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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   

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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 suggest 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 have 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

  1. 15 ml sterile conical tubes (Corning, catalog number: 430055 )
  2. 50 ml sterile conical tubes (Corning, catalog number: 430829 )
  3. Microslide glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22-230-900 )
  4. 0.22 μm syringe filter (Pall, catalog number: PN4192 )
  5. 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)
  6. Minimum essential medium-alpha (Thermo Fisher Scientific, GibcoTM, catalog number: 12571 )
  7. Dulbecco’s phosphate buffered saline without calcium & magnesium (Mediatech, catalog number: 21-031-CVR )
  8. TrypLETM Express enzyme (Thermo Fisher Scientific, GibcoTM, catalog number: 12605 )
  9. 4% paraformaldehyde solution (Biosesang, catalog number: P2031 )
  10. Ethanol
  11. DAKO EnVisionSystem Peroxidase (DAB) Kit (Agilent Technologies, catalog number: K4006 )
  12. DAKO Protein block, serum-free (Agilent Technologies, catalog number: X0909 )
  13. Antibody for type II collagen (EMD Millipore, catalog number: MAB8887 , antibody dilution 1:100)
  14. Antibody for type X collagen (Thermo Fisher Scientific, Invirtrogen, catalog number: MA5-14268 , antibody dilution 1:100)
  15. Antibody for RUNX2 (Abcam, catalog number: ab76956 , antibody dilution 1:1500)
  16. Antibody for phospho-GSK-3β(Ser9) (Cell Signaling Technology, catalog number: 5558 , antibody dilution 1:1000)
  17. Antibody for β-Catenin (Cell Signaling Technology, catalog number: 9582 , antibody dilution 1:1000)
  18. Antibody for phosphor-Smad1(Ser206) (Cell Signaling Technology, catalog number: 5753 , antibody dilution 1:1000)
  19. Antibody for GAPDH (Abcam, catalog number: ab9485 , antibody dilution 1:4000)
  20. Tris-HCl
  21. BSA
  22. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  23. Mayer’s hematoxylin (Agilent Technologies, catalog number: S3309 )
  24. Shandon Xylene Substitute (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6764506 )
  25. Shandon Xylene Substrate Mountant (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 9999122 )
  26. Fetal bovine serum, certified grade, US origin (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-044 )
  27. Gentamicin (Thermo Fisher Scientific, GibcoTM, catalog number: 15750 )
  28. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965 )
  29. BMP-6 (R&D Systems, catalog number: 507-BP-020/CF )
  30. TGF-β3 (R&D Systems, catalog number: 243-B3-002/CF )
  31. ITS+ (Corning, catalog number: 354352 )
  32. Ascorbic acid (Sigma-Aldrich, catalog number: A8960 )
  33. Dexamethasone (Sigma-Aldrich, catalog number: D2915 )
  34. L-proline (Sigma-Aldrich, catalog number: P5607 )
  35. Sodium pyruvate (Sigma-Aldrich, catalog number: P8574 )
  36. Triiodothyronine (Sigma-Aldrich, catalog number: T6397 )
  37. Primers for Runx2
    Forward 5’-CGG AGT GGA TGA GGC AAG AG-3’
    Reverse 5’-GGC TCA GGT AGG AGG GGT AA-3’
  38. Primers for GAPDH
    Forward 5’- CTT CTT TTG CGT CGC CAG CCG A-3’
    Reverse 5’-TGG CCA GGG GTG CTA AGC AGT-3’
  39. Complete culture media (see Recipes)
  40. In vitro chondrogenesis-inducing media (see Recipes)
  41. In vitro hypertrophy-inducing media (see Recipes)

