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Jun 2016

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Isolation of Multipotent Mesenchymal Stem Cells from Human Extraocular Muscle Tissue
人眼外肌组织多能间充质干细胞的分离   

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Abstract

Mesenchymal stem cells (MSCs) have attracted significant attention as potential therapeutic cells to treat various diseases ranging from tissue injuries, graft versus host disease, degenerative diseases and cancer. Since the initial discovery of MSCs in the bone marrow cells, MSCs have been successfully isolated from various adult and neo-natal tissues, albeit the procedures are often coupled with difficulties in harvesting tissue and produce low yield of cells, requiring extensive expansion in vitro. Here, we explored extra-ocular muscle tissues obtained from patients as a novel source of MSCs which express characteristic cell surface markers of MSCs and show multilineage differentiation potential with high proliferation capacity.

Keywords: Mesenchymal stem cells (间充质干细胞), Stem cells (干细胞), Osteogenesis (成骨作用), Adipogenesis (脂肪形成), Chondrogenesis (软骨形成), Extraocular muscle (眼外肌)

Background

Mesenchymal stem cells (MSCs) were originally identified as plastic adherent, fibroblastic cells derived from bone marrow and showed multi-lineage differentiation potential (Friedenstein et al., 1974a and 1974b). Over the years, MSCs have been implicated to play a role in a wide range of biological processes such as hematopoiesis (Friedenstein et al., 1974a), immune-modulation (Klyushnenkova et al., 2005; Crop et al., 2010), tissue repair, angiogenesis, tumorigenesis (Yagi and Kitagawa, 2013) and chemoresistance (Kumar et al., 2017). MSCs possess a vast secretome and have been heralded as factories of extracellular vesicles and “injury drug store” (Caplan and Correa, 2011) for their ability to produce a myriad number of growth factors and cytokines. The secreted factors of MSCs were found to have roles in tissue repair of renal (Grange et al., 2014; Zhang et al., 2014), neural (Koc et al., 2002; Zappia et al., 2005), liver (Li et al., 2013), lung (Lee et al., 2012), myocardial (Timmers et al., 2008; Bian et al., 2014) as well as ischemic injuries (Zhang et al., 2012). Therefore, MSCs are being explored as a therapeutic option for ameliorating various diseases such as myocardial infarction, respiratory disorders, Crohn’s disease, graft versus host disease, diabetes, bone disorders, as well as liver cirrhosis (Uccelli et al., 2008; Battiwalla and Hematti, 2009; Luk et al., 2015).

Cells with similar properties of bone marrow derived MSCs (BM-MSCs) have since been isolated from placenta, amniotic fluid (Tsai et al., 2004), umbilical cord blood (Bieback et al., 2004), mobilized peripheral blood, adipose tissue (Kim et al., 2013), connective tissue, skeletal muscle (Young et al., 2001), dental (Gronthos et al., 2000), fetal tissue (Shin et al., 2009) and extra-ocular muscle tissue (Mawrie et al., 2016). This protocol explores extraocular muscle as a novel source of MSCs which is generally excised and discarded during strabismus correction surgery. Strabismus surgery is the third most common eye surgery in US with up to 1.2 million cases per year. The incised tissue is discarded after procedure, which can be used as a source of MSCs. Extraocular muscles are unaffected during Duchenne's muscular dystrophy (Kaminski et al., 1992; Khurana et al., 1995) and contain 15 times more side population stem cells than skeletal muscle (Pacheco-Pinedo et al., 2009). The extraocular muscle derived MSCs (EOM-MSCs) expressed CD73, CD90 and CD105 (Mawrie et al., 2016) which are characteristic cell surface markers for MSCs (Dominici et al., 2006) and could differentiate into all the three meseodermal lineages (Mawrie et al., 2016). Moreover, EOM-MSCs are relatively easy to isolate, have high proliferation capacity, neuroectodermal differentiation potential and might be a good candidate for stem cell based therapy for treating neurodegenerative disorders (Mawrie et al., 2016).

Materials and Reagents

  1. Bio-hazard waste container (Tarsons, catalog number: 583254)
  2. Cell culture dish, 35 x 10 mm (Eppendorf, catalog number: 0030700112)
  3. Cryo-vials (Tarsons, catalog number: 523182)
  4. Cryo-tags (Tarsons, Cryo-babies, catalog number: 526070)
  5. FACS tubes (Corning, catalog number: 352063)
  6. Graduated centrifuge tubes, 15 ml (Tarsons, catalog number: 546021)
  7. Graduated centrifuge tubes, 20 ml (Tarsons, catalog number: 546041)
  8. Graduated 20 μl micro-tips (Tarsons, catalog number: 521000) 
  9. Graduated 200 μl, 1,000 μl micro-tips (Thermo Fisher Scientific, catalog numbers: 90030100, 90030210-P)
  10. Tissue culture treated, flat bottom 96-well plate (Eppendorf, catalog number: 6030730119)
  11. Whatman filter paper (Whatman, catalog number: 1001 125)
  12. 10 ml serological glass pipettes (Himedia, catalog number: CG316-1x10NO)
  13. Aseptic Petri dish (Tarsons, catalog number: 460051)
  14. Sterile filtration unit (0.22 μm) (Thermo Fisher Scientific, catalog number: 450-0020)
  15. Antibodies (Table 1)

    Table 1. Antibody details
    S. No.
    Marker
    Antibody Manufacturer
    Dilution
    Amount added per 50 μl of FACS buffer
    1. CD29
    Anti-human CD29 PE
    BD Biosciences, catalog number: 555443
    1:100
    0.5 μl
    2. CD34
    Anti-human CD34 FITC
    Thermo Fisher Scientific, catalog number: CD3458101
    1:100
    0.5 μl
    3. CD44
    Anti-human CD44 FITC
    BD Biosciences, catalog number: 555478
    1:100
    0.5 μl
    4. CD49e
    Anti-human CD49e PE
    BD Biosciences, catalog number: 555617
    1:100
    0.5 μl
    5. CD73
    Anti-human CD73 PE
    BD Biosciences, catalog number: 550257
    1:100
    0.5 μl
    6. CD90
    Anti-human CD90 FITC
    BD Biosciences, catalog number: 555595
    1:100
    0.5 μl
    7. CD105
    Anti-human CD105 PE
    BD Biosciences, catalog number: 560839
    1:100
    0.5 μl
    8. HLA1
    Anti-human HLA1 FITC
    BD Biosciences, catalog number: 555552
    1:100
    0.5 μl
    9. Isotype control
    Anti-mouse IgG1 PE
    BD, catalog number: 556561
    1:100
    0.5 μl
    10. Isotype control
    Anti-mouse IgG1 FITC
    BD, catalog number: 554109
    1:100
    0.5 μl

