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Measurement of Mesenchymal Stem Cells Attachment to Endothelial Cells
间充质干细胞附着于内皮细胞的测定   

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Stem Cells
Nov 2015

Abstract

Mesenchymal stem cells (MSCs) have shown profound therapeutic potential in tissue repair and regeneration. However, recent studies indicate that MSCs are largely entrapped in lungs after intravenous delivery and die shortly. The underlying mechanisms have been poorly understood. We have provided evidence to show that excess expression and activation of integrins in culture-expanded MSCs is a critical cause of MSCs adhesion to endothelial cells of the lung microarteries resulting in the entrapment of the cells (Wang et al., 2015). Therefore, it may be meaningful to test the adhesive ability of MSCs to endothelial cells in vitro before intravenous administration to avoid their lung vascular obstructions. Here we report a simple method to measure MSCs attachment to endothelial cells.

Keywords: Mesenchymal stem cells (间充质干细胞), Endothelial cells (内皮细胞), Cell adhesion (细胞粘附), Integrins ( 整合素)

Background

Mesenchymal stem cells (MSCs) are emerging as an extremely promising therapeutic agent, and numerous clinical trials for a variety of diseases are underway (Salem and Thiemermann, 2010). Intravenous infusion of MSCs has been a popular delivery route for MSCs therapies in recent clinical trials because of its convenience and safety (Wu and Zhao, 2012). However, increasing evidence has indicated that MSCs cause considerable vascular obstructions following intravascular injection. Upon intravenous infusion, more than 80% of MSCs are entrapped in the lungs, and only less than 1% of MSCs are detected in the acute ischemic heart or brain (Lee et al., 2009; Toma et al., 2009).

Recent studies suggest that MSCs are largely stuck in the precapillary microvessel after intravenous administration and most of them die shortly of local ischemia (Toma et al., 2009). Therefore, it has become an increasing concern over the safety and efficacy of intravascularly administered MSCs. The mechanisms of vascular obstructions of MSCs have not been fully understood.

Our data have indicated that excess expression of integrins in MSCs is an important cause for their lung entrapment, which leads to attachment of the cells to endothelial cells in the lungs, thus reducing their trafficking and homing to inflamed tissues. Functional blockade of integrins in MSCs, especially after integrin β1 blockade, significantly decreases their attachment to endothelial cells, resulting in a substantial reduction of MSCs entrapped in the lungs, elevated levels of circulating MSCs in the blood, and increased engraftment of the cells to inflamed tissues (Wang et al., 2015). Here we provide a methodology for measuring the attachment of MSCs to endothelial cells in vitro.

Materials and Reagents

  1. 24-well plates (Corning, Costar®, catalog number: 3524 )
  2. 12-well plates (Corning, catalog number: 3512 )
  3. 10 cm plates (Corning, catalog number: 353003 )
  4. Pipette (Corning, catalog number: 4100 )
  5. 15- or 50-ml conical centrifuge tubes (Corning, catalog number: 430052 , 430828 )
  6. Human bone marrow-derived MSCs (Lonza, catalog number: PT-2501 )
  7. Human umbilical vein endothelial cells (HUVECs) (Lonza, catalog number: CC-2517 )
  8. Human lung microvascular endothelial cells (HMVECs-L) (Lonza, catalog number: CC-2527 )
  9. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41966052 )
  10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco TM, catalog number: 10270106 )
  11. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  12. EGM-2 MV SingleQuot Kit Supplements & Growth Factors (Lonza, catalog number: CC-4147 )
  13. EGMTM-2 MV BulletKitTM Medium (Lonza, catalog number: CC-3202 )
  14. Endothelial basal medium-2 (EBM-2) (Lonza, catalog number: CC-3156 )
  15. Fibronectin (Sigma-Aldrich, catalog number: F0556 )
  16. Sterile phosphate buffer saline (PBS), pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 20012068 )
  17. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025092 )
  18. Vitronectin (Sigma-Aldrich, catalog number: V8379 )
  19. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  20. Lipophilic fluorophore 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) (Sigma-Aldrich, catalog number: 468495 )
  21. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  22. Mouse anti-human integrin β1 antibody (Merck, catalog number: MAB1987 )
  23. Mouse anti-human integrin β1 activated antibody (Merck, catalog number: MAB2079Z )
  24. Anti-human integrin α5 (Merck, catalog number: MAB1956Z )
  25. Anti-human CD51/CD61 (integrin αVβ3) purified (Thermo Fisher Scientific, eBioscienceTM, catalog number: 14-0519 )
  26. Mouse isotype IgG (Sigma-Aldrich, catalog number: M6898 )
  27. Tumor necrosis factor-α (TNF-α) (PeproTech, catalog number: 300-01A )
  28. Medium 199 (Sigma-Aldrich, catalog number: M4530 )
  29. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  30. Ficoll-paque Plus solution (GE Healthcare, catalog number: 17-1440-02 )
  31. Dead Cell Apoptosis Kit with Annexin V Alexa FluorTM 488 & Propidium Iodide (PI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: V13241 )
  32. Human leucocytes (see Recipes)

