In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells
体外诱导间充质干细胞分化形成肥大化软骨细胞
发布: 2016年12月05日第6卷第23期 DOI: 10.21769/BioProtoc.2057 浏览次数: 13755
评审: Vivien Jane Coulson-ThomasAnonymous reviewer(s)
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The authors used this protocol in:
Nov 2015
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Abstract
To investigate underlying mechanism of chondrogenic hypertrophy, we need proper in vitro hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of in vitro chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; triiodothyronine) and minimum osteogenic-inducing factors to culture medium, we could induce hypertrophy of hUCB-MSCs in vitro. Hypertrophic induction was validated using immunohistochemical analysis, Western blotting and reverse transcriptase polymerase chain reaction.
Keywords: Mesenchymal stem cell (间充质干细胞)
in vitro chondrogenic hypertrophy (体外软骨形成性肥大)
Chondrogenic differentiation (软骨形成分化)
Triiodothyronine (三碘甲状腺原氨酸)
Background
Several studies have shown that expression of hypertrophy-associated genes, including type X collagen, alkaline phosphatase, and parathyroid hormone-related protein receptor (PTHrPR) in chondrogenic differentiation of MSCs. The expression of these genes suggests that chondrogenic differentiation in MSCs inevitably induce chondrogenic hypertrophy stage which is typical of endochondral ossification. In addition, it is known that the activation of the parathyroid hormone-related protein (PTHrP) pathway induces MSC transition to an osteogenic phenotype (Guo et al., 2006). Based on these reports, Mueller et al. suggested that depletion of TGF-β, low concentration of dexamethasone, and addition of triiodothyronine (T3) was important for hypertrophic induction of bone marrow-derived MSCs (Mueller et al., 2008). In Mueller’s protocol, β-glycerophosphate and dexamethasone are necessary to induce higher hypertrophy status. However, their results indicated that treatment of β-glycerophosphate is not essential to induce hypertrophic morphology of chondrocytes. In addition, a recent report showed that dexamethasone has inhibitory effects on hypertrophic induction of MSCs dependent experimental conditions (Shintani et al., 2011). Thus, the use of these agents is not necessarily required to induce hypertrophy. We established a simpler hypertrophy-inducing protocol by withdrawal of β-glycerophosphate and dexamethasone from hypertrophy-inducing culture medium.
Materials and Reagents
- 15 ml sterile conical tubes (Corning, catalog number: 430055 )
- 50 ml sterile conical tubes (Corning, catalog number: 430829 )
- Microslide glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22-230-900 )
- 0.22 μm syringe filter (Pall, catalog number: PN4192 )
- Umbilical cord blood-derived mesenchymal stem cells (Neonatal umbilical cord blood was collected from umbilical veins, with informed maternal consent. For UCB collection, a 16-gauge needle was inserted into the umbilical vein, and UCB was allowed to flow by gravity; See Yang et al. [2004] for the protocol of isolation and maintenance of cells)
- Minimum essential medium-alpha (Thermo Fisher Scientific, GibcoTM, catalog number: 12571 )
- Dulbecco’s phosphate buffered saline without calcium & magnesium (Mediatech, catalog number: 21-031-CVR )
- TrypLETM Express enzyme (Thermo Fisher Scientific, GibcoTM, catalog number: 12605 )
- 4% paraformaldehyde solution (Biosesang, catalog number: P2031 )
- Ethanol
- DAKO EnVisionSystem Peroxidase (DAB) Kit (Agilent Technologies, catalog number: K4006 )
- DAKO Protein block, serum-free (Agilent Technologies, catalog number: X0909 )
- Antibody for type II collagen (EMD Millipore, catalog number: MAB8887 , antibody dilution 1:100)
- Antibody for type X collagen (Thermo Fisher Scientific, Invitrogen, catalog number: MA5-14268 , antibody dilution 1:100)
- Antibody for RUNX2 (Abcam, catalog number: ab76956 , antibody dilution 1:1500)
- Antibody for phospho-GSK-3β(Ser9) (Cell Signaling Technology, catalog number: 5558 , antibody dilution 1:1000)
- Antibody for β-Catenin (Cell Signaling Technology, catalog number: 9582 , antibody dilution 1:1000)
- Antibody for phosphor-Smad1(Ser206) (Cell Signaling Technology, catalog number: 5753 , antibody dilution 1:1000)
- Antibody for GAPDH (Abcam, catalog number: ab9485 , antibody dilution 1:4000)
- Tris-HCl
- BSA
- Tween 20 (Sigma-Aldrich, catalog number: P1379 )
- Mayer’s hematoxylin (Agilent Technologies, catalog number: S3309 )
- Shandon Xylene Substitute (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6764506 )
- Shandon Xylene Substrate Mountant (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 9999122 )
- Fetal bovine serum, certified grade, US origin (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-044 )
- Gentamicin (Thermo Fisher Scientific, GibcoTM, catalog number: 15750 )
- Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965 )
- BMP-6 (R&D Systems, catalog number: 507-BP-020/CF )
- TGF-β3 (R&D Systems, catalog number: 243-B3-002/CF )
- ITS+ (Corning, catalog number: 354352 )
- Ascorbic acid (Sigma-Aldrich, catalog number: A8960 )
- Dexamethasone (Sigma-Aldrich, catalog number: D2915 )
- L-proline (Sigma-Aldrich, catalog number: P5607 )
- Sodium pyruvate (Sigma-Aldrich, catalog number: P8574 )
- Triiodothyronine (Sigma-Aldrich, catalog number: T6397 )
- Primers for Runx2
Forward 5’-CGG AGT GGA TGA GGC AAG AG-3’
Reverse 5’-GGC TCA GGT AGG AGG GGT AA-3’ - Primers for GAPDH
Forward 5’- CTT CTT TTG CGT CGC CAG CCG A-3’
Reverse 5’-TGG CCA GGG GTG CTA AGC AGT-3’ - Complete culture media (see Recipes)
- In vitro chondrogenesis-inducing media (see Recipes)
- In vitro hypertrophy-inducing media (see Recipes)
Equipment
- 175T flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 159910 )
- Centrifuge with swinging-bucket rotor and adaptors for 15 ml conical tubes
- Humidified cell culture incubator set to 37 °C and 5% CO2
- Hemacytometer (VWR, INCYTO C-ChipTM, catalog number: DHCN015 )
- Cryostat (Leica Biosystems Nussloch, model: CM1850 )
- OCT compound (VWR, Tissue-Tek®, catalog number: 25608-930 )
- Microscope for immunohistochemical staining image analysis (Nikon ECLIPSE 50i with DS-Fi1 digital microscope camera head) (Nikon Instruments, model: ECLIPSE 50i )
Software
- ImageJ program
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jeong, S. Y., Lee, M., Choi, S. J., Oh, W. and Jeon, H. B. (2016). In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells. Bio-protocol 6(23): e2057. DOI: 10.21769/BioProtoc.2057.
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分类
干细胞 > 成体干细胞 > 间充质干细胞
细胞生物学 > 细胞分离和培养 > 细胞分化
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