) Regulating activation of transplanted cells controls tissue regeneration. Proc. Natl. Acad. Sci. U.S.A. 103, 2494-2499. https://doi.org/10.1073/pnas.0506004103
  22. Hyun, I., Hochedlinger, K., Jaenisch, R. and Yamanaka, S. (2007) New advances in iPS cell research do not obviate the need for human embryonic stem cells. Cell Stem Cell 1, 367-368. https://doi.org/10.1016/j.stem.2007.09.006
  23. Le Blanc, K., Frassoni, F., Ball, L., Locatelli, F., Roelofs, H., Lewis, I. et al. (2008) Mesenchymal stem cells for treatment of steroidresistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371, 1579-1586. https://doi.org/10.1016/S0140-6736(08)60690-X
  24. Lee, E. J., Park, S. J., Kang, S. K., Kim, G. H., Kang, H. J., Lee, S. W., Jeon, H. B. and Kim, H. S. (2012) Spherical bullet formation via Ecadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction. Mol. Ther. 20, 1424-1433. https://doi.org/10.1038/mt.2012.58
  25. Li, T. S., Cheng, K., Lee, S. T., Matsushita, S., Davis, D., Malliaras, K., Zhang, Y., Matsushita, N., Smith, R. R. and Marbán, E. (2010) Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell-matrix interactions, rationalizing their enhanced functional potency for myocardial repair. Stem Cells 28, 2088-2098. https://doi.org/10.1002/stem.532
  26. Li, W., Ma, N., Ong, L. L., Nesselmann, C., Klopsch, C., Ladilov, Y., Furlani, D., Piechaczek, C., Moebius, J. M., Lützow, K., Lendlein, A., Stamm, C., Li, R. K. and Steinhoff, G. (2007) Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells 25, 2118-2127. https://doi.org/10.1634/stemcells.2006-0771
  27. Limbourg, A., Korff, T., Napp, L. C., Schaper, W., Drexler, H. and Limbourg, F. P. (2009) Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat. Protoc. 4, 1737-1746. https://doi.org/10.1038/nprot.2009.185
  28. Park, E. and Patel, A. N. (2010) Changes in the expression pattern of mesenchymal and pluripotent markers in human adipose-derived stem cells. Cell Biol. Int. 34, 979-984. https://doi.org/10.1042/CBI20100124
  29. Peterson, K. M., Aly, A., Lerman, A., Lerman, L. O. and Rodriguez-Porcel, M. (2011) Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress. Life Sci. 88, 65-73. https://doi.org/10.1016/j.lfs.2010.10.023
  30. Potapova, I. A., Brink, P. R., Cohen, I. S. and Doronin, S. V. (2008) Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells. J. Biol. Chem. 283, 13100-13107. https://doi.org/10.1074/jbc.M800184200
  31. Rehman, J., Traktuev, D., Li, J., Merfeld-Clauss, S., Temm-Grove, C. J., Bovenkerk, J. E., Pell, C. L., Johnstone, B. H., Considine, R. V. and March, K. L. (2004) Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 109, 1292-1298. https://doi.org/10.1161/01.CIR.0000121425.42966.F1
  32. Rodriguez-Lozano, F. J., Bueno, C., Insausti, C. L., Meseguer, L., Ramirez, M. C., Blanquer, M., Marín, N., Martínez, S. and Moraleda, J. M. (2011) Mesenchymal stem cells derived from dental tissues. Int. Endod. J. 44, 800-806. https://doi.org/10.1111/j.1365-2591.2011.01877.x
  33. Rosova, I., Dao, M., Capoccia, B., Link, D. and Nolta, J. A. (2008) Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 26, 2173-2182. https://doi.org/10.1634/stemcells.2007-1104
  34. Salem, H. K. and Thiemermann, C. (2010) Mesenchymal stromal cells: current understanding and clinical status. Stem Cells 28, 585-596.
  35. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S. and Jones, J. M. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147. https://doi.org/10.1126/science.282.5391.1145
  36. Tolar, J., Le Blanc, K., Keating, A. and Blazar, B. R. (2010) Concise review: hitting the right spot with mesenchymal stromal cells. Stem Cells 28, 1446-1455. https://doi.org/10.1002/stem.459
  37. Tseng, T. C. and Hsu, S. H. (2014) Substrate-mediated nanoparticle/ gene delivery to MSC spheroids and their applications in peripheral nerve regeneration. Biomaterials 35, 2630-2641. https://doi.org/10.1016/j.biomaterials.2013.12.021
  38. Wang, C. C., Chen, C. H., Hwang, S. M., Lin, W. W., Huang, C. H., Lee, W. Y., Chang, Y. and Sung, H. W. (2009a) Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty. Stem Cells 27, 724-732. https://doi.org/10.1634/stemcells.2008-0944
  39. Wang, W., Itaka, K., Ohba, S., Nishiyama, N., Chung, U. I., Yamasaki, Y. and Kataoka, K. (2009b) 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials 30, 2705-2715. https://doi.org/10.1016/j.biomaterials.2009.01.030
  40. Yamaguchi, Y., Ohno, J., Sato, A., Kido, H. and Fukushima, T. (2014) Mesenchymal stem cell spheroids exhibit enhanced in-vitro and invivo osteoregenerative potential. BMC Biotechnol. 14, 105. https://doi.org/10.1186/s12896-014-0105-9
  41. Yeh, H. Y., Liu, B. H., Sieber, M. and Hsu, S. H. (2014) Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes. BMC Genomics 15, 10. https://doi.org/10.1186/1471-2164-15-10
  42. Ylostalo, J. H., Bartosh, T. J., Coble, K. and Prockop, D. J. (2012) Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype. Stem Cells 30, 2283-2296. https://doi.org/10.1002/stem.1191
  43. Yoon, H. H., Bhang, S. H., Shin, J. Y., Shin, J. and Kim, B. S. (2012) Enhanced cartilage formation via three-dimensional cell engineering of human adipose-derived stem cells. Tissue Eng. Part A 18, 1949-1956. https://doi.org/10.1089/ten.tea.2011.0647
  44. Zhang, M., Methot, D., Poppa, V., Fujio, Y., Walsh, K. and Murry, C. E. (2001) Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J. Mol. Cell. Cardiol. 33, 907-921. https://doi.org/10.1006/jmcc.2001.1367
  45. Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., Alfonso, Z. C., Fraser, J. K., Benhaim, P. and Hedrick, M. H. (2002) Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13, 4279-4295. https://doi.org/10.1091/mbc.E02-02-0105
  46. Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J., Benhaim, P., Lorenz, H. P. and Hedrick, M. H. (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7, 211-228. https://doi.org/10.1089/107632701300062859

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