Equipment

  1. 175T flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 159910 )
  2. Centrifuge with swinging-bucket rotor and adaptors for 15 ml conical tubes
  3. Humidified cell culture incubator set to 37 °C and 5% CO2
  4. Hemacytometer (VWR, INCYTO C-ChipTM, catalog number: DHCN015 )
  5. Cryostat (Leica Biosystems Nussloch, model: CM1850 )
  6. OCT compound (VWR, Tissue-Tek®, catalog number: 25608-930 )
  7. Microscope for immunohistochemical staining image analysis (Nikon ECLIPSE 50i with DS-Fi1 digital microscope camera head) (Nikon Instruments, model: ECLIPSE 50i )

Software

  1. ImageJ program

Procedure

  1. Plating cells for proliferation
    1. Seed 5,000 mesenchymal stem cells per cm2 in a 175T flask with complete culture media (50 ml).
    2. Next day, replace half the medium (25 ml) with fresh complete culture media.
    3. Exchange the culture medium with fresh minimum essential medium-alpha culture media every 2-3 days until cells are at least 70-80% confluent.
    4. At 70-80 % confluency, wash the cells with DPBS once and then detach the cells with TrypLETM Express enzyme.

  2. Induction of in vitro chondrogenesis
    1. For chondrogenesis procedure, wash cells with 30 ml of in vitro chondrogenesis-inducing media (without BMP-6, TGF-β3, and ITS+) twice in a 50 ml tube. For washing, centrifuge suspended cells in the 50 ml tube at 500 x g for 5 min at room temperature.
    2. Count the cells using a hemacytometer.
    3. Suspend the cells with in vitro chondrogenesis-inducing media in a 15 ml tube at a final concentration of 2 x 105/400 μl.
    4. Centrifuge the cells in the 15 ml tube at 500 x g for 5 min at room temperature.
    5. Be careful not to shake the tube, culture the cell pellet with in vitro chondrogenesis-inducing media in the incubator. Loosen the cap of the tube for gas exchange and keep the tube upright (Figure 1).
    6. Culture the cell pellet in in vitro chondrogenesis-inducing medium for 2 weeks. Replace the medium with fresh in vitro chondrogenesis-inducing media twice a week (first media change after 3 days and second media change after 4 days).


      Figure 1. Pellet culture using a 15 ml tube in a CO2 incubator. After centrifugation, the 15 ml tube was carefully moved into a CO2 incubator. During culturing period, the 15 ml tube should be placed upright using a conical tube rack.

  3. Induction of in vitro hypertrophy
    1. For hypertrophy induction, wash the cell pellet with in vitro hypertrophy-inducing media twice.
    2. Add 400 μl of fresh in vitro hypertrophy-inducing media into the 15 ml tube
    3. Culture the cell pellet with in vitro hypertrophy-inducing media for 2 weeks. Replace the medium with fresh in vitro hypertrophy-inducing media twice a week (first media change after 3 days and second media change after 4 days).

  4. Immunohistological analysis
    1. Wash the cell pellet using DPBS and fix using 4% paraformaldehyde solution for 2 h at room temperature.
    2. Freeze the cell pellet in OCT compound at -20 °C. Cut into 5 μm thick cryostat sections and place on a charged glass microslide.
    3. Frozen sections are dehydrated for 10 min in ethanol and briefly immersed in distilled water to remove OCT compound.
    4. To block activity of peroxidase, apply enough Peroxidase Block solution (Component of DAKO DAB Kit) to cover specimen and incubate for 5 min at room temperature. Rinse gently with distilled water.
    5. To block nonspecific antibody-binding sites, apply enough Protein Block solution to cover specimen and incubate for 30 min at room temperature. Rinse gently with distilled water.
    6. After washing, the slides are incubated overnight at 4 °C with primary antibody against type X collagen diluted in 0.05 M Tris-HCl with 1% BSA.
    7. Wash the slides with 500 ml of DPBS (containing 0.1% Tween 20) using a staining jar for 10 min. Repeat one more time.
    8. Apply enough Labelled Polymer-HRP anti-mouse solution (Component of DAKO DAB Kit) to cover specimen and incubate for 30 min at room temperature.
    9. Wash the slides with 500 ml of DPBS (containing 0.1% Tween 20) using a staining jar for 10 min. Repeat one more time.
    10. Wash the slides with 500 ml of DPBS using a staining jar for 10 min. Repeat one more time.
    11. Apply enough DAB + Chromogen solution (Component of DAKO DAB Kit) to cover specimen.
    12. Rinse gently with distilled water.
    13. For counter staining, apply enough Hematoxylin solution to cover specimen and incubate for 1 min.
    14. Wash the slides with 500 ml of distilled water using a staining jar for 2 min. Repeat two more time.
    15. Dehydrate the slides consecutively in 70%, 95%, and 100% ethanol for 1 min in each solution.
    16. Set the slides in 100% ethanol for 2 min and repeat one more time.
    17. Set the slides in Shandon Xylene Substitute for 2 min and repeat two more time.
    18. Mount the slides with Shandon Xylene Substrate Mountant. 

