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Jul 2019

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Isolation of Stem Cells, Endothelial Cells and Pericytes from Human Infantile Hemangioma
人婴幼儿血管瘤中干细胞、内皮细胞和周细胞的分离   

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Abstract

Infantile hemangioma (IH) is a vascular tumor noted for its excessive blood vessel formation during infancy, glucose-transporter-1 (GLUT1)-positive staining of the blood vessels, and its slow spontaneous involution over several years in early childhood. For most children, IH poses no serious threat because it will eventually involute, but a subset can destroy facial structures and impair vision, breathing and feeding. To unravel the molecular mechanism(s) driving IH-specific vascular overgrowth, which to date remains elusive, investigators have studied IH histopathology, the cellular constituents and mRNA expression. Hemangioma endothelial cells (HemEC) were first isolated from surgically removed IH specimens in 1982 by Mulliken and colleagues (Mulliken et al., 1982). Hemangioma stem cells (HemSC) were isolated in 2008, hemangioma pericytes in 2013 and GLUT1-positive HemEC in 2015. Indeed, as we describe here, it is possible to isolate HemSC, GLUT1-positive HemEC, GLUT1-negative HemEC and HemPericytes from a single proliferating IH tissue specimen. This is accomplished by sequential selection using antibodies against specific cell surface markers: anti-CD133 to select HemSC, anti-GLUT1 and anti-CD31 to select HemECs and anti-PDGFRβ to select HemPericytes. IH-derived cells proliferate well in culture and can be used for in vitro and in vivo vasculogenesis and angiogenesis assays.

Keywords: Hemangioma (血管瘤), Endothelial cells (内皮细胞), Pericytes (周细胞), Vascular tumor (血管肿瘤), Angiogenesis (血管生成)

Background

IH occurs in 4-5% of infants; it follows a unique life-cycle of rapid vascular growth, called the proliferating phase, followed by a slow spontaneous involuting phase (Leaute-Labreze et al., 2017). The proliferating phase contains immature vascular endothelial growth factor receptor-2 (VEGFR2)+ cells that appear to be in the process of vasculogenesis–the assembly of new vessels from stem/progenitor cells (Yu et al., 2004; Boscolo and Bischoff, 2009). The involuting phase begins after 12 months of age; well-defined vascular channels become evident yet little is known about mechanisms of involution except that endothelial apoptosis increases in the involuting phase (Mancini and Smoller 1996; Iwata et al., 1996; Razon et al., 1998). The residuum in the involuted phase is characterized by sparse vessels, adipocytes and connective tissue. This natural life-cycle of endothelial maturation and involution distinguishes IH from other vascular tumors and malformations, which do not regress and can grow at any time in a patient’s life. In 2008, we isolated a primitive mesenchymal cell from proliferating phase IH that can differentiate into endothelial cells, pericytes and adipocytes and form hemangioma-like (GLUT1-positive) vessels within 7 days after being implanted sub-cutaneously into immune-deficient mice (Khan et al., 2008). We designated these hemangioma stem cells (HemSC); subsequent studies validated HemSCs as the IH-initiating cell (Greenberger et al., 2010; Itinteang et al., 2011; Xu et al., 2011; Mai et al., 2013; Harbi et al., 2016; Edwards et al., 2017). Here we describe in detail how to isolate HemSC from proliferating phase IH, and at the same time isolate GLUT1-positive HemEC, GLUT1-negative HemEC and HemPericytes; this allows detailed studies on patient-derived cells that represent the vascular cellular constituents of IH. In principal, the strategy could be broadly applied to other types of vascular tumors and vascular malformations, in particular isolation of endothelial cells from venous malformations (Goines et al., 2018), capillary malformations (Huang et al., 2017), arteriovenous malformations (Couto et al., 2017) and lymphatic malformations (Boscolo et al., 2015).

Materials and Reagents

  1. Disposable scalpel
  2. 15 ml and 50 ml sterile Falcon® conical tubes
  3. 1.7 ml sterile microcentrifuge tube
  4. Tissue culture-treated 24-well and 6-well plates
  5. Falcon® 100 µm Cell Strainer (Corning, catalog number: 352360)
  6. Pre-Separation Filters (70 µm) (Miltenyi Biotec catalog number: 130-095-823)
  7. Nalgene Rapid-Flow disposable Filter Units with PES (polyethersulfone) membrane, 0.2 µm (Thermo Scientific, catalog number: 564-0020)
  8. MS Columns (Miltenyi Biotec, catalog number: 130-042-201)
  9. DynabeadsTM CD31 Endothelial Cell, human (Thermo Fisher, catalog number: 11155D)
  10. LiberaseTM (Roche, catalog number: 5401119001)
  11. Dispase (Corning, catalog number: 354235)
  12. Fetal Bovine Serum (HyClone, catalog number: SH30396.03)
  13. EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM (Lonza, catalog number: CC-3162)
  14. DMEM, high glucose, GlutaMAXTM Supplement (Thermo Fisher, catalog number: 10566-016)
  15. Fibronectin (FN) (Chemicon, catalog number: FC010; stock= 1 µg/µl)
  16. Phosphate buffered saline (PBS) 10x, without calcium and magnesium (Lonza, catalog number: 17-517Q)
  17. 100x GPS (L-Glutamine-Penicillin-Streptomycin, Corning, catalog number: 30-009-CI)
  18. 0.05% Trypsin 0.53 mM EDTA, 1x (Corning, catalog number: 25-052-CI)
  19. Red blood cell lysis buffer (Roche, catalog number: 11814389001)
  20. FcR Blocking Reagent, human (Miltenyi Biotec, catalog number: 130-059-901)
  21. CD133 MicroBead Kit (Miltenyi Biotec, catalog number: 130-050-801)
  22. DynabeadsTM Pan Mouse IgG (Thermo Fisher, catalog number: 11041)
  23. Dynabeads® M-270 Streptavidin (Thermo Fisher, catalog number: 65305)
  24. Human GLUT1 Antibody (R&D Systems, catalog number: MAB1418) 
  25. Human PDGFRβ Biotinylated Antibody (R&D Systems, catalog number: BAF385)
  26. CaCl2·2H2O
  27. MgSO4·7H2O
  28. Glucose
  29. Sodium citrate
  30. Citric acid
  31. BSA
  32. Na2CO3
  33. DMSO
  34. Heat inactivated FBS (hiFBS) (see Recipes)
  35. LiberaseTM stock (0.5 mg/ml) (see Recipes)
  36. Dispase stock (50 U/ml, 100 ml) (see Recipes)
  37. 10x Calcium and Magnesium (Ca2+/Mg2+) solution (see Recipes)
  38. Collection Medium (see Recipes)
  39. Digestion Buffer (see Recipes)
  40. 6% Citrate Dextrose Solution, Solution A (ACD-A) (see Recipes)
  41. Buffer A (see Recipes)
  42. Buffer B (see Recipes)
  43. EGM-2 medium (see Recipes)
  44. Fibronectin (FN)-coating buffer (see Recipes)
  45. Coating culture plates with fibronectin (FN) (see Recipes)
  46. Quenching/Thawing medium (see Recipes)
  47. Freezing medium (see Recipes)

Equipment

  1. Forcep
  2. Pipettes
  3. Pestle (Thomas Scientific, catalog number: 3431D94; the smooth pestle is 44 mm x 22 mm)
  4. Refrigerator
  5. Centrifuge (Eppendorf, model: 5804)
  6. Water Bath (Fisher Scientific, model: 2223)
  7. MiniMACS Separator (Miltenyi Biotec, catalog number: 130-042-102)
  8. DynaMagTM-2 Magnet (Thermo Fisher, catalog number: 12321D)
  9. -80 °C freezer
  10. -20 °C non-defrost freezer

Procedure

IH specimens should be obtained under an institutional review board (IRB)-approved human subject protocol. The clinical diagnosis should be confirmed by histopathology. Biosafety Level 2 procedures for working with human tissue should be followed. Sterile technique and sterile solutions should be used at each step of tissue homogenization, antibody-mediated selection and cell culture.