  16. 10x Trypsin (2.5%) (Thermo Fisher Scientific, catalog number: 15090-046)
  17. Alizarin Red S (Sigma-Aldrich, catalog number: 199962)
  18. Ascorbic acid-2-phosphate (Sigma-Aldrich, catalog number: A8960)
  19. β-Glycerophosphate (Sigma-Aldrich, catalog number: G9891)
  20. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418-250ML)
  21. Dexamethasone (Sigma-Aldrich, catalog number: D2915)
  22. Fibronectin solution (Sigma-Aldrich, Fibronectin human plasma, catalog number: F0895)
  23. Formaldehyde solution 37-41% (w/v) (Merck, catalog number: 61780805001730) 
  24. Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: 55021C)
  25. IBMX (Sigma-Aldrich, catalog number: I-5879)
  26. Indomethacin (Sigma-Aldrich, catalog number: I-7378)
  27. Insulin (Sigma-Aldrich, catalog number: 91077C)
  28. Isopropanol (Merck, catalog number: 1.07022.2521)
  29. Oil Red O (Sigma-Aldrich, catalog number: O-0625)
  30. Potassium chloride (KCl) (Merck, catalog number: 61779205001730)
  31. Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 60487305001730)
  32. Propidium Iodide (Sigma-Aldrich, catalog number: P4170)
  33. Safranin O (Sigma-Aldrich, catalog number: S8884)
  34. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761)
  35. Sodium chloride (NaCl) (Merck, catalog number: 1.93206.0521)
  36. Sodium dihydrogen phosphate (NaH2PO4) (Merck, catalog number: 61787405001730)
  37. DMEM-low glucose with L-Glutamine medium (Sigma-Aldrich, catalog number: D2902-1L)
  38. DMEM-high glucose with L-Glutamine medium (Sigma-Aldrich, catalog number: D5648-1L)
  39. Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 10270)
  40. 100x Penicillin (10,000 Units/ml)-Streptomycin (10,000 Units/ml) antibiotic (Thermo Fisher Scientific, catalog number: 15140-122)
  41. StemPro chondrogenesis differentiation kit (Thermo Fisher Scientific, catalog number: A10071-01)
  42. De-ionized water (dH2O) (Merck, Elix Type 2 pure water) 
  43. Trypan blue (Sigma-Aldrich, catalog number: T6146)
  44. Liquid nitrogen (99.999%)*
  45. Sodium hydroxide (NaOH) (Merck, catalog number: 61843805001730)
  46. Hydrochloric acid 35% (HCl) (Merck, catalog number: 61762505001730)
  47. DMEM medium (see Recipes)
    1. DMEM-LG (low glucose)/DMEM-HG (high glucose) basal medium
    2. MSC growth medium
  48. Tissue collection medium (see Recipes)
  49. Fibronectin solution (see Recipes)
  50. Phosphate buffered saline (PBS) (see Recipes)
    1. 10x PBS
    2. 1x PBS
    3. PBS with 1x antibiotic
  51. 1x Trypsin (0.25%) (see Recipes)
  52. 0.4% trypan blue (see Recipes)
  53. Heat-inactivated FBS (see Recipes)
  54. Freezing media (see Recipes)
  55. FACS buffer (see Recipes)
  56. Propidium iodide staining solution (see Recipes)
  57. Osteogenesis induction media (see Recipes)
  58. Adipogenesis induction media (see Recipes)
  59. 4% formaldehyde solution (see Recipes)
  60. Alizarin red staining solution (see Recipes)
  61. Oil Red O solution (see Recipes)
    1. 1% Oil Red O stock solution
    2. Oil red O staining solution
  62. Chondrogenic differentiation medium (see Recipes)
  63. 0.1% Safranin O staining solution (see Recipes)

Equipment

  1. Cryo-cooler (Tarsons, catalog number: 525000)
  2. Sterilized sharp-tip forceps and scalpel
  3. Analytical balance (Sartorius, Quintix Analytical Balance 60, 120 g x 0.01, 0.1 mg)
  4. Cryogenic cell storage container (International Cryogenics, model: D-2000C)
  5. Biosafety level 2 cabinet (Thermo Fisher Scientific, model: 1300 series A2) 
  6. CO2 incubator (Thermo Fisher Scientific, Hera Cell 150i)
  7. Flow cytometer (Becton Dickinson, FACS calibur and BD cell quest software)
  8. Hemocytometer (Bright line hemocytometer) (Sigma-Aldrich, catalog number: Z359629)
  9. Inverted microscope with camera (Zeiss, Axio Vert. A1)
  10. Pipette controller (Socorex, Profiller 446)
  11. Single-channel pipettes, 0.5-20 μl, 20-200 μl, and 100-1,000 μl (Gilson, Pipetman classic)
  12. Refrigerated centrifuge (Thermo Fisher Scientific, Sorvall legend X1R)
  13. Water bath (Thermo Fisher Scientific, Labline water bath)
  14. Ultra-low temperature freezer (-80 °C freezer) (Thermo Fisher Scientific, Forma 88000 series)
  15. 4 °C refrigerator*
  16. -20 °C freezer*
  17. Autoclave*

*Note: These items can be ordered from any qualified company.

Software

  1. FlowJo software (FlowJo, LLC)

Procedure

  1. Isolation of extra ocular muscle derived MSCs (EOM-MSCs)
    1. Collect excised EOM tissue (approximately 3-8 mm3) from the patient and transfer it to a vial with 3-4 ml of collection media (see Recipes). Use ice packs to maintain the tissue at low temperature during transportation.

    Note: The following steps are to be performed inside a biosafety level 2 cabinet following aseptic culture techniques.
    1. Before processing the tissue sample, coat the isolation dish with fibronectin at a concentration of 20 ng/cm2. To a 35 mm cell culture dish, add 500 μl of fibronection solution (400 ng/ml; see Recipes) and incubate the dish at 37 °C in a CO2 incubator for 1 h. Remove the excess fibronectin and rinse the dish once with 3 ml of 1x PBS (see Recipes). 
    2. Using sterile forceps, transfer the tissue to an aseptic Petri dish containing 10 ml of PBS with 1x antibiotic (see Recipes). Incubate the tissue at room temperature for 5 min to disinfect and remove excess blood.
    3. Carefully transfer the tissue to a sterile 35 mm cell culture dish coated with fibronectin and tear it into small pieces using sterile forceps or if required cut the tissue into small pieces with a sterile scalpel. 
    4. Add 500 μl of warm fresh MSC growth medium (see Recipes) to the dish and allow the tissue pieces to attach by incubating the dish for 2 h in a CO2 incubator maintaining 5% CO2 and 37 °C. 
    5. After 2 h, add additional 2 ml of warm MSC growth medium without disturbing the tissue pieces and return the dish to the incubator (Figure 1).
      Note: Handle the plate gently during this time since the tissue can be dislodged easily by excessive shaking.


      Figure 1. Procedure of processing EOM tissue

    6. After 48 h, carefully aspirate the medium and replenish with 1.5-2 ml of fresh MSC growth medium (see Recipes) without disturbing/dislodging the tissue.
      Note: MSC growth medium has to be replaced every 72 h thereafter. 
    7. After 7-10 days, check for attached cells around the explants using an inverted microscope (Figure 2). Ensure that the culture is free of any contamination at this point. 
    8. Once a sufficient number of attached cells (200-300 cells) are observed around the tissue pieces, gently remove the tissue explants using a fine tipped forceps without scrubbing the tissue over the dish surface (Figure 2).
      Notes:
      1. Since most of the MSCs attach below the explants, care must be taken not to disturb the cells while removing the tissue.
      2. Discard the tissue in a bio-hazard waste container. All the liquid waste should be appropriately disposed of after treatment with sodium hypochlorite.


      Figure 2. Microscopic image of isolated EOM-MSCs. A. Phase-contrast image showing the emergence of MSCs from the tissue explant within one week of isolation. White arrows represent the cells that have migrated out of the tissue. B. Phase contrast image of EOM-MSCs at passage 0. Scale bars: 200 μm.

  2. Maintenance of culture
    Once the cell colonies reach 50%-80% confluence, sub-culture them as follows.
    1. Aspirate the medium (spent medium) and collect it in a sterile 15 ml centrifuge tube. Wash the cells twice with PBS. 
    2. Add 500 μl of 1x trypsin (see Recipes) to the dish and incubate at 37 °C for 5-7 min.
      Notes: 
      1. Keep trypsin at room temperature for 5-10 min after retrieving from 4 °C before use.
      2. Monitor the cell detachment intermittently under the microscope. Gentle taps on the side of the dish might be required to dislodge the rounded cells. Do not leave the cells in trypsin for more than 10 min.
    3. Add 1-2 ml of spent medium to neutralize trypsin and transfer the cells to a labeled 15 ml centrifuge tube. 
    4. Centrifuge at 300 x g for 5 min at 4 °C to obtain the cell pellet. Discard the supernatant and re-suspend the pellet in 1 ml of fresh MSC growth medium. 
    5. Aliquot a small volume (~50 μl) of cells and add an equal volume of 0.4% trypan blue. Mix properly and count the viable cells using a hemocytometer. 
    6. Seed cells at 1-2 x 103 cells/cm2 in a tissue culture dish and add 2 ml of fresh MSC growth medium. Incubate the dish in 5% CO2 incubator at 37 °C. 
    7. Change the MSC growth medium every 72 h until the cells attain 70%-80% confluence. Cells can be passaged up to 10-12 times by repeating the Steps B1-B6. 