Data analysis

In in vitro chondrogenesis-induction of MSCs, chondrogenic differentiation status was easily estimated by pellet size. After 28 days under chondrogenesis- and hypertrophy-inducing conditions, we measured the size of pellet using an mm scale ruler (Figure 2). The chondrogenic-differentiated (left, Figure 2) and hypertrophic-differentiated (right, Figure 2) pellets are > 1 mm, 0.7-0.8 mm in diameter size, respectively. Our results showed that hypertrophic-differentiated pellet had a smaller size than that of chondrogenic-differentiated pellet of MSCs.


Figure 2. Pellet size comparison. Left pellet of UCB-MSCs was cultured in in vitro chondrogenesis-inducing medium for 4 weeks. Right pellet was cultured in in vitro chondrogenesis-inducing medium for 2 weeks and consecutively cultured in in vitro hypertrophy-inducing medium for 2 weeks. The lower part of picture shows a graduation of mm scale ruler.

To check the hypertrophic induction of pellets under each conditions, we detected expression of type X collagen by immunohistochemical staining (Figure 3). The pellets obtained under chondrogenic-inducing conditions showed a weak positive reactivity to type X collagen antibody (Figure 3A). The pellets cultured in hypertrophy-inducing medium showed a dark brown color that indicated high expression levels of type X collagen (Figure 3B). Collectively, we could observe a higher expression of type X collagen in hypertrophic-differentiated pellet.


Figure 3. Type X collagen expression in pellets. To validate the induction of hypertrophy in the hUCB-MSCs, the pellets were sectioned and expression of collagen type X was detected by immunohistochemical staining. A. For 4 weeks, pellet was induced into chondrogenic differentiation. B. After 2 weeks chondrogenic differentiation, pellet was induced into hypertrophic differentiation for 2 weeks. Scale bar = 250 μm.

For additional validation of hypertrophic induction, we evaluated the expression of the hypertrophy marker, runt-related transcription factor 2 (Runx2), using Western blotting and RT-PCR (Figure 4). Under hypertrophy-inducing conditions, the protein and mRNA expression level of the osteogenic transcription factor Runx2 were 2.4-fold and 3.1-fold higher than those in pellets under chondrogenic-inducing conditions, respectively. The expression of Runx2 was quantified using ImageJ program.


Figure 4. Osteogenic transcription factor expression in pellets. To validate of hypertrophic differentiation in UCB-MSCs, the expression of transcription factor, Runx2, was assessed using Western blotting (A) and reverse transcriptase polymerase chain reaction (B).

To characterize the signal pathway related to hypertrophic induction by our protocol, we evaluated the expression of phosphorylated (p)GSK-3β, β-catenin, and pSMAD1 in the WNT/β-catenin and TGF-β/BMP osteogenic signaling pathways using Western blotting (Figure 5). In hypertrophic-differentiated pellets, pGSK-3β expression was 3.6-fold higher than that of pellet under chondrogenic-inducing conditions. The β-catenin and pSMAD1 expression levels were 2.1-fold and 2.8-fold higher, respectively, than those in chondrogenic-differentiated pellets. The expression of each proteins were quantified using ImageJ program.