  1. Tissue collection and digestion
    1. Transfer IH tissue into a sterile container filled with 5-10 ml Collection Medium as soon as possible after resection. Place the tissue on ice and bring it to the lab.
    2. Rinse the tissue twice with sterile PBS to remove surface blood.
    3. If necessary, use a sterile scalpel and forcep to separate the inner soft IH tissue from the outer rough epidermis.
    4. Mince the IH tissue into ~2 mm3 pieces with a sterile, disposable scalpel.
    5. Transfer minced IH tissue into a 50 ml conical tube and mix with freshly-made Digestion Buffer using gentle pipetting. Use 5 volumes of Digestion Buffer per gram of tumor tissue (i.e., 5 ml Digestion Buffer/1 gram of minced IH).
    6. Incubate mixture at 37 °C. Mix every 5-10 min by tapping the tube. The tissue should appear soft and the solution should become turbid after 40-50 min. Do not over-digest (i.e., longer than 1 h) because the sample will become viscous due to cell lysis and DNA release.
    7. Gently homogenize the IH tissue digest with a Teflon pestle. Move pestle up and down and twist while squeezing the 50 ml tube. Let tumor pieces settle to the bottom of tube and transfer the supernatant to a new tube on ice.
    8. Add 5 ml Collection Medium to the remaining tissue in the original tube and repeat the homogenization; repeat once more. Combine the three supernatants.
    9. Filter the combined supernatants through a 100 µm sterile cell strainer.
    10. Wash cell strainer with 5-10 ml Collection Medium to flush cells through the strainer.
    11. Centrifuge cell suspension at 282 x g, room temperature (RT) for 5 min.
    12. Carefully aspirate supernatant and re-suspend the cell pellet with 5-10 ml Buffer A.
    13. Centrifuge cell suspension at 240 x g, RT for 5min. Aspirate the supernatant carefully.
    14. Optional: Lysis of red blood cells (RBCs) for the very red (i.e., blood filled) specimen
      1. Resuspend cells in 1 ml PBS and mix well with 7 ml ice-cold RBC lysis buffer.
      2. Incubate at 4 °C for 10 min with gentle shaking.
      3. Centrifuge at 240 x g, RT for 5 min. Aspirate the supernatant.
    15. Resuspend the cell pellet in 1 ml Buffer A. Determine cell number. Expect 1 x 106 -2 x 106 cells from 0.5 cm3 IH specimen. Use half of the cells for HemSC Purification (Procedure B) and the other for HemEC and HemPericyte Purification (Procedures C-E). See Figure 1 for a schematic of the cell purification strategy.


      Figure 1. Cell isolation using sequential antibody-coated magnetic beads. Schematic shows work flow for isolating four cell types from IH tissue.

  2. HemSC purification (see Miltenyi Biotec protocol for the additional information)
    1. Centrifuge cell suspension at 240 x g, RT for 5 min. Aspirate supernatant.
    2. Resuspend the cells in 300 µl ice-cold Buffer A.
    3. Add 100 µl human FcR blocking reagent, mix well by pipetting up and down 2-3 times.
    4. Add 100 µl CD133 Microbeads, mix well.
    5. Incubate for 30 min in the refrigerator (2-8 °C), gently tap tube every 5-10 min.
    6. Wash cells by adding 9.5 ml ice-cold Buffer A.
    7. Centrifuge cells at 240 x g, RT for 5 min. Aspirate supernatant.
    8. Resuspend cells in 500 µl ice-cold Buffer A.
    9. Pre-wash MS column by placing the MS column in the magnetic field and rinsing with 500 µl Buffer A. Wait until the column reservoir is empty.
    10. Place a 70 µm cell strainer on the top of column reservoir. Load cell suspension and let it run through cell strainer and enter MS column held in the magnet.
    11. Collect the effluent.
    12. Wash the MS column with 4x 500 µl Buffer A.
    13. Collect all effluent fractions and combine together; save as CD133-negative cells.
    14. Remove the column from the magnetic separator and place it on a 1.7 ml sterile microcentrifuge tube.
    15. Pipette 1 ml Buffer A onto the top of column and collect CD133-positive cells by firmly pushing the plunger (supplied) into the column.
    16. Optional: To increase the purity of CD133-positive cells, load the released cell fraction over a second MS column and repeat CD133 selection.
    17. Determine the number of cells in both CD133-positive and CD133-negative cell fractions. Centrifuge cell suspensions at 240 x g, RT for 5min. Remove supernatant. The number of CD133+ cells varies from 0.2% to 2% of total digested tumor cells, as reported by Yu et al., 2004. The yield depends on the size of the specimen, the enzyme digestion and batch of anti-CD133-coated beads.
    18. Resuspend CD133-positive cells in 1 ml EGM-2 media. Plate them in one-well of 24-well plate pre-coated with 1 µg/cm2 FN. The CD133-positive HemSC will start to grow rapidly in culture after 7-10 days and will exhibit a mesenchymal morphology (Figure 3) (Khan et al., 2008).
    19. Plate CD133-negative cells in one well of 6-well plate pre-coated with 1 µg/cm2 FN. After primary culture, cryopreserve cells for future use (Procedure H), for example the CD133-negative fraction can be used for selection of CD31+ and/or PDGFRβ+ cells.