Equipment

  1. Traceable Nano Timer (Fisher Scientific, catalog number: 14-649-83 )
  2. Centrifuge (Eppendorf, catalog number: 5810 R )
  3. Hemocytometer (Hirschmann Instruments, catalog number: 8100103 )
  4. CO2 incubator (Panasonic, model: MCO-19AIC (UV))
  5. Fluorescence microscope (Leica Microsystems, catalog number: Leica DMI6000 B )

Procedure

  1. Cell culture and single cell suspensions
    1. hMSCs culture
      Human bone marrow-derived MSCs (hMSCs) were purchased from Lonza. Culture hMSCs in a growth medium consisting of DMEM, 10% FBS, and 1% penicillin and streptomycin for 72 h.
    2. Culture of human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVECs-L)
      1. Culture HUVECs in endothelial growth medium (EGM)-2 plus 2% FBS and supplements for 72 h.
      2. Culture HMVECs-L in EGMTM-2 MV BulletKitTM Medium for 72 h.
        Note: EGMTM-2 MV BulletKitTM Medium includes endothelial basal medium-2 (EBM-2, Lonza) and the following growth supplements: human epidermal growth factor (hEGF); vascular endothelial growth factor (VEGF); R3-insulin-like growth factor-1 (R3-IGF-1); ascorbic acid; hydrocortisone; human fibroblast growth factor-Beta (hFGF-β); fetal bovine serum (FBS) and gentamicin/amphotericin-B (GA).

  2. Optimization of blocking antibody concentrations and cell adhesion assay
    1. Coat 24-well plates with 10 μg/ml fibronectin (diluting in sterile HBSS or PBS solutions) or 0.4-1 μg/ml vitronectin (diluting in sterile water) for 1 h at room temperature.
    2. Remove fibronectin or vitronectin buffer without washing, air dry for 1-2 h in a hood.
    3. Block the plates with 3% BSA in PBS for 1 h at 37 °C.
    4. Gently wash the plates with PBS once, air dry in a hood.
    5. Use 3% BSA in PBS alone-coated wells as a negative control.
    6. Pre-label hMSCs and freshly isolated human peripheral blood leucocytes with fluorescent label DiI (Lipophilic fluorophore 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) (Wu et al., 2006).
      1. For cells in suspension, wash with PBS and incubate with DiI at a concentration 2 μg/ml in DMEM basal medium (serum-free) for 20-30 min at 37 °C. Then incubate with fresh growth medium (DMEM supplemented with 10% FBS) for 30 min. After two washes with PBS (400 x g, 5 min), re-suspend the cells in fresh growth medium.
      2. For cells in adhesion, wash with PBS and incubate with DiI at a concentration of 2 μg/ml in DMEM basal medium (serum-free) for 20-30 min at 37 °C. Then replace the medium with fresh growth medium and incubate for 30 min. Trypsinize the cells with 0.25% trypsin-EDTA for 2 min and suspend cells in DMEM supplemented with 10% FBS.
    7. Pre-incubate hMSCs in suspension with different concentrations of integrin β1, integrin α5 or integrin αVβ3 blocking to determine the lowest concentrations of the blocking antibodies that achieve maximum inhibition to hMSCs attachment (Wang et al., 2015).
      1. Incubate hMSCs with anti-integrin β1 blocking mAb at concentrations of 0, 2, 4, 6, 8, 10 and 20 μg/ml in DMEM containing 2% FBS for 30 min at 37 °C.
      2. Incubate hMSCs with anti-integrin α5 blocking mAb at concentrations of 0, 2.5, 5, 10, 20 and 40 μg/ml in DMEM containing 2% FBS for 30 min at 37 °C.
      3. Incubate hMSCs with anti-integrins αVβ3 blocking mAb at concentrations of 0, 2.5, 5, 10 and 20 μg/ml for 30 min at 37 °C.
      4. Incubate hMSCs with isotype IgG antibody as a control.
      5. Wash once with PBS after incubations.
    8. Seed 5 x 104 per well of hMSCs after blockade with integrin β1, integrin α5 or isotype control IgG, or 5 x 104 per well freshly isolated human peripheral blood leucocytes on fibronectin-coated plates.
    9. Seed 5 x 104 hMSCs per well after blockade with integrin αVβ3 or isotype control IgG on vitronectin-coated plates.
    10. Incubate the cells in DMEM plus 10% FBS for 30, 60, 90 and 120 min at 37 °C.
    11. Collect non-adherent cells every 30 min (wash once with PBS).
    12. Centrifuge non-adherent cells at 400 x g, 5 min.
    13. Count the non-adherent cells with a hemocytometer.
    14. Photograph the DiI-labelled hMSCs in attachment by fluorescence microscopy.
    15. The experiment must be performed in quadruplicate for each variable.