Figure 5. Osteogenic signaling pathway during hypertrophic differentiation of hUCB-MSCs. After hypertrophic cultivation, the pellets were harvested and lysed. The expression of GSK-3β, β-catenin, and SMAD1 was analyzed by Western blotting.

Notes

  1. If you observe a lower hypertrophy degree of pellets, increase number of washing step in induction of in vitro hypertrophy.
  2. Use fresh ITS+ supplement within two months of production.
  3. Avoid repeated freezing and thawing of T3.

Recipes

  1. Complete culture media (Prepared media can be kept at 4 °C for 2 weeks)
    Minimium essential medium-alpha
    10% fetal bovine serum (FBS)
    50 μg/ml gentamicin
  2. In vitro chondrogenesis-inducing media (Use prepared media immediately for cytokine activity)
    500 ml Dulbecco’s modified Eagle medium (DMEM)
    500 ng/ml BMP-6
    10 ng/ml TGF-β3
    1% ITS+
    50 μg/ml ascorbic acid
    0.6 μg/ml dexamethasone
    40 μg/ml L-proline
    100 μg/ml sodium pyruvate
    50 μg/ml gentamicin
  3. In vitro hypertrophy-inducing media (Use prepared media immediately for cytokine activity)
    500 ml Dulbecco’s modified Eagle medium (DMEM)
    10% ITS+
    50 μg/ml ascorbic acid
    40 μg/ml L-proline
    1 nM Triiodothyronine
    50 μg/ml gentamicin

Acknowledgments

This protocol was modified from our previous work (Jeong et al., 2015) and method of Jason A. Burdick group (Bian et al., 2012) and was supported by the National Research Foundation (2015M3D6A1065098).

References

  1. Bian, L., Zhai, D. Y., Zhang, E. C., Mauck, R. L. and Burdick, J. A. (2012). Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels. Tissue Eng Part A 18(7-8): 715-724.
  2. Guo, J., Chung, U., Yang, D., Karsenty, G., Bringhurst, F. R., Kronenberg H. M. (2006). PTH/PTHrP receptor delays chondrocyte hypertrophy via both Runx2-dependent and –independent pathways. Dev Biol 292(1): 116-128.
  3. Jeong, S. Y., Ha, J., Lee, M., Jin, H. J., Kim, D. H., Choi, S. J., Oh, W., Yang, Y. S., Kim, J. S., Kim, B. G., Chang, J. H., Cho, D. H. and Jeon, H. B. (2015). Autocrine action of thrombospondin-2 determines the chondrogenic differentiation potential and suppresses hypertrophic maturation of human umbilical cord blood-derived mesenchymal stem cells. Stem Cells 33(11): 3291-3303.
  4. Mueller, M. B. and Tuan, R. S. (2008). Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis Rheum 58(5): 1377-1388.
  5. Shintani, N. and Hunziker, E. B. (2011). Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor. Eur Cell Mater 22: 302-319; discussion 319-320.
  6. Yang, S. E., Ha, C. W., Jung, M., Jin, H. J., Lee, M., Song, H., Choi, S., Oh, W. and Yang, Y. S. (2004). Mesenchymal stem/progenitor cells developed in cultures from UC blood. Cytotherapy 6(5): 476-486.