  3. GLUT1-positive endothelial cell purification (see Pan mouse IgG Dynabeads protocol for the additional information)
    1. Centrifuge the other half of cell suspension at 240 x g, RT for 5 min.
    2. Resuspend cells (up to 106) in 80 µl ice-cold Buffer A.
    3. Add 20 µl human FcR blocking reagent.
    4. Incubate for 10 min in the refrigerator (2-8 °C).
    5. Add 1 µg of mouse anti-human GLUT1 antibody to 100 µl cell suspension–1 µg antibody/106 cells/100 µl buffer.
    6. Incubate for 30 min in the refrigerator (2-8 °C). Mix by gently tapping the tube every 5-10 min.
    7. Add 10 ml ice-cold Buffer A, mix well.
    8. Centrifuge at 240 x g, RT for 5min. Resuspend the cell pellet in 100 µl ice-cold Buffer B.
    9. Pre-wash Dynabeads: Transfer 5 µl of the Pan mouse IgG Dynabeads to a 1.7 ml sterile microcentrifuge tube. Add 1 ml ice-cold Buffer B and mix. Place the tube in a magnet for 1 min and aspirate supernatant.
    10. Remove the tube from the magnet and resuspend the washed Dynabeads with 5 µl ice-cold Buffer B.
    11. Add washed beads to anti-GLUT1 treated cells at 1.5 µl beads/106 cells/100 µl buffer. Mix well and incubate for 10 min in the refrigerator (2-8 °C). The bead to cell ratio can be increased to 2.5 µl beads/106 cells/100 µl buffer but avoid increasing the incubation time because this may increase non-specific binding.
    12. Add 1 ml ice-cold Buffer B. Place the tube in the magnet for 1 min. Anti-GLUT1-bound cells will move towards the magnet leaving the GLUT1-negative cells free in suspension. Gently collect the GLUT1-negative cell fraction into a new 15 ml conical tube.
    13. Remove the tube from the magnet. Add 1 ml ice-cold buffer B and mix well. Place the tube back to the magnet, collect and combine the un-bound cells in the same 15 ml tube.
    14. Repeat this wash step twice more.
    15. After the final wash, remove the tube from the magnet to release the bead-bound GLUT1-positive cells by adding 1 ml EGM-2 media to resuspend cells. Determine the cell number. Plate cells in one well of 24-well plate precoated with 1 µg/cm2 FN.
    16. The GLUT1-negative cells will be further purified using anti-CD31 magnetic beads selection as described in the next section.
      Optional: To increase the yield of GLUT1-positive cells, a second round of anti-mouse IgG beads selection can be applied in GLUT1-negative cell fraction. Repeat magnetic separation and plate cells in a new well of 24-well plate. Do not combine cells from the 1st selection. The GLUT1-positive endothelial cells will start to proliferate after 10-12 days in culture (Huang et al., 2015).
      Note: Almost all of GLUT1-positive cells in proliferating IH are endothelial cells (see Huang et al., 2015 Figure 1C), that is, they express endothelial markers CD31, VE-Cadherin and VEGRR2. After 2-3 weeks in vitro culture and expansion, the cells transition to a mesenchymal phenotype (Figure 3) (Huang et al., 2015).

  4. GLUT1-negative endothelial cells purification (see anti-human CD31 Dynabeads protocol for the additional information)
    1. Determine the number of cells in the GLUT1-negative fraction. Centrifuge cell suspension at 240 x g, RT for 5 min. Aspirate the supernatant.
    2. Resuspend the cells in 200 µl ice-cold buffer B.
    3. Pre-wash Dynabeads: Transfer 5 µl anti-human CD31 Dynabeads to a 1.7 ml sterile microcentrifuge tube. Add 1 ml ice-cold Buffer B and mix. Place the tube in a magnet for 1 min and aspirate supernatant.
    4. Remove the tube from the magnet and resuspend washed beads with 5 µl ice-cold Buffer B Add 2 µl washed anti-human CD31 Dynabeads to the cells and incubate for 10 min in the refrigerator (2-8 °C).
      Note: 5-8 beads per cell is optimal for selection (Figure 2). If too many beads bind, it will reduce endothelial cell attachment to the FN-coated dish. Do not incubate for longer than 10 min as this might result in non-specific binding and decreased purity.


      Figure 2. Anti-CD31-bead bound cells

    5. Add 1 ml ice-cold Buffer B. Place the tube in the magnet and hold for 1 min. Anti-CD31 Dynabead bound cells will move towards the magnet (accumulating on the wall of the tube) leaving the unbound cells, the CD31-negative cell fraction, free in suspension. Gently collect the CD31-negative cell fraction into a new 15 ml conical tube.
    6. Remove the tube from the magnet. Add 1 ml ice-cold buffer B and mix well. Repeat magnetic separation. Collect and combined bead un-bound cells into the same 15 ml tube.
    7. Repeat wash step two more times.
    8. After the final wash, remove the tube from the magnet and release the bead-bound CD31-positive cells in 1 ml EGM-2 media (Figure 2). Determine the cell number and plate cells in one well of 24-well plate precoated with 1 µg/cm2 FN.
    9. GLUT1-negative HemECs will require 7-10 days to begin rapid proliferation. If needed, repeat the anti-CD31 selection to increase the endothelial cell purity. GLUT1-negative HemEC show typical endothelial morphology (Figure 3).
      Optional: To increase the yield of CD31-positive cells, a second round of anti-CD31 beads selection can be applied in the CD31-negative cell fraction. Repeat magnetic separation and plate cells in a new well of 24-well plate. Do not combine cells from the first selection.


      Figure 3. IH cells in the primary culture

  5. PDGFRβ-positive cell purification (see Dynabeads® M-270 Streptavidin protocol for the additional information)
    1. Determine the number of cells in the CD31-negative cell fraction. Centrifuge the cell suspension at 240 x g, RT for 5 min. Aspirate the supernatant.
    2. Resuspend the cells in 80 µl ice-cold buffer A.
    3. Add 20 µl human FcR blocking reagent.
    4. Incubate for 10 min in the refrigerator (2-8 °C).
    5. Add 1 µg of biotinylated anti-PDGFRβ antibody to the 100 µl cell suspension.
    6. Incubate for 30 min in the refrigerator (2-8 °C). Mix by gently tapping tube every 5-10 min.
    7. Add 10 ml ice-cold buffer A, mix well.
    8. Centrifuge cell suspension at 240 x g, RT for 5min. Aspirate supernatant and resuspend cells in 100 µl ice-cold Buffer B.
    9. Pre-wash Dynabeads: Transfer 5 µl Streptavidin-coupled Dynabeads to a 1.7 ml microcentrifuge tube. Add 1 ml ice-cold Buffer B and mix. Place the tube in a magnet for 1 min and aspirate supernatant. Remove the tube from the magnet and resuspend the washed beads with 5 µl ice-cold Buffer B.
    10. Add 1.5 µl washed Streptavidin-coupled Dynabeads to the cells, mix well and incubate for 10 min in the refrigerator (2-8 °C).
    11. Add 1ml ice-cold Buffer B. Place the tube in the magnet for 1 min. PDGFRβ-positive cells will move towards the magnet leaving the PDGFRβ-negative cell fraction free in suspension. Gently collect the PDGFRβ-negative cell fraction into a new 15 ml conical tube.
    12. Remove the tube from the magnetic field. Add 1 ml ice-cold buffer B and mix gently by pipetting. Place the tube back to the magnet, collect and combine the un-bound cells in the same 15ml tube.
    13. Repeat wash step two more times, collect and combine PDGFRβ-negative cells.
    14. After the final wash, collect PDGFRβ-positive cells in 1 ml EGM-2 media, determine cell number and plate into one well of 24-well plate precoated with 1 µg/cm2 FN.
      Note: PDGFRβ-positive cells–HemPericytes–will start to expand after 5-7 days (Figure 3). 
    15. Determine the number of PDGFRβ-negative cells. Plate cells into one well of 6-well plate precoated with 1 µg/cm2 FN and filled with 2ml EGM-2 media. These are stromal cells.
    16. Alternatively, the Hem Pericytes can be plated on a non-coated dish in 10% FBS-DMEM as described in Boscolo et al., 2013.