  3. hMSCs attachment to endothelial cells
    1. Culture HUVECs and HMVECs-L in 12-well plates with endothelial growth medium (EGM)-2 and EGMTM-2 MV BulletKitTM Medium, respectively, to confluence. The confluence density is approximate percentage of 80%-90%. Cells were treated with or without of 10 ng/ml TNF-α for 24 h immediately before the addition of hMSCs or leucocytes. TNF-α treatment, which is able to activate endothelial cells (Ko et al., 2009), increased the attachment of hMSCs and leucocytes to endothelial cells.
    2. Isolate leucocytes from freshly human peripheral blood (Boyum, 1976; Chacko et al., 2013).
    3. Prepare hMSCs: trypsinize hMSCs from 80%-90% confluent 10 cm plates.
    4. Label hMSCs and leucocytes with fluorescent DiI.
    5. Suspend DiI-labeled leucocytes at a density of 1 x 106 in M199 plus 0.1% BSA.
    6. Suspend DiI-labeled hMSCs at a density of 0.5 x 106 in DMEM plus 0.1% BSA.
    7. Find out the appropriate time-point to detect hMSCs attachment to endothelial cells.
      1. Add DiI-labeled hMSCs to 80%-90% confluent monolayers of HUVECs and HMVECs-L in 12-well plates and incubate cells at 37 °C and 5% CO2.
      2. Detect hMSCs attached to endothelial cells every 15 min for 60 min.
      3. Incubation for 30 min is normally sufficient for strong adhesion of hMSCs to endothelial cells.
    8. Incubate hMSCs which have been treated with blocking antibodies against integrin β1 at 4 μg/ml, integrin α5 at 10 μg/ml, integrin αVβ3 at 5 μg/ml, or control IgG at 10 μg/ml for 30 min at 37 °C.
    9. Wash samples once with PBS.
    10. Re-suspend samples with fresh growth medium and incubate for 30 min at 37 °C.
    11. Add cells to 80%-90% confluent monolayers of HUVECs or HMVECs-L in 12-well plates.
    12. Incubate culture seeded with leucocytes for 1 h, and culture seeded with hMSCs for 30 min.
    13. Wash the cultures twice with HBSS to remove non-adherent cells.
    14. Count the non-adherent cells with a hemocytometer.
    15. Fix the adherent cells with 1% PFA.
    16. Acquire images of adherent cells under a fluorescence microscope (200x), and 10 fields per well were imaged (Figure 1).
    17. Count the number of cells per field. Six duplicate wells are used for each condition.


      Figure 1. Adhesion of hMSCs to HMVEC-L. A. DiI-labeled single hMSCs derived from monolayers (2D) were pre-incubated with blocking antibodies against integrin α5, integrin β1, or integrin αVβ3, seeded on HMVECs-L monolayers and incubated for 30 min. hMSCs and leucocytes pre-incubated with isotype IgG were used as controls. Scale bars = 250 µm. B. The non-adherent cells were removed and counted, and the adherent DiI-hMSCs were photographed. Six replicate wells were used for each condition, and the experiment was repeated three times, * P < 0.05; ** P < 0.01. Abbreviation: MSC, mesenchymal stem cells.

