Differentiation potential of canine mesenchymal stem cells on hydrogel scaffold-based three-dimensional environment

하이드로젤 지지체 기반 3차원 환경에서 개 간엽줄기세포의 분화능 분석

  • Gu, Na-Yeon (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Park, Mi Jeong (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Lee, Jienny (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Byeon, Jeong Su (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Jeong, Da-Un (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Cho, In-Soo (Viral Disease Research Division, Animal and Plant Quarantine Agency) ;
  • Cha, Sang-Ho (Viral Disease Research Division, Animal and Plant Quarantine Agency)
  • 구나연 (농림축산검역본부 바이러스질병과) ;
  • 박미정 (농림축산검역본부 바이러스질병과) ;
  • 이지현 (농림축산검역본부 바이러스질병과) ;
  • 변정수 (농림축산검역본부 바이러스질병과) ;
  • 정다운 (농림축산검역본부 바이러스질병과) ;
  • 조인수 (농림축산검역본부 바이러스질병과) ;
  • 차상호 (농림축산검역본부 바이러스질병과)
  • Received : 2018.08.31
  • Accepted : 2018.12.10
  • Published : 2018.12.31


Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy. Physiological cell environment not only connects cells to each other, but also connects cells to the extracellular matrix that provide mechanical support, thus exposing the entire cell surface and activating signaling pathways. Hydrogel is a polymeric material that swells in water and maintains a distinct 3-dimensional (3D) network structure by cross linking. In this study, we investigated the optimized cellular function for canine adipose tissue-derived MSCs (cAD-MSCs) using hydrogel. We observed that the expression levels of Ki67 and proliferating cell nuclear antigen, which are involved in cell proliferation and stemness, were increased in transwell-hydrogel (3D-TN) compared to the transwell-normal (TN). Also, transforming growth factor-${\beta}1$ and SOX9, which are typical bone morphogenesis-inducing factors, were increased in 3D-TN compared to the TN. Collagen type II alpha 1, which is a chondrocyte-specific marker, was increased in 3D-TN compared to the TN. Osteocalcin, which is a osteocyte-specific marker, was increased in 3D-TN compared to the TN. Collectively, preconditioning cAD-MSCs via 3D culture systems can enhance inherent secretory properties that may improve the potency and efficacy of MSCs-based therapies for bone regeneration process.

TSSOBU_2018_v58n4_211_f0001.png 이미지

Fig. 1. Characterization of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). (A) The cAD-MSCs obtained from canine adipose tissue were able to attach to the culture plates and expand in vitro. 40× (left), 100× (right). (B) Flow cytometry histograms show the expression of surface markers (CD34, CD44 and CD90) by cAD-MSCs populations compared with controls.

TSSOBU_2018_v58n4_211_f0002.png 이미지

Fig. 3. Effects of TN and 3D-TN culture systems on cell phenotype in cAD-MSCs. (A) Schematic diagram of TN and 3D-TN culture systems. (B) Cellular morphology of TN and 3D-TN-cultured cells. 40×. mRNA expressions of (C) cell surface markers (CD29, CD34, CD44, CD45 and CD90), (D) Ki-67 and PCNA were confirmed by qRT-PCR and normalized to the GAPDH level. GAPDH was used as a housekeeping control gene. Each data from three independent experiments was evaluated and expressed as the mean ± SD (***p < 0.0001).

TSSOBU_2018_v58n4_211_f0003.png 이미지

Fig. 2. Effects of 2-dimentional (2D) and transwell (TN) culture systems on cell phenotype in cAD-MSCs. (A) Schematic diagram of 2D culture and TN culture system. (B) mRNA expression of proliferation genes (Ki-67 and PCNA) were confirmed by qRT-PCR and normalized to the GAPDH level. GAPDH was used as a housekeeping control gene. Each data from three independent experiments was evaluated and expressed as the mean ± SD (*p < 0.05, ***p < 0.0001).

