Optimization of Microenvironments Inducing Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Endothelial Cell-Like Cells

  • Se-Young Oh
  • Da Hyeon Choi
  • Yoon Mi Jin
  • Yeonsil Yu
  • Ha Yeong Kim
  • Gyungah Kim
  • Yoon Shin ParkEmail author
  • Inho JoEmail author
Original Article



Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments.


TMSCs were preconditioned with Dulbecco’s Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4 days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers.


Preconditioning TMSCs in EGM for 4 days and then replating them on Matrigel induced the formation of a capillary-like network in 3 h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells.


This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4 days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.


Tonsils Mesenchymal stem cells Endothelial cells Differentiation Microenvironments 



This study was supported by the Basic Science Research (NRF-2017M3A9B3063636 and NRF-2017R1A2B4002611), the Bio & Medicial Technology Development Program (NRF-2016M3A9B4919639) and Small Grant for Exploratory Research (NRF-2018R1D1A1A02085696) programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning. The paper was also supported by RP-Grant 2019 of Ewha Womans University.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Ethical statement

The study protocol was approved by the Institutional Review Board of Ewha Womans University Medical Center (IRB No. ECT-11-53-02). Informed consent was confirmed by the IRB.


  1. 1.
    World Health Organization. World health statistics 2018: monitoring health for the SDGs [Internet]. 2018 [cited 2019 May 18]. Available from:
  2. 2.
    Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000;87:840–4.PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Widlansky ME, Gokce N, Keaney JF Jr, Vita JA. The clinical implications of endothelial dysfunction. J Am Coll Cardiol. 2003;42:1149–60.PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Su JB. Vascular endothelial dysfunction and pharmacological treatment. World J Cardiol. 2015;7:719–41.PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Khan OF, Sefton MV. Endothelialized biomaterials for tissue engineering applications in vivo. Trends Biotechnol. 2011;29:379–87.PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Barabaschi GD, Manoharan V, Li Q, Bertassoni LE. Engineering pre-vascularized scaffolds for bone regeneration. Adv Exp Med Biol. 2015;881:79–94.PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Sethe S, Scutt A, Stolzing A. Aging of mesenchymal stem cells. Ageing Res Rev. 2006;5:91–116.PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Son Y. Recent advances in stem cell researches and their future perspectives in regenerative medicine. Tissue Eng Regen Med. 2017;14:641–2.PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Ryu KH, Cho KA, Park HS, Kim JY, Woo SY, Jo I, et al. Tonsil-derived mesenchymal stromal cells: evaluation of biologic, immunologic and genetic factors for successful banking. Cytotherapy. 2012;14:1193–202.PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Chen J, Park HC, Addabbo F, Ni J, Pelger E, Li H, et al. Kidney-derived mesenchymal stem cells contribute to vasculogenesis, angiogenesis and endothelial repair. Kidney Int. 2008;74:879–89.PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Janeczek Portalska K, Leferink A, Groen N, Fernandes H, Moroni L, van Blitterswijk C, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One. 2012;7:e46842.PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Liu C, Tsai AL, Li PC, Huang CW, Wu CC. Endothelial differentiation of bone marrow mesenchyme stem cells applicable to hypoxia and increased migration through Akt and NFκB signals. Stem Cell Res Ther. 2017;8:29.PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Lee SH, Lee Y, Chun YW, Crowder SW, Young PP, Park KD, et al. In situ crosslinkable gelatin hydrogels for vasculogenic induction and delivery of mesenchymal stem cells. Adv Funct Mater. 2014;24:6771–81.PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Oh SY, Choi YM, Kim HY, Park YS, Jung SC, Park JW, et al. Concise review: application of tonsil-derived mesenchymal stem cells in tissue regeneration. Stem Cells. 2019;37:1252–60.CrossRefGoogle Scholar