Data analysis

  1. Adherent cells (%) = (number of attached cells)/[total cell number] x 100%.
  2. The lowest concentrations of the blocking antibodies that achieved maximum inhibition to hMSCs attachment were chosen for experiments: integrin β1 at 4 μg/ml, integrin α5 at 10 μg/ml, and integrin αVβ3 at 5 μg/ml.

Notes

  1. Incubation for 30 min was the best time point for detection of hMSCs attachment to endothelial cells. Shorter incubation reduced the number of hMSCs in attachment, but longer incubation did not increase the number of hMSCs attached to endothelial cells.
  2. Pre-incubation with blocking antibodies against integrin β1, integrin α5, or integrin αVβ3 significantly decreased the number of hMSCs attached to the endothelial cells (P < 0.05), with integrin β1 blockade resulting in the most evident reduction (by ~40%).
  3. The non-adherent cells were analyzed using Dead Cell Apoptosis Kit by flow cytometry. At the above concentrations, blockade of integrin β1, but not integrin α5 nor integrin αVβ3 caused a modest increase of hMSCs apoptosis (2-3%) (P < 0.05).
  4. Our in vitro attachment experiments showed that functional blockade of these integrins in hMSCs significantly reduced their attachment to endothelial monolayers, but there were still more hMSCs attached to endothelial cells than leucocytes did. These results suggest that more adhesive molecules may be involved in hMSCs attachment to endothelial cells (Schenkel et al., 2004; Wang et al., 2015).
  5. We detected fibronectin, the major ligand of integrin α5β1, and vitronectin, the major ligand of integrin αVβ3, on cultured HMVECs-L and the normal endothelium in lungs.

Recipes

  1. Human leucocytes
    Note: Leucocytes were isolated from the whole peripheral blood using Ficoll-paque Plus solution.
    1. Place fresh blood into 15- or 50-ml conical centrifuge tubes
    2. Use a sterile pipet to add an equal volume of room-temperature PBS and mix well
    3. Slowly pipette the Ficoll-paque Plus solution by placing the tip of the pipette at the bottom of the sample tube containing blood/PBS mixture. Use 5 ml Ficoll-paque solution per 10 ml blood/PBS mixture
    4. Centrifuge for 30 min at 400 x g, 4 °C
    5. Use a sterile pipet, remove the upper layer that contains the plasma and most of the platelets
    6. Use another pipet to transfer the mononuclear cell layer to another centrifuge tube
    7. Wash cells by adding excess HBSS (~3 times the volume of the mononuclear cell layer) and centrifuge for 5 min at 400 x g (1,300 rpm), 4 °C
    8. Remove supernatant, re-suspend cells in HBSS, and repeat the wash once to remove most of the platelets

Acknowledgments

This protocol was adapted from previously published papers (Luu et al., 2013; Wang et al., 2015; Wu et al., 2006). This work was supported by grants from the National Natural Science Foundation of China Natural Science Foundation of China (Nos. 31371404, 31571429), Natural Science Foundation of Guangdong (2015A030311041), and Shenzhen Science and Technology Innovation Committee (JCY20160301150838144).
Disclosure of potential conflicts of interest: The authors indicate no potential conflicts of interest.