  3. Freezing and thawing of EOM-MSCs
    1. After Step B5, re-suspend 1 x 106 cells in 0.5 ml of pre-chilled sterile FBS (see Recipes). 
    2. Transfer the cell suspension to a labeled cryo-vial and add an equal volume of pre-chilled freezing media (see Recipes). Gently mix and transfer the vial immediately to a cryo-cooler.
    3. Keep the cryo-cooler in a -80 °C freezer for 24 h before transferring the vial to a cryogenic cell storage container with liquid nitrogen for long-term storage.
    4. For reviving the cells, thaw by placing the cryo-vial immediately into a water bath at 37 °C for 45-60 s.
    5. Add 0.5 ml of fresh warm MSC growth medium to the vial and transfer the cell suspension to a sterile 15 ml centrifuge tube. Add additional 4 ml of MSC growth medium drop-wise.
    6. Centrifuge the cells at 300 x g for 5 min at 4 °C. Re-suspend the cell pellet in 1 ml of fresh MSC growth medium to remove any remaining DMSO. 
    7. Count the viable cells as described in Step B5 and seed 2-3 x 105 cells in a 35 mm cell culture dish with 2 ml fresh MSC growth medium. 
    8. Replace the growth medium after 12 h and maintain the cells according to Step B7 thereafter. 

  4. Cell surface marker staining and flow cytometry
    1. Trypsinize and count the EOM-MSCs as described in Procedure B.
      Note: Keep the cells on ice during the whole procedure to maintain viability and reduce internalization of surface markers.
    2. For each marker to be analyzed, distribute 1 x 105 cells per FACS tube for staining with fluorophore-labeled antibody. For each type of antibody used, keep additional FACS tube to be stained with a non-specific isotype control antibody as control.
    3. Centrifuge the cells and wash the pellet twice with 3 ml PBS.
      Note: All centrifugation steps are to be done at 300 x g for 5 min at 4 °C.
    4. Resuspend the cells in 50 μl of FACS buffer (see Recipes). Add 2 μl of diluted antibody (refer to antibody details in Table 1) followed by short gentle vortexing at low RPM. Incubate the tubes at 4 °C for 30 min in the dark. 
    5. Wash the cells with 1 ml of FACS buffer and re-suspend in 300 μl of propidium iodide staining buffer (see Recipes). Analyze the samples in a flow cytometer using proper gain/amp settings for the channels (Figure 3).


      Figure 3. Cell surface marker expression of EOM-MSCs. A. Plots showing the gating for flow cytometry analysis; live cells were gated (G1) on the basis of propidium iodide (PI) negative (FL3 low) population in FL3 vs. FSC plot followed by gating (G2) in FSC vs. SSC plot to remove the cell debris. Cells in gate (G2) were further analyzed for their fluorescence intensity. B. EOM-MSCs were stained with fluorescently conjugated antibodies against CD29, CD34, CD44, CD49E, CD73, CD90, CD105 and HLA class I, and the expression was analyzed by flow cytometry. The histogram in red represents the isotype control and blue represents the stained sample. Y-axis represents the number of events/counts and X-axis represents the fluorescence intensity of the mentioned cell surface marker.

  5. Osteogenic and adipogenic differentiation 
    1. Trypsinize and count the EOM-MSCs as described in Procedure B.
    2. Seed EOM-MSCs at a density of 5,000 cells/cm2 for osteogenesis and 20,000 cells/cm2 for adipogenesis induction in a 96-well plate with adequate MSC growth medium (see Recipes).
    3. After 24 h, add osteogenesis or adipogenesis induction media (see Recipes) to the osteogenic and adipogenic conditions respectively. 
    4. Replace the induction media every 3-4 days and allow the cells to differentiate for 14-21 days. 

  6. Alizarin red staining for assessing calcium deposition during osteogenesis
    1. Aspirate the medium and wash the cells twice with PBS.
    2. Fix the cells in 4% formaldehyde solution (see Recipes) for 1 h at room temperature. 
    3. Wash the cells twice with dH2O.
    4. Stain with freshly prepared Alizarin red staining solution (see Recipes) for 10 min at room temperature. 
    5. Wash cells five times with dH2O followed by a wash with PBS for 15 min to remove excess stain. Add PBS to the cells to prevent them from drying. 
    6. Proceed to imaging the cells under an inverted microscope with a camera (Figure 4A). 

  7. Oil red O staining for assessing lipid accumulation during adipogenesis
    Note: Prepare Oil Red O staining solution (see Recipes) at least 1 h prior to staining. 
    1. Aspirate the medium and wash the cells twice with PBS.
    2. Fix the cells in 4% formaldehyde solution (see Recipes) for 1 h at room temperature. 
    3. Remove the fixing solution and rinse cells with 60% isopropanol in dH2O. 
    4. Stain the cells with Oil Red O staining solution for 10 min.
    5. Wash cells five times with dH2O. Add PBS to the cells to prevent them from drying.
    6. Proceed to imaging the cells under an inverted microscope with a camera (Figure 4B). 

  8. Chondrogenic differentiation 
    1. Trypsinize and count the EOM-MSCs as described in Procedure B.
    2. Re-suspend the pellet in an appropriate volume to generate a cell suspension of 1.6 x 107 cells/ml. Add a 5 μl droplet of this cell suspension to the center of each well in a 96-well plate and allow cells to attach by incubating the plate for 2 h in a 5% CO2 incubator at 37 °C.
    3. After 2 h, add adequate (100 μl) chondrogenic differentiation media (see Recipes) to the wells without disturbing the cell micromass and return the plate to the incubator.
    4. Replace the chondrogenic media every 2 days and allow cells to differentiate for > 14 days.

  9. Safranin O staining for detection of cartilage, mucin and mast cell granules
    1. Aspirate the medium and wash the cells twice with PBS.
    2. Fix the cells with 4% formaldehyde solution (see Recipes) for 1 h at room temperature. 
    3. Wash the cells twice with dH2O.
    4. Stain with 0.1% safranin O solution (see Recipes) for 5 min at room temperature.
    5. Wash cells with dH2O. Add PBS to the cells to prevent them from drying. 
    6. Proceed to imaging the cells under an inverted microscope with a camera (Figure 4C).


      Figure 4. Differentiation of EOM-MSCs into osteogenic, adipogenic and chondrogenic cells. EOM-MSCs were seeded in the respective cell density and induction media for differentiation into osteogenic, adipogenic and chondrogenic lineage cells. The differentiated cells were stained with (A) alizarin red, (B) oil red o (C) safranin o for determining osteogenic, adipogenic and chondrogenic differentiation respectively. Scale bars: 100 μm.

Data analysis

The flow cytometry data for the expression of various surface markers on EOM-MSCs was analyzed using FlowJo software. Live cells were gated on the basis of propidium iodide negative (FL3 low) population in FL3 vs. FSC plot followed by gating in FSC vs. SSC plot to remove the cell debris. Histograms of signal in FL1 (for FITC-conjugated antibodies) and FL2 (for PE-conjugated antibodies) were plotted to define negative (unstained) and positive (stained) population using appropriate isotype-matched control antibody. This protocol shows representative data for only one of the EOM-MSCs samples and does not include any statistical analysis.