TSSOBU_2018_v58n4_211_f0004.png 이미지

Fig. 4. Differentiation potentials of TN and 3D-TN culture systems in cAD-MSCs. (A) Using the specific staining kit, chondrocytes (Alcian Blue) and osteocytes (Alizarin Red) were positively stained in two groups. 100×. (B) The mRNA expressions of chondrocyte (COL2A1 and SOX9) and osteocyte (OC and TGF-β1) related genes were detected by qRT-PCR and normalized to the GAPDH level. GAPDH was used as a housekeeping control gene. Each data from three independent experiments was evaluated and expressed as the mean ± SD (**p < 0.001, ***p < 0.0001).

Table 1. List of primers used for quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)

TSSOBU_2018_v58n4_211_t0001.png 이미지

Table 2. Composition of differentiation media

TSSOBU_2018_v58n4_211_t0002.png 이미지


Supported by : 농림축산검역본부


  1. Barsby T, Bavin EP, Guest DJ. Three-dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of equine embryo-derived stem cells. Tissue Eng Part A 2014, 20, 2604-2613.
  2. Breslin S, O'Driscoll L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 2013, 18, 240-249.
  3. Byeon JS, Lee J, Kim DH, Lee GB, Kim HR, Gu NY, Cho IS, Cha SH. [Canine mesenchymal stem cells immunomodulate atopic dermatitis through the induction of regulatory T cells in an ex vivo experimental study]. J Prev Vet Med 2016, 40, 12-21. Korean.
  4. Cha BH, Kim JH, Kang SW, Do HJ, Jang JW, Choi YR, Park H, Kim BS, Lee SH. Cartilage tissue formation from dedifferentiated chondrocytes by codelivery of BMP-2 and SOX-9 genes encoding bicistronic vector. Cell Transplant 2013, 22, 1519-1528.
  5. Chen G, Deng C, Li YP. $TGF-{\beta}$ and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci 2012, 8, 272-288.
  6. Chen X, Martin BD, Neubauer TK, Linhardt RJ, Dordick JS, Rethwisch DG. Enzymatic and chemoenzymatic approaches to synthesis of sugar-based polymer and hydrogels. Carbohydr Polym 1995, 28, 15-21.
  7. Chesnutt BM, Yuan Y, Buddington K, Haggard WO, Bumgardner JD. Composite chitosan/nano-hydroxyapatite scaffolds induce osteocalcin production by osteoblasts in vitro and support bone formation in vivo. Tissue Eng Part A 2009, 15, 2571-2579.
  8. Debnath T, Ghosh S, Potlapuvu US, Kona L, Kamaraju SR, Sarkar S, Gaddam S, Chelluri LK. Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel. PLoS One 2015, 10, e0120803.
  9. Dufrane D. Impact of age on human adipose stem cells for bone tissue engineering. Cell Transplant 2017, 26, 1496-1504.
  10. El-Sherbiny IM, Yacoub MH. Hydrogel scaffolds for tissue engineering: progress and challenges. Glob Cardiol Sci Pract 2013, 2013, 316-342.
  11. English K, French A, Wood KJ. Mesenchymal stromal cells: facilitators of successful transplantation? Cell Stem Cell 2010, 7, 431-442.
  12. Fenger JM, London CA, Kisseberth WC. Canine osteosarcoma: a naturally occurring disease to inform pediatric oncology. ILAR J 2014, 55, 69-85.
  13. Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci 2010, 123, 4195-4200.
  14. Hamidi M, Azadi A, Rafiei P. Hydrogel nanoparticles in drug delivery. Adv Drug Deliv Rev 2008, 60, 1638-1649.
  15. Kill IR. Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component. J Cell Sci 1996, 109, 1253-1263.
  16. Kim S, Chang KA, Kim J, Park HG, Ra JC, Kim HS, Suh YH. The preventive and therapeutic effects of intravenous human adipose-derived stem cells in Alzheimer's disease mice. PLoS One 2012, 7, e45757.
  17. Koundrioukoff S, Jonsson ZO, Hasan S, de Jong RN, van der Vliet PC, Hottiger MO, Hubscher U. A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation. J Biol Chem 2000, 275, 22882-22887.