References

  1. Bartosh, T. J., Ylostalo, J. H., Mohammadipoor, A., Bazhanov, N., Coble, K., Claypool, K., Lee, R. H., Choi, H. and Prockop, D. J. (2010). Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107(31): 13724-13729.
  2. Boyum, A. (1976). Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol Suppl 5: 9-15.
  3. Chacko, B. K., Kramer, P. A., Ravi, S., Johnson, M. S., Hardy, R. W., Ballinger, S. W. and Darley-Usmar, V. M. (2013). Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood. Lab Invest 93(6): 690-700.
  4. Guo, L., Zhou, Y., Wang, S. and Wu, Y. (2014). Epigenetic changes of mesenchymal stem cells in three-dimensional (3D) spheroids. J Cell Mol Med 18(10): 2009-2019.
  5. Ip, J. E., Wu, Y., Huang, J., Zhang, L., Pratt, R. E. and Dzau, V. J. (2007). Mesenchymal stem cells use integrin β1 not CXC chemokine receptor 4 for myocardial migration and engraftment. Mol Biol Cell 18(8): 2873-2882.
  6. Ko, I. K., Kean, T. J. and Dennis, J. E. (2009). Targeting mesenchymal stem cells to activated endothelial cells. Biomaterials 30(22): 3702-3710.
  7. Lee, R. H., Pulin, A. A., Seo, M. J., Kota, D. J., Ylostalo, J., Larson, B. L., Semprun-Prieto, L., Delafontaine, P. and Prockop, D. J. (2009). Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5(1): 54-63.
  8. Luu, N. T., McGettrick, H. M., Buckley, C. D., Newsome, P. N., Rainger, G. E., Frampton, J. and Nash, G. B. (2013). Crosstalk between mesenchymal stem cells and endothelial cells leads to downregulation of cytokine-induced leukocyte recruitment. Stem Cells 31(12): 2690-2702.
  9. Salem, H. K. and Thiemermann, C. (2010). Mesenchymal stromal cells: current understanding and clinical status. Stem Cells 28(3): 585-596.
  10. Schenkel, A. R., Mamdouh, Z. and Muller, W. A. (2004). Locomotion of monocytes on endothelium is a critical step during extravasation. Nat Immunol 5(4): 393-400.
  11. Toma, C., Wagner, W. R., Bowry, S., Schwartz, A. and Villanueva, F. (2009). Fate of culture-expanded mesenchymal stem cells in the microvasculature: in vivo observations of cell kinetics. Circ Res 104(3): 398-402.
  12. Wang, S., Guo, L., Ge, J., Yu, L., Cai, T., Tian, R., Jiang, Y., Zhao, R. and Wu, Y. (2015). Excess integrins cause lung entrapment of mesenchymal stem cells. Stem Cells 33(11): 3315-3326.
  13. Wu, Y., Ip, J. E., Huang, J., Zhang, L., Matsushita, K., Liew, C. C., Pratt, R. E. and Dzau, V. J. (2006). Essential role of ICAM-1/CD18 in mediating EPC recruitment, angiogenesis, and repair to the infarcted myocardium. Circ Res 99(3): 315-322.
  14. Wu, Y. and Zhao, R. C. (2012). The role of chemokines in mesenchymal stem cell homing to myocardium. Stem Cell Rev 8(1): 243-250.

简介

间充质干细胞(MSCs)在组织修复和再生中显示出深远的治疗潜力。 然而,最近的研究表明,MSCs在静脉内递送后很大程度上被截留在肺中并且很快死亡。 基本的机制一直不甚了解。 我们提供的证据表明培养扩增的MSCs中整联蛋白的过量表达和活化是MSCs与肺微动脉的内皮细胞粘附的关键原因,导致细胞的包埋(Wang等人 >,2015)。 因此,在静脉给药之前测试MSC对体外内皮细胞的粘附能力以避免它们的肺血管阻塞可能是有意义的。 在这里,我们报告了一种简单的方法来衡量MSC与内皮细胞的附着。

【背景】间充质干细胞(MSCs)正在成为一种极具潜力的治疗药物,许多临床试验正在进行中(Salem和Thiemermann,2010)。由于MSC的方便性和安全性,静脉输注MSCs已成为近期临床试验中MSCs治疗的流行途径(Wu and Zhao,2012)。然而,越来越多的证据表明,MSCs在血管内注射后引起相当大的血管阻塞。在静脉内输注时,超过80%的MSC被包埋在肺中,并且在急性缺血性心脏或脑中仅检测到少于1%的MSC(Lee等人,2009; Toma等人,等人,2009年)。

最近的研究表明,MSCs在静脉内给药后大部分停留在前毛细血管微血管中,并且其中大部分在短期内局部缺血死亡(Toma等人,2009)。因此,血管内给药的MSC的安全性和有效性已成为人们日益关注的问题。尚未完全了解MSCs血管阻塞的机制。

我们的数据表明,MSCs中整联蛋白的过量表达是其肺包埋的重要原因,其导致肺细胞附着于肺内的内皮细胞,从而减少它们的贩运和归巢至发炎的组织。整合素在MSCs中的功能性阻断,尤其是在整合素β1阻断后,显着降低它们与内皮细胞的附着,导致包埋在肺中的MSCs大量减少,血液中循环MSC的水平升高,并且增加细胞移植到发炎组织(Wang等人,2015)。在这里,我们提供了一种方法来测量MSCs在体外对内皮细胞的附着 。

关键字:间充质干细胞, 内皮细胞, 细胞粘附, 整合素

材料和试剂

  1. 24孔板(Corning,Costar ®,产品目录号:3524)
  2. 12孔板(Corning,目录号:3512)
  3. 10厘米板(Corning,目录号:353003)
  4. 移液器(康宁,目录号:4100)