  6. Expanding bead-selected IH cells
    1. Forty-eight hours after plating, carefully remove media and non-adherent cells with a pipette. Add fresh EGM-2 media into each well. You should see attached single cells and/or cell clusters.
    2. Change the media every 2-3 days.

  7. Passaging IH-derived cell
    1. To passage cells, first prepare FN-coated plates at concentration 0.1 µg/cm2.
      Note: This is 10 fold lower than the amount of FN coated on plates used for the initial plating.
    2. Wash plates with sterile PBS twice before use. The PBS from the 2nd wash should be removed right before the cells are ready to be plated.
    3. Wash cell monolayer with PBS twice.
    4. Aspirate PBS and add pre-warmed trypsin-EDTA (0.8 ml/p60, 1.5 ml/p100, 5 ml/p150). Gently rock the plates to evenly distribute the trypsin-EDTA.
    5. Incubate in the 37 °C incubator for 1-2 min. Gently tap the plates to detach cells.
    6. Inactivate the trypsin with quenching/thawing medium (5 ml/p60, 8 ml/p100; 15 ml/p150), transfer the trypsinized cells to a sterile tube.
    7. Recover remaining cells from the plate by adding quenching/thawing medium to rinse the plate. Collect the rinse and combine the cells into the same tube.
    8. Determine cell number, centrifuge the cell suspension at 240 x g, RT for 5min, and aspirate the supernatant.
    9. Resuspend the cells in EGM-2 media and plate cells on a FN-coated, tissue culture-treated dish at a cell density 5,000 cells/cm2 (sparse) or 10,000 cells/cm2.
      1. HemSC, GLUT1-positive HemEC and HemPericyte are usually plated at 5,000 cells/cm2. A 90% confluent p100 plate has 5 x 106-6 x 106 cells.
      2. GLUT1-negative HemEC are usually plated at 10,000 cells/cm2. A 90% confluent p100 plate has 3 x 106-4 x 106 cells.

  8. Freezing IH-derived cells
    1. When cells are confluent, wash the monolayer twice with PBS.
    2. Trypsinize the cells, collect the cell suspension and determine cell number.
    3. Centrifuge cell suspension at 240 x g, RT for 5 min.
    4. Label a cryovial with the passage number (+1), cell number, and date.
    5. Suspend the cell pellet at 1 x 106-5 x 106 cells in 1 ml freezing medium and aliquot 0.5-1 ml per cryovial.
    6. Put cryovials into the “Mr. Frosty” and place at -80 °C overnight.
    7. The next day, move cryovials to the liquid nitrogen tank for long-term storage.

  9. Thawing IH-derived cells
    1. Prepare FN-coated, tissue culture-treated plates (0.1 µg/cm2), pre-warmed EGM2 medium and quenching/thawing medium.
    2. Prepare 15 ml Falcon tubes. Add 5 ml quenching/thawing medium to each tube.
    3. Wash FN-coated plates with PBS twice.
    4. Quickly thaw frozen cells directly after removing from liquid nitrogen by placing in a 37 °C water bath for 1-2 min. Gently swirl the tube to facilitate thawing.
    5. Transfer cells into the 15 ml tube pre-filled with quenching/thawing medium (avoid vigorous pipetting). Determine cell number.
    6. Centrifuge cell suspension at 240 x g, RT for 5 min.
    7. Aspirate quenching/thawing medium. Resuspend cells in EGM-2 media and plate on FN-coated, tissue culture-treated dish (3 ml/p60; 10 ml/p100; 25 ml/p150).
    8. Label the coated plates with cell type, passage number, date, and initials.
    9. Cells should attach within 4-6 h after plating. Add fresh media every 2-3 days.

Data analysis

Analyze cellular phenotype and purity by flow cytometry, immunofluorescence and qPCR at passage 2-3. HemSC are positive for CD90 and VEGFR1 and negative for CD31 (Khan et al., 2008). GLUT1+ HemECs are initially positive for CD31, VE-cadherin and VEGFR2 by qPCR but with time in culture, these endothelial markers are no longer detected but instead the cells express the mesenchymal marker CD90 (see Huang et al., 2015). GLUT1-negative/CD31+ HemEC express CD31, VE-cadherin and VEGFR2 and do not express CD90 (Huang et al., 2015), HemPericytes express PDGFRβ, NG2, calponin,α-smooth muscle actin, NOTCH3 but not CD31 (Boscolo et al., 2013). Cells are re-analyzed as needed, for example before an in vivo experiment, to verify the phenotype. We typically use the cells between passages 4 and 10.

Notes

  1. The percentage of CD133+ cells varies among different IH specimens (Yu et al., 2004).
  2. The percentage of GLUT1+ endothelial cells is reduced in IH specimens from patients over one year of age (Huang et al., 2015).
  3. 1% gelatin in PBS can be substituted for FN for coating the culture plates.
  4. Always use freshly prepared EGM2-media to insure full and consistent activity of the growth factors in the Single Quots (VEGF-A, basic FGF, EGF and IGF1). Once the media is prepared it can be stored in working aliquots at -20 °C until use. Media stored at 2-8 °C should be used within 3-4 days of preparation or thawing.