Recipes

  1. DMEM medium
    1. DMEM-LG (low glucose)/DMEM-HG (high glucose) basal medium
      10 g DMEM-LG or DMEM-HG media powder
      3.7 g sodium bicarbonate
      10 ml 100x Penicillin-Streptomycin antibiotic solution
      Make up the volume to 1 L with autoclaved deionized H2O
      Adjust pH to ~7.0 as pH tends to increase after filtration
      Sterilize by filtration using a sterile filtration unit (0.22 μm)
      Can be stored at 4 °C for up to 2 months
    2. MSC growth medium
      10% FBS in DMEM-LG basal medium
      Sterilize by filtration using a sterile filtration unit (0.22 μm)
      Store at 4 °C and use within two weeks
  2. Tissue collection medium
    2x Penicillin-Streptomycin antibiotic in DMEM-LG basal medium or HBSS solution
  3. Fibronectin solution (400 ng/ml)
    Prepare working stock of fibronectin by diluting 40 μl of 1 mg/ml stock solution in 100 ml dH2O
    Sterilize by filtration using a sterile filtration unit (0.22 μm) and store at 4 °C
  4. Phosphate buffered saline (PBS)
    1. 10x PBS
      80 g NaCl
      2 g KCl
      14.4 g Na2HPO4
      2.4 g KH2PO4
      Make up the volume to 1 L with autoclaved dH2O
      Sterilize by filtration using a sterile filtration unit (0.22 μm)
      Store at room temperature
    2. 1x PBS
      Dilute 10x PBS ten times (1 ml in 10 ml) in H2O
      Adjust pH to 7.4 and autoclave at 121 °C for 40 min
    3. PBS with 1x antibiotic
      100 μl of 100x Penicillin-Streptomycin antibiotic in 10 ml of 1x PBS
  5. 1x Trypsin (0.25%)
    Dilute 10x trypsin ten times in sterile cold 1x PBS (1 ml of 10x trypsin in 9 ml of PBS) and prepare 5 ml aliquots for use
    Store aliquots at -20 °C for long term storage. Thaw at room temperature and store at 4 °C after use for up to 1 week
  6. 0.4% trypan blue
    Dissolve 50 mg of Trypan blue (dye composition 40%) in 5 ml of 1x PBS
    Sterilize by filtration using a sterile filtration unit (0.22 μm)
    Store as aliquots at 4 °C 
  7. Heat inactivated FBS
    Heat inactivate FBS by incubating at 56 °C for 1 h in a water bath and store at -20 °C as 50 ml aliquots
  8. Freezing media
    20% DMSO in heat-inactivated FBS (1 ml DMSO in 4 ml of FBS)
  9. FACS buffer
    2% heat-inactivated FBS in 1x PBS (200 μl FBS in 9.8 ml of PBS)
  10. Propidium iodide staining solution
    2 μg/ml of propidium iodide in FACS buffer
  11. Osteogenesis induction media
    DMEM-high glucose basal medium
    1x Penicillin-Streptomycin antibiotic
    10% FBS
    10 mM β-Glycerophosphate
    0.1 μM Dexamethasone
    0.05 mM Ascorbic acid-2-phosphate
    Note: Osteogenesis induction media can be stored for up to 4 weeks at 4 °C.
  12. Adipogenesis induction media
    DMEM-high glucose basal medium
    1x Penicillin-Streptomycin antibiotic
    10% FBS
    1 μM Dexamethasone
    0.2 mM Indomethacin (add 2 drops of 5 M NaOH to dissolve)
    0.5 mM IBMX
    0.01 M Insulin (add 2 drops of 5 M HCl to dissolve)
    Note: Adipogenesis induction media can be stored for up to 4 weeks at 4 °C.
  13. 4% formaldehyde solution
    Dilute 37%-41% Formaldehyde solution 10 times in 1x PBS
    1 ml of 37%-41% Formaldehyde solution
    9 ml of 1x PBS
  14. Alizarin red staining solution
    0.19 g Alizarin Red S
    10 ml dH2O
    Adjust pH to 4.2
  15. Oil Red O solution
    1. 1% Oil Red O stock solution
      1 g Oil Red O
      100 ml isopropanol
      Warm in a water bath at 56 °C for 30-60 min. Not all of Oil Red O will dissolve
      Can be stored at room temperature for up to 6 months
    2. Oil red O staining solution
      3 parts of 1% Oil Red O stock solution
      2 parts of dH2O
      Mix and let stand for 1 h
      Filter the solution with Whatman filter paper
  16. Chondrogenic differentiation medium
    10 ml 10x Stem Pro Chondrogenesis supplement
    90 ml Stem Pro Osteocyte/Chondrocyte Differentiation Basal Medium
    1x Penicillin-Streptomycin antibiotic
    Note: Chondrogenic differentiation media can be stored for up to 4 weeks at 4 °C.
  17. 0.1% Safranin O staining solution
    0.1 g Safranin O
    100 ml dH2O
    Stir to help dissolve

Acknowledgments

This work was supported by funds from Indian Council for Medical Research (ICMR), Govt. of India. Protocol was adapted from Mawrie et al. (2016). The authors thank Trishna Anand and Vishnu for their assistance in imaging.

Competing interests

The authors declare no competing interests.

Ethics

The study was approved by ethics committee of IIT Guwahati and samples were collected after written informed consent from the patients.