  5. 15或50毫升锥形离心管(Corning,目录号:430052,430828)
  6. 人类骨髓来源的MSC(Lonza,目录号:PT-2501)
  7. 人脐静脉内皮细胞(HUVEC)(Lonza,目录号:CC-2517)
  8. 人肺微血管内皮细胞(HMVECs-L)(Lonza,目录号:CC-2527)
  9. 达尔伯克改良伊格尔培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:41966052)
  10. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  11. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  12. EGM-2 MV SingleQuot套件补充&amp;生长因子(Lonza,目录号:CC-4147)
  13. EGM TM -2 MV BulletKit TM Medium(Lonza,目录号:CC-3202)
  14. 内皮基础培养基-2(EBM-2)(Lonza,目录号:CC-3156)
  15. 纤连蛋白(Sigma-Aldrich,目录号:F0556)

  16. 无菌磷酸缓冲盐水(PBS),pH7.2(赛默飞世尔科技公司,目录号:20012068)
  17. Hank平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14025092)
  18. 玻连蛋白(Sigma-Aldrich,目录号:V8379)
  19. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  20. 亲脂性荧光团1,1'-双十八烷基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐(DiI)(Sigma-Aldrich,目录号:468495)
  21. 胰蛋白酶-EDTA(0.25%),酚红(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  22. 小鼠抗人整合素β1抗体(Merck,目录号:MAB1987)
  23. 小鼠抗人整合素β1激活的抗体(Merck,目录号:MAB2079Z)
  24. 抗人整合素α5(Merck,目录号:MAB1956Z)
  25. 纯化的抗人CD51 / CD61(整联蛋白αVβ3)(Thermo Fisher Scientific,eBioscience TM,目录号:14-0519)
  26. 小鼠同种型IgG(Sigma-Aldrich,目录号:M6898)
  27. 肿瘤坏死因子-α(TNF-α)(PeproTech,目录号:300-01A)
  28. Medium 199(Sigma-Aldrich,目录号:M4530)
  29. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  30. Ficoll-paque Plus解决方案(GE Healthcare,目录号:17-1440-02)
  31. 具有膜联蛋白V Alexa Fluor TM 488&amp;碘化丙锭(PI)(Thermo Fisher Scientific,Invitrogen TM,目录号:V13241)
  32. 人类白细胞(见食谱)

设备

  1. 可溯源纳米计时器(Fisher Scientific,目录号:14-649-83)
  2. 离心机(Eppendorf,目录号:5810 R)
  3. 血球计(Hirschmann Instruments,目录号:8100103)
  4. CO 2培养箱(Panasonic,型号:MCO-19AIC(UV))
  5. 荧光显微镜(Leica Microsystems,目录号:Leica DMI6000 B)

程序

  1. 细胞培养和单细胞悬液
    1. hMSCs培养
      人骨髓来源的MSC(hMSC)购自Lonza。在由DMEM,10%FBS和1%青霉素和链霉素组成的生长培养基中培养hMSC 72小时。
    2. 人脐静脉内皮细胞(HUVECs)和人肺微血管内皮细胞(HMVECs-L)
      1. 在内皮生长培养基(EGM)-2加2%FBS培养HUVEC并补充72小时。
      2. 在EGM TM-2 MV BulletKit TM培养基中培养HMVECs-L 72小时。
        注:EGM TM -2 MV BulletKit TM
  2. 阻断抗体浓度和细胞粘附分析的优化
    1. 在室温下用10μg/ ml纤连蛋白(在无菌HBSS或PBS溶液中稀释)或0.4-1μg/ ml玻连蛋白(在无菌水中稀释)包被24孔板1小时。