Recipes

  1. Heat inactivated FBS (hiFBS)
    1. Thaw a 500 ml bottle of HyClone FBS in warm water (for several hours) or at 4 °C overnight
    2. Put the thawed FBS into the 56 °C pre-heated water bath for 30 min (make sure the water covers all of FBS in the bottle. Mix the FBS in the bottle by shaking/inverting every 10 min to achieve an even temperature throughout the 500 ml bottle
    3. Allow FBS to cool to room temperature
    4. Aliquot 45 ml into sterile 50 ml tubes and store in a -20 °C non-defrost freezer
    5. Thaw a 45 ml frozen aliquot of heat-inactivated (hi)-FBS at 37 °C for about 20 min or at 4 °C overnight before use
  2. LiberaseTM Stock (0.5mg/ml)
    5 mg lyophilized LiberaseTM
    1. Dissolve in 10 ml sterile ddH2O
    2. Make 0.5 ml aliquots and store at -20 °C
  3. Dispase stock (50 U/ml, 100 ml)
    Make 5 ml aliquots and store at -20 °C
  4. 10x Ca2+/Mg2+ solution, 500 ml
    927 mg CaCl2·2H2O (final concentration: 1.26 mM)
    1.0 g MgSO4·7H2O (final concentration: 0.8 mM)
    1. Dissolve in sterile ddH2O to a final volume of 500 ml
    2. Filter (0.2 μm) and store at room temperature
  5. Collection medium, 100 ml
    87 ml DMEM (high glucose, GlutaMAXTM Supplement)
    10 ml 10x Ca2+/Mg2+ Solution
    2 ml hi-FBS
    1 ml 100x GPS
    1. Mix and filter (0.2 μm)
    2. Aliquot to 5 or 10 ml/tube and store at -20 °C for up to 3 months
  6. Digestion buffer, 6 ml (make fresh) 
    1. Thaw 5 ml Collection Medium
    2. Add 0.5 ml LiberaseTM (0.5 mg/ml) (1:10; working concentration: 50 µg/ml)
    3. Add 0.5 ml Dispase (50 U/ml) (1:10; working concentration: 5 U/ml)
  7. 6% ACD-A solution, 1 L
    22.3 g Glucose
    22.0 g Sodium citrate
    8.0 g Citric acid
    1. Dissolve in ddH2O to a final volume of 1 L
    2. Filter (0.2 μm) and store at 4 °C
  8. Buffer A (PBS/0.6% ACD-A/0.5% BSA), 500 ml
    2.5 g BSA
    50 ml 6% ACD-A solution
    450 ml PBS
    Dissolve, filter (0.2 μm) and store at 4 °C
  9. Buffer B (PBS/0.6% ACD-A/0.1% BSA), 500 ml
    0.5 g BSA
    50 ml 6% ACD-A solution
    450 ml PBS
    Dissolve, filter (0.2 μm) and store at 4 °C
  10. EGM-2 Medium, 500 ml
    445 ml EBM-2
    50 ml hiFBS
    5 ml 100x GPS
    EGM-2 Single Quot supplements (all except hydrocortisone)
    Filter (0.2 μm), aliquot (45 ml/tube) and store at -20 °C until needed or store at 4 °C and use within 3-4 days
  11. Fibronectin (FN)-coating buffer (0.1 M Na2CO3), 500 ml
    5.3 g of Na2CO3
    1. Dissolve in ddH2O to a final volume of 500 ml
    2. Adjust pH to 9.4 with 12 N HCl
    3. Filter (0.2 µm) and store at room temperature
  12. Coating culture plates with fibronectin (FN)
    1. Calculate the amount of FN needed; the stock is 1 µg/µl
    2. Use 1 μg/cm2 for freshly-isolated cells and 0.1 μg/cm2 for expansion of primary culture cells
    3. Dilute FN into just enough FN-coating buffer to cover the plate(s)
    4. Mix well and add to plates with a sterile pipette
    5. Incubate in the 37 °C incubator for 30 min or up to overnight (add more coating buffer for overnight incubation in case of evaporation)
  13. Quenching/Thawing medium (500 ml)
    445 ml DMEM (high glucose, GlutaMAXTM Supplement)
    50 ml hiFBS (final 10%)
    5 ml 100x GPS
    Filter (0.2 µm) and store at 4 °C
    EGM-2 media can also be used to quench but it is more expensive than DMEM
  14. Freezing medium (make fresh)
    95% hiFBS
    5% DMSO
    Filter (0.2 µm) and store at 4 °C

Acknowledgments

The development of the methods described in this manuscript was supported by the NHLBI of the National Institutes of Health under award number R01 HL096384 to J.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Competing interests

The authors have no financial and non-financial competing interests to disclose.

Ethics

IH tissue used for these isolation procedures was obtained under an IRB-approved protocol from the Committee on Clinical Investigation at Boston Children’s Hospital (04-12-175R; 11/11/2010 – 6/6/2020). Informed consent was obtained from parents or guardians of the subjects.

References

  1. Boscolo, E. and Bischoff, J. (2009). Vasculogenesis in infantile hemangioma. Angiogenesis 12(2): 197-207.
  2. Boscolo, E., Mulliken, J. B. and Bischoff, J. (2013). Pericytes from infantile hemangioma display proangiogenic properties and dysregulated angiopoietin-1. Arterioscler Thromb Vasc Biol 33: 501-509.
  3. Boscolo, E., Coma, S., Luks, V. L., Greene, A. K., Klagsbrun, M., Warman, M. L. and Bischoff, J. (2015). AKT hyper-phosphorylation associated with PI3K mutations in lymphatic endothelial cells from a patient with lymphatic malformation. Angiogenesis 18 (2): 151-162.
  4. Couto, J. A., Huang, A. Y., Konczyk, D. J., Goss, J. A., Fishman, S. J., Mulliken, J. B., Warman, M. L. and Greene, A. K. (2017). Somatic MAP2K1 mutations are associated with extracranial arteriovenous malformation. Am J Hum Genet 100(3): 546-554.
  5. Edwards, A. K., Glithero, K., Grzesik, P., Kitajewski, A. A., Munabi, N. C., Hardy, K., Tan, Q. K., Schonning, M., Kangsamaksin, T., Kitajewski, J. K., Shawber, C. J. and Wu, J. K. (2017). NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma. JCI Insight 2(21).
  6. Goines, J., Li, X., Cai, Y., Mobberley-Schuman, P., Metcalf, M., Fishman, S. J., Adams, D. M., Hammill, A. M. and Boscolo, E. (2018). A xenograft model for venous malformation. Angiogenesis 21(4): 725-735.
  7. Greenberger, S., Boscolo, E., Adini, I., Mulliken, J. B. and Bischoff, J. (2010). Corticosteroid suppression of VEGF-A in infantile hemangioma-derived stem cells. N Engl J Med 362(11): 1005-1013.
  8. Harbi, S., Wang, R., Gregory, M., Hanson, N., Kobylarz, K., Ryan, K., Deng, Y., Lopez, P., Chiriboga, L. and Mignatti, P. (2016). Infantile hemangioma originates from a dysregulated but not fully transformed multipotent stem cell. Sci Rep 6: 35811.
  9. Huang, L., Couto, J. A., Pinto, A., Alexandrescu, S., Madsen, J. R., Greene, A. K., Sahin, M. and Bischoff, J. (2017). Somatic GNAQ mutation is enriched in brain endothelial cells in sturge-weber syndrome. Pediatr Neurol 67: 59-63.
  10. Huang, L., Nakayama, H., Klagsbrun, M., Mulliken, J. B. and Bischoff, J. (2015). Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells. Stem Cells 33(1): 133-145.
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  15. Mai, H. M., Zheng, J. W., Wang, Y. A., Yang, X. J., Zhou, Q., Qin, Z. P. and Li, K. L. (2013). CD133 selected stem cells from proliferating infantile hemangioma and establishment of an in vivo mice model of hemangioma. Chin Med J (Engl) 126(1): 88-94.
  16. Mancini, A. J. and Smoller, B. R. (1996). Proliferation and apoptosis within juvenile capillary hemangiomas. Am J Dermatopathol 18(5): 505-514.
  17. Mulliken, J. B., Zetter, B. R. and Folkman, J. (1982). In vitro characteristics of endothelium from hemangiomas and vascular malformations. Surgery 92(2): 348-353.
  18. Razon, M. J., Kraling, B. M., Mulliken, J. B. and Bischoff, J. (1998). Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation 5(2-3): 189-195.
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简介

[摘要] 婴儿血管瘤(IH)是一个血管瘤注意到对于其过度血液血管形成期间婴儿期,葡萄糖转运蛋白1 (GLUT1)阳性染色的的血液血管,并且其缓慢自发复旧超过几年中早期童年。对于大多数孩子来说,IH 不会构成严重威胁,因为 它最终将渐开线,但其中一部分会破坏面部结构并损害视力,呼吸和进食。为了解开的分子机制(多个)驱动IH-特定血管过度生长,这给日期遗体难以捉摸,研究者已经研究了IH 组织病理学,所述蜂窝成分和mRNA的表达。血管瘤ë ndothelial 细胞(HemEC )进行第一分离从手术取出IH 试样在1982年由马利肯和同事小号(马利肯等人,1982 )。于2008年分离出血管瘤干细胞(HemSC ),2013年分离出血管瘤周细胞,2015年分离出GLUT1阳性的HemEC 。事实上,正如我们在这里描述,有可能分离HemSC ,GLUT1- 正HemEC ,GLUT1阴性HemEC 和HemPericytes 从一个单一的增殖IH 组织标本。这是实现通过顺序选择使用的抗体针对特定细胞表面标记物:抗CD133 到选择HemSC ,抗GLUT1 和抗CD31 来选择HemECs 和抗PDGFRβ 到选择HemPericytes 。IH衍生的细胞增殖以及在培养和可被用于为在体外和体内血管生成和血管生成测定。

