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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

Abstract

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.

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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.

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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).

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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).

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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)

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Table 2. Composition of differentiation media

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Acknowledgement

Supported by : 농림축산검역본부

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