References

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  5. Crop, M. J., Baan, C. C., Korevaar, S. S., Ijzermans, J. N. M., Weimar, W. and Hoogduijn, M. J. (2010). Human adipose tissue-derived mesenchymal stem cells induce explosive T-cell proliferation. Stem Cells Dev 19(12): 1843-1853. 
  6. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F. C., Krause, D. S., Deans, R. J., Keating, A., Prockop, D. J. and Horwitz, E. M. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8(4): 315-317. 
  7. Friedenstein, A. J., Chailakhyan, R. K., Latsinik, N. V., Panasyuk, A. F. and Keilissb.Iv (1974a). Stromal cells responsible for transferring microenvironment of hematopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 17(4): 331-340. 
  8. Friedenstein, A. J., Deriglasova, U. F., Kulagina, N. N., Panasuk, A. F., Rudakowa, S. F., Luria, E. A. and Rudakow, I. A. (1974b). Precursors for fibroblasts in different populations of hematopoietic cells as detected by in vitro colony assay method. Exp Hematol 2(2): 83-92. 
  9. Grange, C., Tapparo, M., Bruno, S., Chatterjee, D., Quesenberry, P. J., Tetta, C. and Camussi, G. (2014). Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging. Int J Mol Med 33(5): 1055-1063. 
  10. Gronthos, S., Mankani, M., Brahim, J., Robey, P. G. and Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97(25): 13625-13630.
  11. Kaminski, H. J., al-Hakim, M., Leigh, R. J., Katirji, M. B. and Ruff, R. L. (1992). Extraocular muscles are spared in advanced Duchenne dystrophy. Ann Neurol 32(4): 586-588.
  12. Khurana, T. S., Prendergast, R. A., Alameddine, H. S., Tome, F. M., Fardeau, M., Arahata, K., Sugita, H. and Kunkel, L. M. (1995). Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing. J Exp Med 182(2): 467-475.
  13. Kim, J., Escalante, L. E., Dollar, B. A., Hanson, S. E. and Hematti, P. (2013). Comparison of breast and abdominal adipose tissue mesenchymal stromal/stem cells in support of proliferation of breast cancer cells. Cancer Invest 31(8): 550-554. 
  14. Klyushnenkova, E., Mosca, J. D., Zernetkina, V., Majumdar, M. K., Beggs, K. J., Simonetti, D. W., Deans, R. J. and McIntosh, K. R. (2005). T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci 12(1): 47-57. 
  15. Koc, O. N., Day, J., Nieder, M., Gerson, S. L., Lazarus, H. M. and Krivit, W. (2002). Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 30(4): 215-222. 
  16. Kumar, A., Bhattacharyya, J. and Jaganathan, B. G. (2017). Adhesion to stromal cells mediates imatinib resistance in chronic myeloid leukemia through ERK and BMP signaling pathways. Sci Rep 7(1): 9535.
  17. Lee, C., Mitsialis, S. A., Aslam, M., Vitali, S. H., Vergadi, E., Konstantinou, G., Sdrimas, K., Fernandez-Gonzalez, A. and Kourembanas, S. (2012). Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension. Circulation 126(22): 2601-2611. 
  18. Li, T. F., Yan, Y. M., Wang, B. Y., Qian, H., Zhang, X., Shen, L., Wang, M., Zhou, Y., Zhu, W., Li, W. and Xu, W. R. (2013). Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis. Stem Cells Dev 22(6): 845-854. 
  19. Luk, F., de Witte, S. F. H., Bramer, W. M., Baan, C. C. and Hoogduijn, M. J. (2015). Efficacy of immunotherapy with mesenchymal stem cells in man: a systematic review. Expert Rev Clin Immunol 11(5): 617-636. 
  20. Mawrie, D., Kumar, A., Magdalene, D., Bhattacharyya, J. and Jaganathan, B. G. (2016). Mesenchymal stem cells from human extra ocular muscle harbor neuroectodermal differentiation potential. Plos One 11(6): e0156697. 
  21. Pacheco-Pinedo, E. C., Budak, M. T., Zeiger, U., Jorgensen, L. H., Bogdanovich, S., Schroder, H. D., Rubinstein, N. A. and Khurana, T. S. (2009). Transcriptional and functional differences in stem cell populations isolated from extraocular and limb muscles. Physiol Genomics 37(1): 35-42.
  22. Shin, K. S., Na, K. H., Lee, H. J., Kim, D. G., Shin, S. J., Kim, J. K. and Kim, G. J. (2009). Characterization of fetal tissue-derived mesenchymal stem cells. Int J Stem Cells 2(1): 51-58. 
  23. Timmers, L., Lim, S. K., Arslan, F., Armstrong, J. S., Hoefer, I. E., Doevendans, P. A., Piek, J. J., El Oakley, R. M., Choo, A., Lee, C. N., Pasterkamp, G. and de Kleijn, D. P. V. (2008). Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium. Stem Cell Res 1(2): 129-137. 
  24. Tsai, M. S., Lee, J. L., Chang, Y. J. and Hwang, S. M. (2004). Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 19(6): 1450-1456. 
  25. Uccelli, A., Moretta, L. and Pistoia, V. (2008). Mesenchymal stem cells in health and disease. Nat Rev Immunol 8(9): 726-736. 
  26. Yagi, H. and Kitagawa, Y. (2013). The role of mesenchymal stem cells in cancer development. Front Genet 4: 261. 
  27. Young, H. E., Steele, T. A., Bray, R. A., Hudson, J., Floyd, J. A., Hawkins, K., Thomas, K., Austin, T., Edwards, C., Cuzzourt, J., Duenzl, M., Lucas, P. A. and Black, A. C. (2001). Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 264(1): 51-62. 
  28. Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, E., Giunti, D., Ceravolo, A., Cazzanti, F., Frassoni, F., Mancardi, G. and Uccelli, A. (2005). Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 106(5): 1755-1761. 
  29. Zhang, G. Y., Zou, X. Y., Miao, S., Chen, J. J., Du, T., Zhong, L., Ju, G. Q., Liu, G. H. and Zhu, Y. J. (2014). The anti-oxidative role of micro-vesicles derived from human Wharton-Jelly mesenchymal stromal cells through NOX2/gp91(phox) suppression in alleviating renal ischemia-reperfusion injury in rats. Plos One 9(3): e92129. 
  30. Zhang, H. C., Liu, X. B., Huang, S., Bi, X. Y., Wang, H. X., Xie, L. X., Wang, Y. Q., Cao, X. F., Lv, J., Xiao, F. J., Yang, Y. and Guo, Z. K. (2012). Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo. Stem Cells Dev 21(18): 3289-3297.

简介

间充质干细胞(MSCs)作为治疗各种疾病的潜在治疗细胞引起了极大的关注,所述疾病包括组织损伤,移植物抗宿主病,退行性疾病和癌症。 自骨髓细胞中MSCs的最初发现以来,已经成功地从各种成人和新生儿组织中分离了MSC,尽管这些程序通常伴随着收获组织的困难并且产生低产量的细胞,需要大量扩增 体外。 在这里,我们探索从患者获得的眼外肌肉组织作为MSCs的新来源,其表达MSC的特征性细胞表面标志物并显示具有高增殖能力的多向分化潜能。
【背景】间充质干细胞(MSCs)最初被鉴定为来自骨髓的塑性贴壁,成纤维细胞并且显示出多谱系分化潜能(Friedenstein 等,1974a和1974b)。多年来,MSCs一直参与广泛的生物学过程,如造血(Friedenstein et al。,1974a),免疫调节(Klyushnenkova 等。,2005; Crop et al。,2010),组织修复,血管生成,肿瘤发生(Yagi和Kitagawa,2013)和化疗耐药性(Kumar et al。,2017) )。 MSCs具有巨大的分泌组织,并且已经被预示为细胞外囊泡和“损伤药物储存”(Caplan和Correa,2011)的工厂,因为它们能够产生无数的生长因子和细胞因子。 MSCs的分泌因子被发现在肾脏组织修复中起作用(Grange et al。,2014; Zhang et al。,2014),神经(Koc et al。,2002; Zappia et al。,2005),肝脏(Li et al。,2013),肺(Lee et al。,2012),心肌(Timmers et al。,2008; Bian et al。,2014)以及缺血性损伤(Zhang 等人,2012)。因此,正在探索MSCs作为改善各种疾病的治疗选择,如心肌梗塞,呼吸系统疾病,克罗恩病,移植物抗宿主病,糖尿病,骨骼疾病以及肝硬化(Uccelli 等。,2008; Battiwalla和Hematti,2009; Luk et al。,2015)。

从那时起,骨髓来源的MSCs(BM-MSCs)具有相似特性的细胞从胎盘,羊水(Tsai 等人,2004),脐带血(Bieback et al。 ,2004),动员外周血,脂肪组织(Kim et al。,2013),结缔组织,骨骼肌(Young et al。,2001)牙科(Gronthos et al。,2000),胎儿组织(Shin et al。,2009)和眼外肌肉组织(Mawrie 等。,2016)。该方案探索眼外肌作为MSC的新来源,其通常在斜视矫正手术期间被切除并丢弃。斜视手术是美国第三大常见的眼科手术,每年有120万例。手术后丢弃切开的组织,其可用作MSC的来源。 Duchenne肌营养不良期间眼外肌未受影响(Kaminski et al。,1992; Khurana et al。,1995)并且含有比骨骼肌多15倍的侧群干细胞( Pacheco-Pinedo et al。,2009)。眼外肌来源的MSCs(EOM-MSCs)表达CD73,CD90和CD105(Mawrie et al。,2016),它们是MSCs的特征性细胞表面标志物(Dominici 等。>,2006)并且可以分化为所有三种中胚层谱系(Mawrie et al。,2016)。此外,EOM-MSCs相对容易分离,具有高增殖能力,神经外胚层分化潜能,并且可能是用于治疗神经退行性疾病的干细胞疗法的良好候选者(Mawrie 等人,2016)。

关键字:间充质干细胞, 干细胞, 成骨作用, 脂肪形成, 软骨形成, 眼外肌

材料和试剂

  1. 生物危害废物容器(Tarsons,目录号:583254)
  2. 细胞培养皿,35×10mm(Eppendorf,目录号:0030700112)
  3. Cryo-vials(Tarsons,目录号:523182)
  4. Cryo-tags(Tarsons,Cryo-babies,目录号:526070)
  5. FACS管(Corning,目录号:352063)
  6. 分级离心管,15 ml(Tarsons,目录号:546021)
  7. 分级离心管,20 ml(Tarsons,目录号:546041)
  8. 分级20μl微提示(Tarsons,目录号:521000) 
  9. 分级200μl,1,000μl微尖(Thermo Fisher Scientific,目录号:90030100,90030210-P)
  10. 组织培养处理,平底96孔板(Eppendorf,目录号:6030730119)
  11. Whatman滤纸(Whatman,目录号:1001 125)
  12. 10 ml血清玻璃移液器(Himedia,目录号:CG316-1x10NO)
  13. 无菌培养皿(Tarsons,目录号:460051)
  14. 无菌过滤装置(0.22μm)(Thermo Fisher Scientific,目录号:450-0020)
  15. 抗体(表1)