[ 背景] IH 发生在4-5%的婴儿中;它遵循一个独特的生命周期的快速血管生长,称为的增殖阶段,随后通过一个慢的自发消退相(Leaute-Labreze 等人。,2017) 。该p roliferating相含有不成熟血管内皮生长因子受体-2(VEGFR2)+,似乎是在过程的细胞血管生成-the的组件从干/祖细胞新血管(于等人,2004;和博斯科洛比肖夫, 2009)。在12个月后,年龄消退期开始; 清晰可见的血管通道变得明显,但对内旋机制的了解却很少,只是在内旋期中内皮细胞凋亡增加了(Mancini and Smoller 1996; Iwata 等,1996; Razon 等,1998)。渐开线期的残留物的特征是稀疏的血管,脂肪细胞和结缔组织。内皮成熟和退化的自然生命周期将IH与其他血管肿瘤和畸形区分开来,后者不会消退,并且可以在患者生命中的任何时候生长。在2008年,我们从增殖期IH中分离了原始的间充质细胞,该细胞可分化为内皮细胞,周细胞和脂肪细胞,并在皮下植入免疫缺陷小鼠后7天内形成血管瘤样(GLUT1阳性)血管(Khan 等人,2008)。我们将这些血管瘤干细胞(HemSC )命名为;随后的研究证实HemSCs 是IH起始细胞(Greenberger 等,2010; Itinteang 等,2011; Xu 等,2011; Mai 等,2013; Harbi 等,2016; Edwards 等, 2017)。在这里,我们详细描述如何从增殖期IH 分离HemSC ,同时分离GLUT1阳性HemEC ,GLUT1阴性HemEC 和HemPericytes ;这可以对代表IH血管细胞成分的患者来源细胞进行详细研究。原则上,该策略可广泛应用于其他类型的血管肿瘤和血管畸形,特别是从静脉畸形中分离内皮细胞(Goines 等,2018),毛细血管畸形(Huang 等,2017),动静脉畸形(库托等人。,2017)和淋巴管畸形小号(博斯科洛等人,2015 )。

关键字:血管瘤, 内皮细胞, 周细胞, 血管肿瘤, 血管生成

材料和试剂


 


一次性手术刀
15毫升和50毫升无菌猎鹰® 锥形管
1.7 ml无菌微量离心管
组织培养处理的24孔和6孔板
鹘® 100微米的细胞过滤(Corning公司,目录号:352360)
分离前过滤器(70 µm)(Miltenyi Biotec 目录号:130-095-823)
与PES(NALGENE快速流量一次性过滤单元的聚醚砜)膜,0.2 微米(Thermo Scientific的,Ç :564-0020 atalog数)
MS色谱柱(Miltenyi Biotec ,目录号:130-042-201)
Dynabeads TM CD31内皮细胞,人类(Thermo Fisher,目录号:11155D)
Liberase TM (罗氏(Roche),目录号:5401119001 )
Dispase (Corning,目录号:354235)
胎牛血清(HyClone ,目录号:SH30396.03)
EGM TM -2内皮细胞生长培养基-2 BulletKit TM (Lonza,目录号:CC-3162)
DMEM,高葡萄糖,GlutaMAX TM Supplement(Thermo Fisher,目录号:10566-016)
纤连蛋白(FN)(Chemicon ,目录号:FC010;储备液= 1 µg / µl)
磷酸盐缓冲盐水(PBS)10x,不含钙和镁(Lonza,目录号:17-517Q)
100x GPS(L-谷氨酰胺-青霉素-链霉素,康宁,目录号:30-009-CI)
0.05%胰蛋白酶0.53 mM EDTA,1x(Corning,目录号:25-052-CI)
红细胞裂解缓冲液(罗氏,目录号:11814389001)
FcR 阻断剂,人类(Miltenyi Biotec ,目录号:130-059-901)
CD133 MicroBead 试剂盒(Miltenyi Biotec ,目录号:130-050-801)
Dynabeads TM 泛小鼠IgG(Thermo Fisher,目录号:11041)
的Dynabeads ® M-270链亲和素(赛默飞世,目录号:65305)
人类GLUT1抗体(R&d 小号ystems,目录号:MAB1418)
人类PDGFRβ生物素化抗体(R&d 小号ystems,目录号:BAF385)
氯化钙2 ·2H 2 O
MgSO 4 ·7H 2 O
葡萄糖
柠檬酸钠
柠檬酸
牛血清白蛋白
Na 2 CO 3
二甲基亚砜
              热灭活的FBS(hiFBS )(请参阅食谱)
Liberase TM 储备液(0.5 mg / ml)(请参阅食谱)
分配储备液(50 U / ml,100 ml)(请参阅食谱)
10x钙和镁(Ca 2+ / Mg 2+ )溶液(请参阅食谱)
收集介质(请参见食谱)
消化缓冲液(请参阅食谱)
6%柠檬酸葡萄糖溶液,溶液A(ACD-A)(请参阅食谱)
缓冲区A(请参阅食谱)
缓冲区B(请参阅食谱)
EGM-2培养基(请参阅食谱)
纤连蛋白(FN)涂层缓冲液(请参见食谱)
用纤连蛋白(FN)包被培养板(见食谱)
淬火/解冻介质(请参见食谱)
冷冻介质(请参见食谱)
 


设备


 


镊子
移液器
杵(Thomas Scientific,目录号:3431D94;光滑的杵为44 mm x 22 mm)
冰箱
离心机仪(Eppendorf,米Odel等:5804)
水浴(Fisher Scientific公司,米奥德尔:2223)
MiniMACS 分离器(Miltenyi Biotec ,目录号:130-042-102)
DynaMag TM -2磁铁(Thermo Fisher,目录号:12321D)
-80°C冷冻室
-20°C无霜冰柜




程序


 


IH标本应在机构审查委员会(IRB)批准的人类受试者规程下获得。临床诊断应通过组织病理学证实。应遵循用于人体组织的生物安全2级程序。在组织均质化,抗体介导的选择和细胞培养的每个步骤均应使用无菌技术和无菌溶液。


 