    2. 除去纤维连接蛋白或玻连蛋白缓冲液,不用清洗,在通风橱中风干1-2小时。
    3. 用含3%BSA的PBS在37℃封闭平板1小时。

    4. 用PBS缓缓冲洗平板,在通风橱中风干。
    5. 在PBS单独涂层的孔中使用3%BSA作为阴性对照。
    6. 用荧光标记物DiI(亲脂性荧光团1,1'-双十八烷基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐)预先标记hMSC和新鲜分离的人外周血白细胞(Wu等人 ,2006)。
      1. 对于悬浮的细胞,用PBS洗涤并且在DMEM基础培养基(无血清)中以2μg/ ml的浓度与DiI在37℃孵育20-30分钟。然后与新鲜生长培养基(补充有10%FBS的DMEM)孵育30分钟。用PBS(400×g,5分钟)洗涤两次后,将细胞重新悬浮于新鲜生长培养基中。
      2. 对于粘附细胞,用PBS洗涤并在DMEM基础培养基(无血清)中与浓度为2μg/ ml的DiI在37℃孵育20-30分钟。然后用新鲜生长培养基替换培养基并孵育30分钟。用0.25%胰蛋白酶-EDTA将细胞胰蛋白酶消化2分钟,并将细胞悬浮于补充有10%FBS的DMEM中。
    7. 用不同浓度的整联蛋白β1,整联蛋白α5或整联蛋白αVβ3阻断物预孵育悬浮的hMSC以确定对hMSC附着达到最大抑制的阻断抗体的最低浓度(Wang等人,2015) 。
      1. 在含有2%FBS的DMEM中,在0,2,4,6,8,10和20μg/ ml的浓度下,在37℃下用抗整联蛋白β1阻断mAb孵育hMSC 30分钟。
      2. 在含有2%FBS的DMEM中,在0,2.5,5,10,20和40μg/ ml的浓度下,在37℃下用抗整联蛋白α5阻断mAb孵育hMSC 30分钟。
      3. 在0,2.5,5,10和20μg/ ml的浓度下,用抗整合素αVβ3阻断性mAb在37°C孵育hMSC 30分钟。
      4. 用同种型IgG抗体孵育hMSCs作为对照。
      5. 孵化后用PBS清洗一次。
    8. 在用整联蛋白β1,整联蛋白α5或同种型对照IgG或每孔5×10 4个新鲜分离的人外周血白细胞阻断后,将hMSC的每孔5×10 4个种子接种纤连蛋白包被的平板。
    9. 在用玻连蛋白包被的平板上的整联蛋白αVβ3或同种型对照IgG阻断后,每孔种植5×10 4个hMSCs。
    10. 将细胞在37°C孵育30分钟,60分钟,90分钟和120分钟的DMEM加10%FBS。

    11. 每30分钟收集一次非粘附细胞(用PBS洗一次)

    12. 在400 g x g离心非贴壁细胞5分钟
    13. 用血细胞计数器计数非贴壁细胞。
    14. 通过荧光显微镜拍摄DiI标记的hMSC。
    15. 必须对每个变量一式四份进行实验。

  3. hMSCs附着于内皮细胞
    1. 在分别具有内皮生长培养基(EGM)-2和EGM TM -2 MV BulletKit TM TM Medium的12孔板中培养HUVEC和HMVECs-L以汇合。合流密度约为80%-90%。在加入hMSC或白细胞之前立即用或不用10ng / ml TNF-α处理细胞24小时。能够激活内皮细胞的TNF-α治疗(Ko等人,2009)增加了hMSC和白细胞对内皮细胞的附着。
    2. 从新鲜的人外周血分离白细胞(Boyum,1976; Chacko等人,2013)。
    3. 准备hMSCs:从80%-90%汇合的10cm平板胰蛋白酶消化hMSC。
    4. 用荧光DiI标记hMSCs和白细胞。
    5. 在M199加0.1%BSA中以1×10 6的密度悬浮DiI标记的白细胞。
    6. 在含有0.1%BSA的DMEM中以0.5×10 6的密度悬浮DiI标记的hMSC。
    7. 找出适当的时间点来检测hMSCs与内皮细胞的附着。
      1. 将DiI标记的hMSC加入到12孔板中80%-90%汇合的单层HUVEC和HMVECs-L中,并在37℃和5%CO 2下孵育细胞。

      2. 每15分钟检测附着于内皮细胞的hMSC 60分钟
      3. 孵育30分钟通常足以使hMSCs对内皮细胞产生强烈的粘附。
    8. 温育已经用4μg/ ml的整联蛋白β1,10μg/ ml的整联蛋白α5,5μg/ ml的整联蛋白αVβ3或10μg/ ml的对照IgG的封闭抗体在37℃处理30分钟的hMSC 30分钟。

    9. 用PBS清洗样品一次
    10. 用新鲜生长培养基重新悬浮样品并在37°C孵育30分钟。
    11. 将细胞添加到12孔板中的80%-90%HUVECs或HMVECs-L单层细胞中。
    12. 培养接种白细胞1小时的培养物,培养接种hMSCs 30分钟。
    13. 用HBSS洗涤培养物两次以去除非贴壁细胞。
    14. 用血细胞计数器计数非贴壁细胞。
    15. 用1%PFA固定贴壁细胞。
    16. 在荧光显微镜下(200x)获取贴壁细胞的图像,并对每个孔中的10个图像进行成像(图1)。
    17. 计算每个字段的单元格数量。
      每个条件使用六个复制井