    表1.抗体详情
    bordercolor =“#000000”style =“width:700px;” border =“1”cellspacing =“0”cellpadding =“2”>的 S上。不。
    标记
    <强>抗体 制造商
    稀释
    每50μlFACS缓冲液添加量
    1。 CD29
    抗人CD29 PE
    BD Biosciences,目录编号:555443
    1:100
    0.5μl
    2。 CD34
    抗人类CD34 FITC
    Thermo Fisher Scientific,目录号:CD3458101
    1:100
    0.5μl
    3。 CD44
    抗人CD44 FITC
    BD Biosciences,目录号:555478
    1:100
    0.5μl
    4。 CD49e
    抗人CD49e PE
    BD Biosciences,目录号:555617
    1:100
    0.5μl
    5。 CD73
    抗人CD73 PE
    BD Biosciences,目录号:550257
    1:100
    0.5μl
    6。 CD90
    反人类CD90 FITC
    BD Biosciences,目录号:555595
    1:100
    0.5μl
    7。 CD105
    抗人CD105 PE
    BD Biosciences,目录号:560839
    1:100
    0.5μl
    8。 HLA1
    抗人类HLA1 FITC
    BD Biosciences,目录号:555552
    1:100
    0.5μl
    9。 同型对照
    抗小鼠IgG1 PE
    BD,目录编号:556561
    1:100
    0.5μl
    10。 同型对照
    抗小鼠IgG1 FITC
    BD,目录编号:554109
    1:100
    0.5μl
  16. 10x胰蛋白酶(2.5%)(Thermo Fisher Scientific,目录号:15090-046)
  17. 茜素红S(Sigma-Aldrich,目录号:199962)
  18. 抗坏血酸-2-磷酸酯(Sigma-Aldrich,目录号:A8960)
  19. β-甘油磷酸盐(Sigma-Aldrich,目录号:G9891)
  20. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418-250ML)
  21. 地塞米松(西格玛奥德里奇,目录号:D2915)
  22. 纤连蛋白溶液(Sigma-Aldrich,Fibronectin human plasma,目录号:F0895)
  23. 甲醛溶液37-41%(w / v)(默克,目录号:61780805001730)&nbsp;
  24. 汉克的平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:55021C)
  25. IBMX(Sigma-Aldrich,目录号:I-5879)
  26. 吲哚美辛(Sigma-Aldrich,目录号:I-7378)
  27. 胰岛素(Sigma-Aldrich,目录号:91077C)
  28. 异丙醇(默克,目录号:1.07022.2521)
  29. 油红O(Sigma-Aldrich,目录号:O-0625)
  30. 氯化钾(KCl)(默克,目录号:61779205001730)
  31. 磷酸二氢钾(KH 2 PO 4 )(默克,目录号:60487305001730)
  32. 碘化丙锭(Sigma-Aldrich,目录号:P4170)
  33. Safranin O(西格玛奥德里奇,目录号:S8884)
  34. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  35. 氯化钠(NaCl)(默克,目录号:1.93206.0521)
  36. 磷酸二氢钠(NaH 2 PO 4 )(默克,目录号:61787405001730)
  37. 含有L-谷氨酰胺培养基的DMEM-低葡萄糖(Sigma-Aldrich,目录号:D2902-1L)
  38. 含有L-谷氨酰胺培养基的DMEM-高葡萄糖(Sigma-Aldrich,目录号:D5648-1L)
  39. 胎牛血清(赛默飞世尔科技,目录号:10270)
  40. 100x青霉素(10,000单位/ ml) - 链霉素(10,000单位/ ml)抗生素(Thermo Fisher Scientific,目录号:15140-122)
  41. StemPro软骨形成分化试剂盒(Thermo Fisher Scientific,目录号:A10071-01)
  42. 去离子水(dH 2 O)(Merck,Elix Type 2纯水)&nbsp;
  43. 台盼蓝(Sigma-Aldrich,目录号:T6146)
  44. 液氮(99.999%)*
  45. 氢氧化钠(NaOH)(默克,目录号:61843805001730)
  46. 盐酸35%(HCl)(默克,目录号:61762505001730)
  47. DMEM培养基(见食谱)
    1. DMEM-LG(低葡萄糖)/ DMEM-HG(高葡萄糖)基础培养基
    2. MSC生长培养基
  48. 组织收集培养基(见食谱)
  49. 纤维连接蛋白溶液(见食谱)
  50. 磷酸盐缓冲盐水(PBS)(见食谱)
    1. 10x PBS
    2. 1x PBS
    3. PBS含1x抗生素
  51. 1x胰蛋白酶(0.25%)(见食谱)
  52. 0.4%台盼蓝(见食谱)
  53. 热灭活FBS(见食谱)
  54. 冷冻媒体(见食谱)
  55. FACS缓冲液(见食谱)
  56. 碘化丙锭染色液(见食谱)
  57. 成骨诱导培养基(见食谱)
  58. 脂肪生成诱导培养基(见食谱)
  59. 4%甲醛溶液(见食谱)
  60. 茜素红染色液(见食谱)
  61. 油红O解决方案(见食谱)
    1. 1%油红O原液
    2. 油红O染色液
  62. 软骨分化培养基(见食谱)
  63. 0.1%番红O染色液(见食谱)

设备

  1. 冷冻冷却器(Tarsons,目录号:525000)
  2. 消毒尖尖镊子和手术刀
  3. 分析天平(Sartorius,Quintix Analytical Balance 60,120 g x 0.01,0.1 mg)
  4. 低温细胞储存容器(International Cryogenics,型号:D-2000C)
  5. 生物安全2级柜(赛默飞世尔科技,型号:1300系列A2)&nbsp;
  6. CO 2 培养箱(Thermo Fisher Scientific,Hera Cell 150i)
  7. 流式细胞仪(Becton Dickinson,FACS calibur和BD细胞任务软件)
  8. 血细胞计数器(亮线血细胞计)(Sigma-Aldrich,目录号:Z359629)
  9. 带摄像头的倒置显微镜(Zeiss,Axio Vert.A1)
  10. 移液器控制器(Socorex,Profiller 446)
  11. 单通道移液器,0.5-20μl,20-200μl和100-1,000μl(Gilson,Pipetman classic)
  12. 冷冻离心机(赛默飞世尔科技,Sorvall传奇X1R)
  13. 水浴(Thermo Fisher Scientific,Labline水浴)
  14. 超低温冰箱(-80°C冰箱)(Thermo Fisher Scientific,Forma 88000系列)
  15. 4°C冰箱*
  16. -20°C冰柜*
  17. 高压灭菌器*

*注意:这些物品可以从任何合格的公司订购。

软件

  1. FlowJo软件(FlowJo,LLC)

程序

  1. 分离额外的眼肌来源的MSCs(EOM-MSCs)
    1. 从患者收集切除的EOM组织(约3-8mm 3 )并将其转移至具有3-4ml收集培养基的小瓶中(参见食谱)。在运输过程中使用冰袋将组织保持在低温。

    注意:按照无菌培养技术,在生物安全2级柜内进行以下步骤。
    1. 在处理组织样品之前,用浓度为20ng / cm 2的纤连蛋白包被隔离培养皿。向35mm细胞培养皿中加入500μl纤维连接溶液(400ng / ml;参见配方),并将培养皿在37℃下在CO 2 培养箱中孵育1小时。去除多余的纤维连接蛋白,用3 ml 1x PBS冲洗培养皿一次(参见食谱)。&nbsp;
    2. 使用无菌镊子,将组织转移到含有10毫升含1x抗生素的PBS的无菌培养皿中(见食谱)。在室温下孵育组织5分钟以消毒并除去多余的血液。
    3. 小心地将组织转移到涂有纤连蛋白的无菌35 mm细胞培养皿中,并使用无菌镊子将其撕成小块,或者如果需要,用无菌手术刀将组织切成小块。&nbsp;
    4. 将500μl温热的新鲜MSC生长培养基(参见食谱)加入培养皿中,通过在CO 2 培养箱中培养培养皿2小时使组织块附着,保持5%CO 2 和37°C&nbsp;
    5. 2小时后,再加入2毫升温热的MSC生长培养基,不要打扰组织碎片,将培养皿放回培养箱中(图1)。
      注意:在这段时间内轻轻地握住盘子,因为过度摇晃可以轻易地移除组织。