简介

为了研究软骨形成性肥大的基础机制,我们需要适当的体外间充质干细胞(MSC)的增生模型。该协议描述了我们定义的诱导人脐带血衍生的MSC(hUCB-MSC)的体外软骨形成性肥大的方法。通过将甲状腺激素(T3;三碘甲状腺原氨酸)和最小成骨诱导因子添加到培养基中,我们可以在体外诱导hUCB-MSCs的增生。使用免疫组织化学分析,Western印迹和逆转录酶聚合酶链反应验证肥大性诱导。
关键词:间充质干细胞,体外软骨形成性肥大,成软骨分化,三碘甲腺原氨酸;

[背景] 几项研究表明肥大相关基因的表达,包括X型胶原,碱性磷酸酶和甲状旁腺激素相关蛋白受体(PTHrPR)软骨形成分化。这些基因的表达表明MSC中的软骨形成分化不可避免地诱导软骨形成性肥大阶段,这是典型的软骨内骨化。此外,已知甲状旁腺激素相关蛋白(PTHrP)途径的活化诱导MSC转变成成骨表型(Guo等人,2006)。基于这些报告,Mueller等人。表明去除TGF-β,低浓度的地塞米松和加入三碘甲状腺原氨酸(T3)对于骨髓来源的MSC的肥大诱导是重要的(Mueller等人,2008)。在Mueller的方案中,β-甘油磷酸酯和地塞米松是诱导更高肥大状态所必需的。然而,他们的结果表明,β-甘油磷酸盐的治疗对于诱导软骨细胞的肥大形态不是必需的。此外,最近的报告显示地塞米松对MSCs依赖性实验条件的肥大诱导具有抑制作用(Shintani等人,2011)。因此,使用这些药物不一定需要诱导肥大。我们通过从肥大诱导培养基中撤出β-甘油磷酸酯和地塞米松来建立更简单的肥大诱导方案。

关键字:间充质干细胞, 体外软骨形成性肥大, 软骨形成分化, 三碘甲状腺原氨酸

材料和试剂

  1. 15ml无菌锥形管(Corning,目录号:430055)
  2. 50ml无菌锥形管(Corning,目录号:430829)
  3. 微滑玻璃(Thermo Fisher Scientific,Fisher Scientific,目录号:22-230-900)
  4. 0.22μm注射器过滤器(Pall,目录号:PN4192)
  5. 脐带血源性间充质干细胞(从脐静脉采集新生儿脐带血,并通知母体同意。对于UCB收集,将16号针插入脐静脉,并且通过重力使UCB流动。 Yang等人[2004]关于细胞分离和维持的方案)
  6. 最小必需培养基(Thermo Fisher Scientific,Gibco TM ,目录号:12571)
  7. Dulbecco磷酸盐缓冲盐水,无钙&镁(Mediatech,目录号:21-031-CVR)
  8. TrypLE TM超高速缓冲液酶(Thermo Fisher Scientific,Gibco TM ,目录号:12605)
  9. 4%多聚甲醛溶液(Biosesang,目录号:P2031)
  10. 乙醇
  11. DAKO EnVisionSystem过氧化物酶(DAB)试剂盒(Agilent Technologies,目录号:K4006)
  12. DAKO Protein block,无血清(Agilent Technologies,目录号:X0909)
  13. II型胶原抗体(EMD Millipore,目录号:MAB8887,抗体稀释度1:100)
  14. X型胶原抗体(Thermo Fisher Scientific,Invirtrogen,目录号:MA5-14268,抗体稀释度1:100)
  15. RUNX2抗体(Abcam,目录号:ab76956,抗体稀释度1:1500)
  16. 磷酸-GSK-3β(Ser9)的抗体(Cell Signaling Technology,目录号:5558,抗体稀释度1:1000)
  17. β-联蛋白的抗体(Cell Signaling Technology,目录号:9582,抗体稀释度1:1000)
  18. 磷-SMad1(Ser206)的抗体(Cell Signaling Technology,目录号:5753,抗体稀释度1:1000)
  19. GAPDH抗体(Abcam,目录号:ab9485,抗体稀释度1:4000)
  20. Tris-HCl
  21. BSA
  22. 吐温20(Sigma-Aldrich,目录号:P1379)
  23. Mayer的苏木精(安捷伦科技公司,目录号:S3309)
  24. Shandon二甲苯替代品(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:6764506)
  25. Shandon二甲苯底物封固剂(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:9999122)
  26. 胎牛血清,认证级,美国产地(Thermo Fisher Scientific,Gibco TM ,目录号:16000-044)
  27. 庆大霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15750)
  28. Dulbecco's改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:11965)
  29. BMP-6(R& D Systems,目录号:507-BP-020/CF)
  30. TGF-β3(R& D Systems,目录号:243-B3-002/CF)
  31. ITS +(Corning,目录号:354352)
  32. 抗坏血酸(Sigma-Aldrich,目录号:A8960)
  33. 地塞米松(Sigma-Aldrich,目录号:D2915)
  34. L-脯氨酸(Sigma-Aldrich,目录号:P5607)
  35. 丙酮酸钠(Sigma-Aldrich,目录号:P8574)
  36. 三碘甲状腺原氨酸(Sigma-Aldrich,目录号:T6397)
  37. Runx2的引文
    正向5'-CGG AGT GGA TGA GGC AAG AG-3'
    反向5'-GGC TCA GGT AGG AGG GGT AA-3'
  38. GAPDH的入门
    正向5'-CTT CTT TTG CGT CGC CAG CCG A-3'
    反向5'-TGG CCA GGG GTG CTA AGC AGT-3'
  39. 完成培养基(见配方)
  40. 体外软骨形成诱导培养基(参见配方)
  41. 体外肥大诱导培养基(参见食谱)