      图1. hMSC与HMVEC-L的粘附。 :一种。将来自单层(2D)的DiI标记的单个hMSC与用于整联蛋白α5,整联蛋白β1或整联蛋白αVβ3的阻断抗体预温育,接种在HMVECs-L单层上并温育30分钟。使用与同种型IgG预孵育的hMSC和白细胞作为对照。比例尺= 250μm。 B.去除非贴壁细胞并计数,并贴壁的DiI-hMSC被拍照。对于每种条件使用六个重复孔,并且将该实验重复三次,* P < 0.05; ** P &lt; 0.01。缩写:MSC,间充质干细胞。

数据分析

  1. 贴壁细胞(%)=(贴壁细胞数量)/ [总细胞数量]×100%。
  2. 选择实现对hMSC附着最大抑制的最低浓度的阻断性抗体用于实验:4μg/ ml的整联蛋白β1,10μg/ ml的整联蛋白α5和5μg/ ml的整联蛋白αVβ3。

笔记

  1. 孵育30分钟是检测hMSC与内皮细胞粘附的最佳时间点。孵育时间越短,hMSCs的附着数量越少,但培养时间越长,hMSCs附着于内皮细胞的数量越少。
  2. 用针对整联蛋白β1,整联蛋白α5或整联蛋白αVβ3的阻断性抗体预温育显着降低了与内皮细胞连接的hMSC的数量( P <0.05),其中整联蛋白β1阻断导致最明显的减少(减少约40%)。
  3. 使用死亡细胞凋亡试剂盒通过流式细胞术分析非贴壁细胞。在上述浓度下,整合素β1(而不是整合素α5)和整联蛋白αVβ3的阻断引起hMSC凋亡的适度增加(2-3%)( p <0.05)。
  4. 我们的体外实验显示hMSCs中这些整联蛋白的功能性阻断显着减少了它们与内皮单层的附着,但是与白细胞相比,更多的hMSC与内皮细胞相连。这些结果表明,更多的粘附分子可能参与hMSC与内皮细胞的连接(Schenkel等人,2004; Wang等人,2015)。
  5. 我们检测了整合素α5β1的主要配体纤连蛋白和整合素αVβ3的主要配体玻连蛋白对培养的HMVECs-L和肺内正常内皮的作用。

食谱

  1. 人类白细胞
    注:使用Ficoll-paque Plus溶液从全部外周血中分离白细胞。
    1. 将新鲜血液放入15或50毫升锥形离心管中。
    2. 使用无菌吸管加入等体积的室温PBS并充分混合
    3. 将移液管的尖端放在含有血液/ PBS混合液的样品管底部,慢慢移取Ficoll-paque Plus溶液。
      每10 ml血液/ PBS混合液使用5 ml Ficoll-paque溶液

    4. 在400×g,4℃下离心30分钟
    5. 使用无菌吸管,去除含有血浆和大部分血小板的上层。
    6. 使用另一支移液管将单核细胞层转移到另一个离心管
    7. 通过加入过量的HBSS(约3倍体积的单核细胞层)来洗涤细胞,并在4℃以400×g(1,300rpm)离心5分钟。
    8. 去除上清液,重新悬浮在HBSS中的细胞,并重复洗一次,以删除大部分血小板

致谢

该协议改编自以前发表的论文(Luu et al。,2013; Wang等人,2015; Wu等人,2006,2006 )。这项工作得到了中国国家自然科学基金委(31371404,31571429),广东省自然科学基金(2015A030311041)和深圳市科学技术创新委员会(JCY20160301150838144)的资助。
披露潜在的利益冲突:作者表示没有潜在的利益冲突。

参考

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  2. Boyum,A。(1976)。 分离淋巴细胞,粒细胞和巨噬细胞 Scand J Immunol Suppl < 5:9-15。
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引用:Wang, S., Madsen, C. D. and Wu, Y. (2018). Measurement of Mesenchymal Stem Cells Attachment to Endothelial Cells. Bio-protocol 8(6): e2776. DOI: 10.21769/BioProtoc.2776.
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