  15. 15.
    Kim SY, Kim YR, Park WJ, Kim HS, Jung SC, Woo SY, et al. Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells. Differentiation. 2015;90:27–39.PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Yu Y, Park YS, Kim HS, Kim HY, Jin YM, Jung SC, et al. Characterization of long-term in vitro culture-related alterations of human tonsil-derived mesenchymal stem cells: role for CCN1 in replicative senescence-associated increase in osteogenic differentiation. J Anat. 2014;225:510–8.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Park YS, Lee Y, Jin YM, Kim G, Jung SC, Park YJ, et al. Sustained release of parathyroid hormone via in situ cross-linking gelatin hydrogels improves the therapeutic potential of tonsil-derived mesenchymal stem cells for hypoparathyroidism. J Tissue Eng Regen Med. 2018;12:e1747–56.PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Park YS, Hwang S, Jin YM, Yu Y, Jung SA, Jung SC, et al. CCN1 secreted by tonsil-derived mesenchymal stem cells promotes endothelial cell angiogenesis via integrin αvβ3 and AMPK. J Cell Physiol. 2015;230:140–9.PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Yu Y, Gao Y, Qin J, Kuang CY, Song MB, Yu SY, et al. CCN1 promotes the differentiation of endothelial progenitor cells and reendothelialization in the early phase after vascular injury. Basic Res Cardiol. 2010;105:713–24.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Lin CH, Su JJ, Lee SY, Lin YM. Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med. 2018;12:2099–111.PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Minakawa T. Long-term culture of microvascular endothelial cells derived from Mongolian gerbil brain. Stroke. 1989;20:947–51.PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Um Min Allah N, Berahim Z, Ahmad A, Kannan TP. Biological interaction between human gingival fibroblasts and vascular endothelial cells for angiogenesis: a co-culture perspective. Tissue Eng Regen Med. 2017;14:495–505.PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Smeets EF, von Asmuth EJ, van der Linden CJ, Leeuwenberg JF, Buurman WA. A comparison of substrates for human umbilical vein endothelial cell culture. Biotech Histochem. 1992;67:241–50.PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Wang CY, Tsai AC, Peng CY, Chang YL, Lee KH, Teng CM, et al. Dehydrocostuslactone suppresses angiogenesis in vitro and in vivo through inhibition of Akt/GSK-3β and mTOR signaling pathways. PLoS One. 2012;7:e31195.PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Park YS, Kim HS, Jin YM, Yu Y, Kim HY, Park HS, et al. Differentiated tonsil-derived mesenchymal stem cells embedded in Matrigel restore parathyroid cell functions in rats with parathyroidectomy. Biomaterials. 2015;65:140–52.PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Kim G, Jin YM, Yu Y, Kim HY, Jo SA, Park YJ, et al. Double intratibial injection of human tonsil-derived mesenchymal stromal cells recovers postmenopausal osteoporotic bone mass. Cytotherapy. 2018;20:1013–27.PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Park JH, Jin YM, Hwang S, Cho DH, Kang DH, Jo I. Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: a mechanism for uric acid-induced cardiovascular disease development. Nitric Oxide. 2013;32:36–42.PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Hwang S, Lee HJ, Kim G, Won KJ, Park YS, Jo I. CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis. Free Radic Biol Med. 2015;89:229–40.PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Du WJ, Chi Y, Yang ZX, Li ZJ, Cui JJ, Song BQ, et al. Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, and placenta. Stem Cell Res Ther. 2016;7:163.PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Oswald J, Boxberger S, Jørgensen B, Feldmann S, Ehninger G, Bornhäuser M, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells. 2004;22:377–84.PubMedCrossRefGoogle Scholar
  31. 31.