设备

  1. 175T烧瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:159910)
  2. 带有摆动斗转子和适配器的离心机用于15 ml锥形管
  3. 将加湿的细胞培养箱设定为37℃和5%CO 2/h
  4. 血细胞计数器(VWR,INCYTO C-Chip TM ,目录号:DHCN015)
  5. 低温恒温器(Leica Biosystems Nussloch,型号:CM1850)
  6. OCT化合物(VWR,Tissue-Tek ,目录号:25608-930)
  7. 用于免疫组织化学染色图像分析的显微镜(具有DS-Fi1数字显微镜照相机头的Nikon ECLIPSE 50i)(Nikon Instruments,型号:ECLIPSE 50i)

软件

  1. ImageJ程序

程序

  1. 电镀细胞增殖
    1. 在具有完全培养基(50ml)的175T烧瓶中种子5000个间充质干细胞/cm 2。
    2. 第二天,用新鲜的完全培养基替换一半培养基(25ml)。
    3. 每2-3天更换培养基与新鲜最低必需培养基α培养基,直到细胞至少70-80%汇合。
    4. 在70-80%融合时,用DPBS洗涤细胞一次,然后用TrypLE TM Suppress Express酶分离细胞。

  2. 诱导体外软骨形成
    1. 对于软骨形成程序,用30ml的体外软骨形成诱导培养基(不含BMP-6,TGF-β3和ITS +)在50ml管中洗涤细胞两次。为了洗涤,在室温下将悬浮细胞在500ml管中以500×g离心5分钟。
    2. 使用血球计数器计数细胞
    3. 将细胞用体外软骨诱导介质悬浮在15ml试管中,终浓度为2×10 5/u /400μl。
    4. 在室温下以500×g离心5分钟,将细胞在15ml管中离心。
    5. 小心不摇动试管,用培养箱中的体外软骨诱导培养基培养细胞沉淀。松开用于气体交换的管帽,并保持管直立(图1)。
    6. 在体外软骨形成诱导培养基中将细胞沉淀物培养2周。用新鲜的体外软骨形成诱导培养基每周两次更换培养基(3天后更换培养基,4天后更换培养基)。


      图1.在CO 2培养箱中使用15ml管的沉淀培养物。离心后,将15ml管小心地移至CO 2/>孵化器。在培养期间,15ml试管应使用锥形管架直立放置
  3. 诱导体外肥大
    1. 对于肥大诱导,用体外的肥大诱导培养基洗涤细胞沉淀两次
    2. 将400μl新鲜的体外增殖诱导培养基加入15ml管中
    3. 用体外培养的肥大诱导培养基培养细胞沉淀2周。每周两次用新鲜的体外增强诱导培养基替换培养基(3天后第一次培养基更换,4天后第二次培养基更换)。