      图1.处理EOM组织的程序

    6. 48小时后,小心地吸出培养基并用1.5-2ml新鲜的MSC生长培养基补充(见食谱),不要打扰/移走组织。
      注意:此后72小时内必须更换MSC生长培养基。&nbsp;
    7. 7-10天后,使用倒置显微镜检查外植体周围的附着细胞(图2)。此时确保培养物没有任何污染。&nbsp;
    8. 一旦在组织块周围观察到足够数量的附着细胞(200-300个细胞),使用细尖镊子轻轻移除组织外植体,而不用擦洗培养皿表面上的组织(图2)。
      注意:
      1. 由于大多数MSCs附着在外植体下方,因此在移除组织时必须小心不要打扰细胞。
      2. 丢弃生物危害废物容器中的纸巾。所有液体废物应在用次氯酸钠处理后适当处理。


      图2.分离的EOM-MSC的显微图像。 A.相位对比图像显示在分离后一周内来自组织外植体的MSC的出现。白色箭头表示已从组织迁移出的细胞。 B.第0代时EOM-MSC的相差图像。比例尺:200μm。

  2. 维护文化
    一旦细胞集落达到50%-80%汇合,则按如下方式对其进行亚培养。
    1. 吸出培养基(用过的培养基)并在无菌的15ml离心管中收集。用PBS清洗细胞两次。&nbsp;
    2. 将500μl1x胰蛋白酶(参见食谱)加入培养皿中,37°C孵育5-7分钟。
      注意:&nbsp;
      1. 在使用前从4°C取出后,将胰蛋白酶在室温下保持5-10分钟。
      2. 在显微镜下间歇地监测细胞分离。可能需要在盘子侧面轻柔地敲击圆形细胞。不要将细胞留在胰蛋白酶中超过10分钟。
    3. 加入1-2毫升用过的培养基中和胰蛋白酶,将细胞转移到标记的15毫升离心管中。&nbsp;
    4. 在4℃下以300 x g 离心5分钟以获得细胞沉淀。弃去上清液,将沉淀重新悬浮于1 ml新鲜MSC生长培养基中。&nbsp;
    5. 等分小体积(~50μl)细胞并加入等体积的0.4%台盼蓝。正确混合并使用血细胞计数器计数活细胞。&nbsp;
    6. 在组织培养皿中以1-2×10 3个细胞/ cm 2培养物种细胞,并加入2ml新鲜MSC生长培养基。将培养皿在37℃下在5%CO 2 培养箱中孵育。&nbsp;
    7. 每72小时更换MSC生长培养基直至细胞达到70%-80%汇合。通过重复步骤B1-B6,细胞可传代达10-12次。&nbsp;

  3. EOM-MSCs的冷冻和解冻
    1. 在步骤B5之后,将1×10 6个细胞重新悬浮在0.5ml预冷的无菌FBS中(参见食谱)。&nbsp;
    2. 将细胞悬浮液转移到标记的冷冻小瓶中,并加入等体积的预冷冻结培养基(参见食谱)。轻轻混合并将小瓶立即转移至低温冷却器。
    3. 将冷冻冷却器保持在-80°C冰箱中24小时,然后将小瓶转移到装有液氮的低温细胞储存容器中,以便长期储存。
    4. 为了使细胞复苏,将冷冻小瓶立即置于37℃的水浴中45-60秒进行解冻。
    5. 向小瓶中加入0.5ml新鲜的温热MSC生长培养基,并将细胞悬浮液转移到无菌的15ml离心管中。逐滴加入另外4ml MSC生长培养基。
    6. 将细胞在300℃下离心5分钟,在4℃下离心5分钟。将细胞沉淀重新悬浮于1ml新鲜MSC生长培养基中以除去任何剩余的DMSO。&nbsp;
    7. 如步骤B5中所述计数活细胞,并在具有2ml新鲜MSC生长培养基的35mm细胞培养皿中种子2-3×10 5个细胞。&nbsp;
    8. 12小时后更换生长培养基,然后根据步骤B7维持细胞。&nbsp;

  4. 细胞表面标志物染色和流式细胞术
    1. 如程序B中所述,胰蛋白酶消化并计数EOM-MSC。
      注意:在整个过程中将细胞保持在冰上以保持活力并减少表面标记物的内化。
    2. 对于待分析的每种标志物,每个FACS管分配1×10 5个细胞用于用荧光团标记的抗体染色。对于使用的每种类型的抗体,保持另外的FACS管用非特异性同种型对照抗体作为对照染色。
    3. 离心细胞,用3 ml PBS洗涤沉淀两次。
      注意:所有离心步骤均在300 x g 4°C下进行5分钟。
    4. 将细胞重悬于50μlFACS缓冲液中(参见配方)。加入2μl稀释的抗体(参见表1中的抗体细节),然后在低RPM下短暂温和涡旋。在黑暗中将管在4°C孵育30分钟。&nbsp;
    5. 用1ml FACS缓冲液洗涤细胞,并重悬于300μl碘化丙啶染色缓冲液中(参见配方)。使用适当的通道增益/放大器设置在流式细胞仪中分析样品(图3)。


      图3. EOM-MSC的细胞表面标志物表达。 A.图显示流式细胞仪分析的门控;在FL3对FSC图中基于碘化丙啶(PI)阴性(FL3低)群体门控活细胞(G1),然后在FSC对比SSC图中门控(G2)以除去细胞碎片。进一步分析门(G2)中的细胞的荧光强度。 B.用针对CD29,CD34,CD44,CD49E,CD73,CD90,CD105和HLA I类的荧光缀合的抗体染色EOM-MSC,并通过流式细胞术分析表达。红色的直方图表示同种型对照,蓝色表示染色的样品。 Y轴表示事件/计数的数量,X轴表示所述细胞表面标志物的荧光强度。

  5. 成骨和成脂分化&nbsp;
    1. 如程序B中所述,胰蛋白酶消化并计数EOM-MSC。
    2. 种子EOM-MSCs密度为5,000个细胞/ cm 2 用于成骨,20,000个细胞/ cm 2 用于在具有足够MSC生长培养基的96孔板中诱导脂肪形成(见食谱)。
    3. 24小时后,分别在成骨和脂肪形成条件下添加成骨或脂肪生成诱导培养基(见食谱)。&nbsp;
    4. 每3-4天更换一次感应培养基,让细胞分化14-21天。&nbsp;

  6. 用于评估成骨过程中钙沉积的茜素红染色
    1. 吸出培养基并用PBS洗涤细胞两次。
    2. 将细胞在4%甲醛溶液中(参见食谱)在室温下固定1小时。&nbsp;
    3. 用dH 2 O洗涤细胞两次。
    4. 用新鲜制备的茜素红染色溶液(见食谱)在室温下染色10分钟。&nbsp;
    5. 用dH 2 O洗涤细胞5次,然后用PBS洗涤15分钟以除去过量的染色剂。在细胞中加入PBS以防止它们干燥。&nbsp;
    6. 继续在倒置显微镜下用相机对细胞成像(图4A)。&nbsp;