  18. Lee SJ, Ryu MO, Seo MS, Park SB, Ahn JO, Han SM, Kang KS, Bhang DH, Youn HY. Mesenchymal stem cells contribute to improvement of renal function in a canine kidney injury model. In Vivo 2017, 31, 1115-1124.
  19. Leitinger B, Hohenester E. Mammalian collagen receptors. Matrix Biol 2007, 26, 146-155.
  20. Matsuda T, Takami T, Sasaki R, Nishimura T, Aibe Y, Paredes BD, Quintaniha LF, Matsumoto T, Ishikawa T, Yamamoto N, Tani K, Terai S, Taura Y, Sakaida I. A canine liver fibrosis model to develop a therapy for liver cirrhosis using cultured bone marrow-derived cells. Hepatol Commun 2017, 1, 691-703.
  21. McMillan A, Nguyen MK, Gonzalez-Fernandez T, Ge P, Yu X, Murphy WL, Kelly DJ, Alsberg E. Dual non-viral gene delivery from microparticles within 3D high-density stem cell constructs for enhanced bone tissue engineering. Biomaterials 2018, 161, 240-255.
  22. Muller P, Lemcke H, David R. Stem cell therapy in heart diseases - cell types, mechanisms and improvement strategies. Cell Physiol Biochem 2018, 17, 2607-2655.
  23. Rustad KC, Wong VW, Sorkin M, Glotzbach JP, Major MR, Rajadas J, Longaker MT, Gurtner GC. Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold. Biomaterials 2012, 33, 80-90.
  24. Semino CE, Kasahara J, Hayashi Y, Zhang S. Entrapment of migrating hippocampal neural cells in three-dimensional peptide nanofiber scaffold. Tissue Eng 2004, 10, 643-655.
  25. Shah K, Drury T, Roic I, Hansen P, Malin M, Boyd R, Sumer H, Ferguson R. Outcome of allogeneic adult stem cell therapy in dogs suffering from osteoarthritis and other joint defects. Stem Cells Int 2018, 2018, 7309201.
  26. Toh WS, Lim TC, Kurisawa M, Spector M. Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomaterials 2012, 33, 3835-3845.
  27. Tongers J, Losordo DW, Landmesser U. Stem and progenitor cell-based therapy in ischaemic heart disease: promise, uncertainties, and challenges. Eur Heart J 2011, 32, 1197-1206.
  28. Tsui JH, Lee W, Pun SH, Kim J, Kim DH. Microfluidicsassisted in vitro drug screening and carrier production. Adv Drug Deliv Rev 2013, 65, 1575-1588.
  29. Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J, Pfister S, Eckstein V, Ho AD. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 2008, 3, e2213.
  30. Wang S, Nagrath D, Chen PC, Berthiaume F, Yarmush ML. Three-dimensional primary hepatocyte culture in synthetic selfassembling peptide hydrogel. Tissue Eng Part A 2008, 14, 227-236.
  31. Westhrin M, Xie M, Olderoy MO, Sikorski P, Strand BL, Standal T. Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices. PLoS One 2015, 10, e0120374.
  32. Wichterle O, Lim D. Hydrophilic gels for biological use. Nature 1960, 185, 117-118.
  33. Wu H, Mahato RI. Mesenchymal stem cell-based therapy for type 1 diabetes. Discov Med 2014, 17, 139-143.
  34. Zhang S, Holmes TC, DiPersio CM, Hynes RO, Su X, Rich A. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials 1995, 16, 1385-1393.
  35. Zhang S, Holmes T, Lockshin C, Rich A. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci U S A 1993, 90, 3334-3338.
  36. Zheng, L, Fan HS, Sun J, Chen XN, Wang G, Zhang L, Fan YJ, Zhang XD. Chondrogenic differentiation of mesenchymal stem cells induced by collagen-based hydrogel: an in vivo study. J Biomed Mater Res A 2010, 93, 783-792.
  37. Zhu J, Marchant RE. Design properties of hydrogel tissueengineering scaffolds. Expert Rev Med Devices 2011, 8, 607-626.