  4. 免疫组织化学分析
    1. 使用DPBS洗涤细胞沉淀,并使用4%多聚甲醛溶液在室温下固定2小时。
    2. 在-20℃下冻存OCT化合物中的细胞沉淀。切成5μm厚的低温恒温器切片并置于带电玻璃微滑片上。
    3. 冷冻切片在乙醇中脱水10分钟,并简单地浸入蒸馏水中以除去OCT化合物
    4. 为了阻断过氧化物酶的活性,应用足够的过氧化物酶封闭溶液(DAKO DAB试剂盒的组分)覆盖标本并在室温下孵育5分钟。用蒸馏水轻轻冲洗。
    5. 为了阻断非特异性抗体结合位点,应用足够的蛋白质封闭溶液覆盖标本,并在室温下孵育30分钟。用蒸馏水轻轻冲洗。
    6. 洗涤后,将载玻片在4℃下与在含有1%BSA的0.05M Tris-HCl中稀释的针对X型胶原蛋白的一抗孵育过夜。
    7. 用500ml DPBS(含有0.1%Tween 20)用染色罐洗涤载玻片10分钟。重复一次。
    8. 应用足够的标记聚合物HRP抗小鼠溶液(DAKO DAB套件的组件)覆盖标本,并在室温下孵育30分钟。
    9. 用500ml DPBS(含有0.1%Tween 20)用染色罐洗涤载玻片10分钟。重复一次。
    10. 用500ml DPBS用染色罐洗涤载玻片10分钟。重复一次。
    11. 施用足够的DAB +色原溶液(DAKO DAB试剂盒的组分)以覆盖样品
    12. 用蒸馏水轻轻冲洗。
    13. 对于逆染色,应用足够的苏木精溶液覆盖标本并孵育1分钟
    14. 使用染色罐用500ml蒸馏水洗涤载玻片2分钟。重复两次。
    15. 使载玻片在70%,95%和100%乙醇中连续脱水1分钟。
    16. 将载玻片置于100%乙醇中2分钟,重复一次。
    17. 将幻灯片放在Shandon Xylene Substitute中2分钟,然后重复两次。
    18. 使用Shandon二甲苯底物Mountant安装载玻片。

数据分析

在体外软骨形成诱导的MSC中,软骨形成分化状态容易通过颗粒大小估计。在软骨形成和肥大诱导条件下28天后,我们使用mm刻度尺测量小球的尺寸(图2)。软骨形成分化的(左,图2)和肥厚分化的(右,图2) 1mm,直径尺寸为0.7-0.8mm。我们的研究结果表明,肥厚分化的沉淀小于软骨形成分化的MSCs的沉淀。


图2.丸粒尺寸比较。将UCB-MSC的左侧沉淀在体外软骨形成诱导培养基中培养4周。将右小丸在体外软骨形成诱导培养基中培养2周,并在体外增殖诱导培养基中连续培养2周。图片的下部显示mm刻度尺的刻度。

为了检查在每种条件下颗粒的肥大诱导,我们通过免疫组织化学染色检测X型胶原的表达(图3)。在软骨形成诱导条件下获得的颗粒显示对X型胶原抗体的弱阳性反应性(图3A)。在肥大诱导培养基中培养的沉淀显示暗褐色,表明X型胶原的高表达水平(图3B)。总之,我们可以观察到在肥大分化的沉淀中X型胶原蛋白的更高表达

为了验证hUCB-MSC中肥大的诱导,将切片切片并通过免疫组织化学染色检测X型胶原蛋白的表达。 A. 4周,将沉淀诱导成软骨形成分化。 B.在软骨形成分化2周后,将沉淀诱导为肥大分化2周。比例尺= 250μm。