  7. 用于评估脂肪生成期间脂质积累的油红O染色
    注意:染色前至少1小时准备油红O染色液(见食谱)。
    1. 吸出培养基并用PBS洗涤细胞两次。
    2. 将细胞在4%甲醛溶液中(参见食谱)在室温下固定1小时。&nbsp;
    3. 取出定影液,用60%异丙醇在dH 2 O中冲洗。&nbsp;
    4. 用油红O染色溶液染色细胞10分钟。
    5. 用dH 2 O洗涤细胞五次。向细胞中加入PBS以防止它们干燥。
    6. 继续用倒置显微镜在相机下成像细胞(图4B)。&nbsp;

  8. 软骨细胞分化&nbsp;
    1. 如程序B中所述,胰蛋白酶消化并计数EOM-MSC。
    2. 将沉淀重新悬浮在合适的体积中以产生1.6×10 5个细胞/ ml的细胞悬浮液。将5μl这种细胞悬浮液滴加到96孔板中每个孔的中心,通过将板在5%CO 2 培养箱中37°温育2小时使细胞附着。 C.
    3. 2小时后,向孔中加入足够的(100μl)软骨形成分化培养基(参见食谱),不会干扰细胞微团,并将培养板返回培养箱。
    4. 每2天更换软骨形成培养基,使细胞分化为≥1%。 14天。

  9. Safranin O染色检测软骨,粘蛋白和肥大细胞颗粒
    1. 吸出培养基并用PBS洗涤细胞两次。
    2. 用4%甲醛溶液(见食谱)将细胞在室温下固定1小时。&nbsp;
    3. 用dH 2 O洗涤细胞两次。
    4. 用0.1%番红O溶液(见配方)在室温下染色5分钟。
    5. 用dH 2 O洗涤细胞。在细胞中加入PBS以防止它们干燥。&nbsp;
    6. 继续使用倒置显微镜在相机下对细胞进行成像(图4C)。


      图4. EOM-MSCs分化为成骨细胞,脂肪细胞和软骨细胞。 EOM-MSCs接种于各自的细胞密度和诱导培养基中,用于分化为成骨细胞,脂肪细胞和软骨细胞谱系细胞。分化的细胞用(A)茜素红,(B)油红o(C)番红素染色,分别用于确定成骨,脂肪形成和软骨形成分化。比例尺:100μm。

数据分析

使用FlowJo软件分析用于在EOM-MSC上表达各种表面标志物的流式细胞术数据。在FL3对FSC图中基于碘化丙啶阴性(FL3低)群体对活细胞进行门控,然后在FSC对比SSC图中进行门控以除去细胞碎片。使用适当的同种型匹配的对照抗体,绘制FL1中的信号(对于FITC-缀合的抗体)和FL2(对于PE-缀合的抗体)的直方图以定义阴性(未染色)和阳性(染色)群体。该协议仅显示一个EOM-MSC样品的代表性数据,不包括任何统计分析。

食谱

  1. DMEM媒体
    1. DMEM-LG(低葡萄糖)/ DMEM-HG(高葡萄糖)基础培养基
      10克DMEM-LG或DMEM-HG介质粉末
      3.7克碳酸氢钠
      10毫升100x青霉素 - 链霉素抗生素溶液
      用高压灭菌的去离子H 2 O将体积补足至1L 将pH调节至~7.0,因为过滤后pH趋于增加
      使用无菌过滤装置(0.22μm)过滤灭菌
      可在4°C下储存长达2个月
    2. MSC生长介质
      DMEM-LG基础培养基中10%FBS
      使用无菌过滤装置(0.22μm)过滤灭菌
      储存在4°C并在两周内使用
  2. 组织收集介质
    2x青霉素 - 链霉素抗生素在DMEM-LG基础培养基或HBSS溶液中
  3. 纤连蛋白溶液(400 ng / ml)
    通过在100 ml dH 2 O中稀释40μl的1 mg / ml储备溶液来制备纤连蛋白的工作储备。
    使用无菌过滤装置(0.22μm)通过过滤灭菌并在4℃下储存
  4. 磷酸盐缓冲盐水(PBS)
    1. 10x PBS
      80克NaCl
      2克KCl
      14.4g Na 2 HPO 4
      2.4 g KH 2 PO 4
      用高压灭菌的dH 2 O将体积补足至1L 使用无菌过滤装置(0.22μm)过滤灭菌
      在室温下储存
    2. 1x PBS
      在H 2 O中稀释10倍PBS(10毫升,1毫升)10次 将pH调节至7.4并在121℃下高压灭菌40分钟
      C。 PBS含1x抗生素
      在10ml 1x PBS中的100μl100x青霉素 - 链霉素抗生素
  5. 1x胰蛋白酶(0.25%)
    在无菌冷1x PBS中稀释10倍胰蛋白酶10次(1 ml 10x胰蛋白酶在9 ml PBS中)并准备5 ml等分试样用于
    将等分试样储存在-20°C下长期储存。在室温下解冻并在使用后储存在4°C下长达1周
  6. 0.4%台盼蓝
    将50毫克台盼蓝(染料组合物40%)溶于5毫升1×PBS中 使用无菌过滤装置(0.22μm)过滤灭菌
    在4°C下保存为等分试样&nbsp;
  7. 热灭活FBS
    通过在水浴中于56℃温育1小时来加热灭活FBS,并在-20℃下以50ml等分试样储存
  8. 冻结媒体
    在热灭活的FBS中的20%DMSO(在4ml FBS中的1ml DMSO)
  9. FACS缓冲区
    1x PBS中2%热灭活的FBS(在9.8 ml PBS中200μlFBS)
  10. 碘化丙啶染色液
    在FACS缓冲液中加入2μg/ ml碘化丙啶
  11. 成骨诱导培养基
    DMEM-高葡萄糖基础培养基
    1x青霉素 - 链霉素抗生素
    10%FBS
    10mMβ-甘油磷酸盐
    0.1μM地塞米松
    0.05 mM抗坏血酸-2-磷酸盐
    注意:成骨诱导培养基可在4°C下储存长达4周。
  12. 脂肪生成诱导培养基
    DMEM-高葡萄糖基础培养基
    1x青霉素 - 链霉素抗生素
    10%FBS
    1μM地塞米松
    0.2mM吲哚美辛(加入2滴5M NaOH溶解)
    0.5 mM IBMX
    0.01 M胰岛素(加入2滴5 M HCl溶解)
    注意:脂肪生成诱导培养基可在4°C下储存长达4周。
  13. 4%甲醛溶液
    在1x PBS中稀释37%-41%甲醛溶液10次
    1毫升37%-41%甲醛溶液
    9毫升1x PBS
  14. 茜素红染色液
    0.19克茜素红S
    10毫升dH 2 O
    将pH调节至4.2
  15. 油红O解决方案
    1. 1%油红O原料溶液
      1克油红O
      100毫升异丙醇
      在56°C的水浴中温热30-60分钟。并非所有油红O都会溶解
      可在室温下储存长达6个月
    2. 油红O染色液
      3份1%油红O原液
      2份dH 2 O
      混合并静置1小时
      用Whatman滤纸过滤溶液
  16. 软骨分化培养基
    10 ml 10x Stem Pro软骨细胞生成补充剂
    90 ml Stem Pro骨细胞/软骨细胞分化基础培养基
    1x青霉素 - 链霉素抗生素
    注意:软骨形成分化培养基可在4°C下储存长达4周。
  17. 0.1%番红O染色液
    0.1克Safranin O
    100毫升dH 2 O
    搅拌帮助解散

致谢

这项工作得到了印度医学研究委员会(ICMR)政府的支持。印度协议改编自Mawrie 等人(2016)。作者感谢Trishna Anand和Vishnu在成像方面提供的帮助。

利益争夺

作者宣称没有竞争利益。

伦理

该研究得到了IIT Guwahati伦理委员会的批准,并在患者书面知情同意后收集样本。

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引用:Sharma, A., Mawrie, D., Magdalene, D. and Jaganathan, B. G. (2019). Isolation of Multipotent Mesenchymal Stem Cells from Human Extraocular Muscle Tissue. Bio-protocol 9(4): e3167. DOI: 10.21769/BioProtoc.3167.
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