组织收集和消化
切除后尽快将IH组织转移到装有5-10 ml收集培养基的无菌容器中。将组织放在冰上,并带到实验室。
用无菌PBS冲洗组织两次以去除表面血液。
如有必要,使用无菌手术刀和镊子将内部软IH组织与外部粗糙表皮分开。
用无菌的一次性手术刀将IH组织切成约2 mm 3的小块。
将切碎的IH组织转移到50 ml锥形管中,并用温和的移液器与新鲜制成的Digestion Buffer混合。每克肿瘤组织使用5体积的消化缓冲液(即5毫升消化缓冲液/ 1克切碎的IH)。
在37°C下孵育混合物。轻敲试管,每5-10 分钟混合一次。40-50分钟后,组织应显得柔软,溶液应变浑浊。不要过度消化(即超过1小时),因为样品会由于细胞裂解和DNA释放而变得粘稠。
用铁氟龙杵轻轻匀浆IH组织消化液。挤压50毫升试管时,上下移动杵并扭转。让肿瘤碎片沉淀到试管底部,然后将上清液转移到冰上的新试管中。
5毫升收藏品添加Ñ 中等至剩余的组织在原管和重复的均质化; 再重复一次。结合了3个上清液。
过滤器的组合的上清液通过一个100 微米的无菌细胞滤网。
用5-10 毫升的Colle ction培养基洗涤细胞滤网,以冲洗通过滤网的细胞。
在282 xg ,室温(RT)下将细胞悬浮液离心5分钟。
小心吸出上清液,并用5-10 ml缓冲液A重悬细胞沉淀。
离心细胞悬液在240 xg ,室温下5分钟。小心吸出上清液。
可选:红血细胞(红细胞)的很红的裂解(即,血液填充)样本
重悬的细胞在1 ml的PBS 和混合以及与7 毫升冰冷的RBC裂解缓冲液。
轻轻摇动于4°C孵育10分钟。
离心机在240 X 克,RT FO - [R 5 分钟。吸出上清液。
将细胞沉淀重悬于1 ml缓冲液A中。确定细胞数。可以从0.5 cm 3 IH样本中获得1 x 10 6 -2 x 10 6个细胞。将一半的细胞用于HemSC 纯化(过程B),另一半用于HemEC 和HemPericyte 纯化(过程CE)。有关细胞纯化策略的示意图,请参见图1。
 


D:\陈丹工作\ 1902882--1252 JoyceBischoff 811375 \ Figs jpg \ Fig1.jpg


˚F 使用顺序抗体包被的磁珠igure 1.细胞分离。示意图显示了从IH组织中分离出四种细胞类型的工作流程。


 


HemSC 纯化(有关其他信息,请参见Miltenyi Biotec 方案)
离心细胞悬液于240 x g,室温离心5分钟。吸出上清液。
将细胞重悬于300 µl冰冷的缓冲液A中。
加入100 µl人FcR 封闭剂,上下吹打2-3次以充分混合。
加入100 µl CD133微珠,充分混合。
在冰箱(2-8°C)中孵育30分钟,每5-10 分钟轻轻敲打一次试管。
加入9.5 ml冰冷的Buffer A洗涤细胞。
于240 x g 离心细胞,室温离心5分钟。吸出上清液。
将细胞重悬于500 µl冰冷的缓冲液A中。
预洗MS 柱通过将所述MS 柱中的磁场和漂洗用500 微升缓冲液A. 等待直到所述列储层是空的。
放置一个70 微米的细胞滤网上的顶部的列容器。负载细胞悬液,并让它运行通过细胞过滤器和输入MS 柱举行中的磁铁。
收集的污水。
洗的MS 柱用4× 500 微升缓冲液A.
收集所有废水馏分并合并在一起;保存为CD133阴性细胞。
除去所述柱从所述磁分离器和放置其上一个1.7 毫升无菌微量离心管中。
移液管1 ml的卜FFER甲上到柱上,收集CD133阳性细胞的通过牢固地推动柱塞(提供)到塔的顶部。
可选:要提高CD133阳性细胞的纯度,可将释放的细胞级分加载到第二个MS色谱柱上,然后重复CD133选择。
确定CD133阳性和CD133阴性细胞级分中的细胞数。将细胞悬液以240 x g的速度离心5分钟。除去上清液。如Yu 等人报道,CD133 +细胞的数量在消化的肿瘤细胞总数的0.2%到2%之间变化。,2004。产量取决于样本的大小,酶消化和抗CD133包被的珠子的批次。
在1 ml EGM-2 培养基中重悬CD133阳性细胞。将它们放在预先涂有1 µg / cm 2 FN 的24孔板的一个孔中。CD133阳性的HemSC 将在7-10天后开始在培养物中快速生长,并且将表现出间充质形态(图3)(Khan 等,2008)。
板CD133阴性细胞在一个良好的6-W Ê LL 板预涂覆有1 微克/厘米2 FN。在原代培养后,可以冷冻保存细胞以备将来使用(程序H),例如CD133阴性级分可用于选择CD31 + 和/或PDGFRβ + 细胞。
 


GLUT1阳性内皮细胞纯化(有关更多信息,请参见Pan Mouse IgG Dynabeads 方案)
离心的其它半的细胞悬浮液在240 X 克,RT 5分钟。
在80 µl冰冷的缓冲液A中重悬细胞(最多10 6 )。
加入20 µl 人FcR 封闭剂。
孵育为10 分钟在该冰箱(2-8 ℃)。
添加1 微克的小鼠抗人GLUT1 抗体到100 微升细胞悬浮液- 1 微克抗体/ 10 6 个细胞/ 100 微升缓冲液。
孵育为30 分钟在该冰箱(2-8 ℃)。混合通过轻轻拍打该管每隔5-10 分钟。
加入10 ml 冰冷的缓冲液A,混合均匀。
离心机在240 X 克,RT 为5分钟。重悬的细胞沉淀在100 微升冰冷的缓冲液B.
预洗的Dynabeads :转移5 微升的所述泛小鼠的IgG 的Dynabeads 到一个1.7 毫升无菌微量离心管中。加入1 ml 冰冷的缓冲液B 并混合。放置在管中一个磁体为1 分钟和抽吸上清液。
除去所述管从所述磁体和重悬的洗涤的Dynabeads 用5 微升冰冷的缓冲液B.
将洗涤过的珠子以1.5 µl 珠子/ 10 6 细胞/ 100 µl 缓冲液的量添加到经抗GLUT 1 处理的细胞中。混合好并孵育为10 分钟在该冰箱(2-8 ℃)。所述胎圈到细胞比可以被提高至2.5 微升珠/ 10 6 个细胞/ 100 微升缓冲液但避免增加的温育时间,因为这可能会增加非特异性结合。
添加1 ml的冰冷的缓冲液B. 将所述管在所述磁铁为1 分钟。抗GLUT1结合细胞会移动朝的磁铁离开的GLUT1阴性细胞释放的悬挂。轻轻收集的GLUT1阴性细胞级分成一个新的15 ml的锥形管中。
除去所述管从所述磁体。加入1 ml 冰冷的缓冲液B 并充分混合。放置的管回至所述磁体,收集和结合所述未结合的细胞中的同一15 ml的试管中。
重复此洗涤步骤两次。
后的最终洗涤,除去所述管从所述磁铁到释放的珠结合的GLUT1阳性细胞通过加入1 ml的EGM-2 培养基,以悬浮细胞。确定的细胞数量。板的细胞在一个良好的24孔板预涂用1 微克/厘米2 FN。
的GLUT1阴性细胞将被进一步纯化,使用抗CD31 磁性珠选择如描述在该下一个部分。
可选:为了提高所述收率的GLUT1阳性细胞,一第二轮的抗小鼠IgG抗体磁珠选择可以被应用在GLUT1阴性细胞级分。重复磁分离和板的细胞在一个新井的24孔板。难道不是从第一选择相结合的细胞。培养10-12天后,GLUT1阳性内皮细胞将开始增殖(Huang 等,2015)。