    Cao Y, Sun Z, Liao L, Meng Y, Han Q, Zhao RC. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun. 2005;332:370–9.PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Castelli G, Parolini I, Cerio AM, D’Angiò A, Pasquini L, Carollo M, et al. Conditioned medium from human umbilical vein endothelial cells markedly improves the proliferation and differentiation of circulating endothelial progenitors. Blood Cells Mol Dis. 2016;61:58–65.PubMedCrossRefGoogle Scholar
  33. 33.
    Ponce ML. Tube formation: an in vitro matrigel angiogenesis assay. Methods Mol Biol. 2009;467:183–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Fischer LJ, McIlhenny S, Tulenko T, Golesorkhi N, Zhang P, Larson R, et al. Endothelial differentiation of adipose-derived stem cells: effects of endothelial cell growth supplement and shear force. J Surg Res. 2009;152:157–66.PubMedCrossRefGoogle Scholar
  35. 35.
    Kouroupis D, Churchman SM, English A, Emery P, Giannoudis PV, McGonagle D, et al. Assessment of umbilical cord tissue as a source of mesenchymal stem cell/endothelial cell mixtures for bone regeneration. Regen Med. 2013;8:569–81.PubMedCrossRefGoogle Scholar
  36. 36.
    Lee JH, Laronde S, Collins TJ, Shapovalova Z, Tanasijevic B, McNicol JD, et al. Lineage-specific differentiation is influenced by state of human pluripotency. Cell Rep. 2017;19:20–35.PubMedCrossRefGoogle Scholar
  37. 37.
    Yang YK, Ogando CR, Wang See C, Chang TY, Barabino GA. Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem Cell Res Ther. 2018;9:131.PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Lim JE, Son Y. Endogenous stem cells in homeostasis and aging. Tissue Eng Regen Med. 2017;14:679–98.PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Gu Y, Li T, Ding Y, Sun L, Tu T, Zhu W, et al. Changes in mesenchymal stem cells following long-term culture in vitro. Mol Med Rep. 2016;13:5207–15.PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I. Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol. 2008;36:1176–85.PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Wu Q, Fang T, Chen M, Qi G. Endothelial growth medium suppresses apoptosis of mesenchymal stem cells in vitro via decrease of miR-29a. Mol Med Rep. 2017;16:2675–81.PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601.PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Lee GY, Shin SH, Shin HW, Chun YS, Park JW. NDRG3 lowers the metastatic potential in prostate cancer as a feedback controller of hypoxia-inducible factors. Exp Mol Med. 2018;50:61.PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Li A, Dubey S, Varney ML, Dave BJ, Singh RK. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol. 2003;170:3369–76.PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Contois LW, Nugent DP, Caron JM, Cretu A, Tweedie E, Akalu A, et al. Insulin-like growth factor binding protein-4 differentially inhibits growth factor-induced angiogenesis. J Biol Chem. 2012;287:1779–89.PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Babaei S, Stewart DJ. Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model. Cardiovasc Res. 2002;55:190–200.PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Namkoong S, Kim CK, Cho YL, Kim JH, Lee H, Ha KS, et al. Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling. Cell Signal. 2009;21:906–15.PubMedCrossRefPubMedCentralGoogle Scholar
  48. 48.
    Nguyen L, Bang S, Noh I. Tissue regeneration of human mesenchymal stem cells on porous gelatin micro-carriers by long-term dynamic in vitro culture. Tissue Eng Regen Med. 2019;16:19–28.PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Davidenko N, Schuster CF, Bax DV, Farndale RW, Hamaia S, Best SM, et al. Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry. J Mater Sci Mater Med. 2016;27:148.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society 2019

Authors and Affiliations

  1. 1.Department of Molecular Medicine, College of MedicineEwha Womans UniversitySeoulRepublic of Korea
  2. 2.Ewha Tonsil-derived Mesenchymal Stem Cells Research Center (ETSRC), College of MedicineEwha Womans UniversitySeoulRepublic of Korea
  3. 3.School of Biological Sciences, College of Natural SciencesChungbuk National UniversityCheongjuRepublic of Korea
  4. 4.Department of Otorhinolaryngology-Head and Neck Surgery, College of MedicineEwha Womans UniversitySeoulRepublic of Korea

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