对于肥大诱导的其他验证,我们使用蛋白质印迹和RT-PCR(图4)评估了肥大标记物,表达相关的转录因子2(Runx2)的表达。在肥大诱导条件下,成骨转录因子Runx2的蛋白质和mRNA表达水平分别是软骨形成诱导条件下的沉淀中的2.4倍和3.1倍。使用ImageJ程序定量Runx2的表达

为了验证UCB-MSC中的肥大分化,使用蛋白质印迹(A)和逆转录酶聚合酶链反应(A)来评估转录因子Runx2的表达。 B)。

为了表征与我们的协议肥厚诱导相关的信号通路,我们评估磷酸化(p)GSK-3β,β-连环素和pSMAD1在WNT /β-连环蛋白和TGF-β/BMP成骨信号通路中的表达使用西方印迹(图5)。在肥大分化的小丸中,pGSK-3β表达比软骨形成诱导条件下的小丸高3.6倍。 β-连环蛋白和pSMAD1表达水平分别比软骨形成分化的小丸中的高2.1倍和2.8倍。使用ImageJ程序定量每种蛋白质的表达

图5.hUCB-MSC的肥大分化过程中的成骨信号传导途径。在肥大培养后,收获并裂解沉淀。通过Western印迹分析GSK-3β,β-连环蛋白和SMAD1的表达。

笔记

  1. 如果观察到较低的球粒肥大程度,则在体外诱导的肥大中增加洗涤步骤的数量。
  2. 在生产后两个月内使用新鲜的ITS +补充剂。
  3. 避免T3的反复冻融。

食谱

  1. 完全培养基(制备的培养基可以在4℃保持2周)
    Minimium essential medium-alpha
    10%胎牛血清(FBS)
    50μg/ml庆大霉素
  2. 体外软骨形成诱导培养基(立即使用准备的培养基用于细胞因子活性)
    500ml Dul becco改良的Eagle培养基)
    500ng/ml BMP-6
    10ng/ml TGF-β3 1%ITS +
    50μg/ml抗坏血酸
    0.6μg/ml地塞米松 40μg/ml L-脯氨酸 100μg/ml丙酮酸钠 50μg/ml庆大霉素
  3. 体外肥大诱导培养基(立即使用准备的培养基用于细胞因子活性)
    ulbecco改良的Eagle培养基(DMEM培养基) 500ml 致谢


    该协议从我们先前的工作(Jeong等人,2015)和Jason A.Burdick小组(Bian等人,2012)的方法修改并且得到支持国家研究基金会(2015M3D6A1065098)。

    参考文献

    1. Bian,L.,Zhai,DY,Zhang,EC,Mauck,RL和Burdick,JA(2012)。  血小板反应蛋白的自分泌作用-2确定软骨形成细胞分化潜能并抑制人脐带血源性间充质干细胞的肥大成熟。干细胞 33(11):3291-3303。
    2. Mueller,MB和Tuan,RS(2008)。  功能表征人间质干细胞软骨形成中的肥大。关节炎风湿 58(5):1377-1388。
    3. Shintani,N.和Hunziker,EB(2011)。  地塞米松对间充质基质细胞软骨形成的差异作用:微环境,组织起源和生长因子的影响.Eur Cell Mater 22:302-319;讨论319-320。
    4. Yang,SE,Ha,CW,Jung,M.,Jin,HJ,Lee,M.,Song,H.,Choi,S.,Oh,W.and Yang,YS(2004) "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15512914"target ="_ blank">来自UC血液的培养物中发育的间充质干/祖细胞。 Cytotherapy 6(5):476-486。
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引用:Jeong, S. Y., Lee, M., Choi, S. J., Oh, W. and Jeon, H. (2016). In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells. Bio-protocol 6(23): e2057. DOI: 10.21769/BioProtoc.2057.
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