注意:增殖的IH中几乎所有GLUT1阳性细胞都是内皮细胞(参见Huang等人,2015 图1C),即它们表达内皮标记物CD31,VE-钙黏着蛋白和VEGRR2。在2-3周的体外培养和扩增后,细胞转变为间充质表型(图3)(Huang等,2015 )。


 


GLUT1阴性内皮细胞纯化(有关其他信息,请参见抗人CD31 Dynabeads 方案)
Determi 甲肾上腺素的在GLUT1阴性级分的细胞的数目。于240 x g 离心细胞悬浮液,室温离心5分钟。吸出上清液。
重悬细胞我Ñ 200 微升冰冷的缓冲液B.
预洗的Dynabeads :传输5 微升抗人CD31 的Dynabeads 到一个1.7 毫升无菌微量离心管中。加入1 ml 冰冷的缓冲液B 并混合。放置在管中一个磁体为1 分钟和抽吸上清液。
除去所述管从所述磁体和重悬洗涤珠与5 微升冰冷的缓冲液乙加入2μl洗涤的抗人CD31 的Dynabeads 至细胞中并在冰箱(2-8℃)孵育10分钟。
注意:每个细胞5-8个珠子是选择的最佳选择(图2)。如果有太多的珠子结合,它将减少内皮细胞与FN涂层培养皿的附着。孵育时间不要超过10分钟,因为这可能会导致非特异性结合并降低纯度。


 


D:\陈丹工作\ 1902882--1252 JoyceBischoff 811375 \ Figs jpg \ Fig2.jpg


˚F igure 2.抗CD31珠结合的细胞


 


添加1 ml的冰冷的缓冲液B. 将所述管在所述磁体和保持为1 分钟。抗CD31 Dynabead 结合细胞将移动朝向所述磁体(累积上的壁的所述管)离开的未结合的细胞,所述CD31阴性细胞级分,游离在悬浮液中。轻轻收集的CD31阴性细胞级分成一个新的15 ml的锥形管中。
除去所述管从所述磁体。加入1 ml 冰冷的缓冲液B 并充分混合。重复磁分离。收集和组合珠未结合的细胞进入的同一15 ml的试管中。
重复洗涤步骤2 更多倍。
后的最终洗涤,除去所述管从所述磁体和释放的珠结合的CD31阳性细胞在1 ml的EGM-2 培养基(图2) 。确定的细胞数和板细胞在一个良好的24孔板预涂用1 微克/厘米2 FN。
GLUT1负HemECs 将需要7-10 天,以开始迅速扩散。如果需要的话,重复的抗CD31 的选择,以增加对内皮细胞的纯度。GLUT1阴性的HemEC 显示典型的内皮形态(图3)。
可选:为了提高所述收率的CD31阳性细胞,一第二轮的抗CD31 珠选择可以被应用在对CD31阴性细胞级分。重复磁分离和板的细胞在一个新井的24孔板。不要不结合细胞从的第一选择。


 


D:\陈丹工作\ 1902882--1252 JoyceBischoff 811375 \ Figs jpg \ Fig3.jpg


˚F igure 3. IH细胞在原代培养物


 


 


PDGFRβ阳性细胞的纯化(参见磁珠® 用于附加信息M-270链霉亲和协议)
确定的数目的细胞中的CD31阴性细胞级分。离心该细胞悬浮液在240 X 克,RT 为5 分钟。吸出上清液。
重悬的细胞在80 微升冰冷的缓冲液A.
加入20 µl 人FcR 封闭剂。
孵育为10 分钟在该冰箱(2-8 ℃)。
添加1 微克的生物素化的抗PDGFRβ 抗体到所述100 微升细胞悬浮液。
孵育为30 分钟在该冰箱(2-8 ℃)。混合通过轻轻拍打管每隔5-10 分钟。
加入10 ml 冰冷的缓冲液A,混合均匀。
在240 x g ,RT f 或5min下离心细胞悬液。吸上清液和重悬的细胞在100 微升冰冷的缓冲液B.
预洗的Dynabeads :传输5 微升链霉亲和耦合的Dynabeads 到一个1.7 ml的微量离心管中。加入1 ml 冰冷的缓冲液B 并混合。放置在管中一个磁体为1 分钟和抽吸上清液。除去所述管从所述磁体和重悬的洗涤珠粒与5 微升冰冷的缓冲液B.
添加1.5 微升洗涤链亲和素偶联的Dynabeads 到所述细胞中,混合好并孵育为10 分钟在该冰箱(2-8 ℃)。
添加1 ml的冰冷的缓冲液B. 将所述管在所述磁铁为1 分钟。PDGFRβ阳性细胞将移动朝向所述磁体离开的PDGFRβ阴性细胞级分游离在悬浮液中。轻轻收集的PDGFRβ阴性细胞级分成一个新的15 ml的锥形管中。
除去所述管从所述磁场。加入1 ml 冰冷的缓冲液B,然后通过移液轻轻混合。放置的管回至所述磁体,收集和结合所述未结合的细胞中的同一15 ml的试管中。
重复洗涤步骤2 更多的时间,收集并结合PDGFRβ阴性细胞。
后的最终洗涤,收集PDGFRβ阳性细胞在1 ml的EGM-2 培养基,确定细胞数和板到一个良好的24孔板预涂用1 微克/厘米2 FN。
注:PDGFRβ阳性细胞- HemPericytes - 将会开始后5-7天(图3)扩大。


确定的数目的PDGFRβ阴性细胞。板的细胞进入一个孔的6孔平板precoate d用1微克/厘米2 FN和填充有2毫升EGM-2培养基。这些是基质细胞。
备选地,如Boscolo 等人,2013中所述,可将下周周膜平板接种在10%FBS-DMEM中的未涂覆的皿上。
 


Ë xpanding 珠选择的IH细胞
铺板后48小时,用移液管小心除去培养基和非贴壁细胞。在每个孔中加入新鲜的EGM-2培养基。您应该看到附着的单个单元格和/或单元格群集。
每2-3 天更换一次介质。
 


 


IH传代细胞
到通道单元,第一制备FN涂覆的板在浓度0.1 微克/厘米2 。
注意:这比用于初始电镀的板上的FN量低10倍。


洗板用无菌PBS 两次之前使用。在PBS 从在第二洗应该被删除的权利之前的细胞都准备好要进行电镀。
用PBS 洗涤细胞单层两次。
吸PBS 和添加预热的胰蛋白酶-EDTA (0.8 ml的/ P60,1.5 ml的/ P100,5 ml的/ P150)。轻轻摇动该板,以均匀地分布的胰蛋白酶-EDTA。
孵育在该37 ℃的培养箱中进行1-2 分钟。轻轻敲击所述板到分离细胞。
灭活的胰蛋白酶与淬火/解冻介质(5 ml的/ P60,8 ml的/ P100; 15 ml的/ P150),转移的胰蛋白酶处理细胞,以一个无菌管中。
回收剩余的细胞从所述板通过加入淬火/解冻介质来漂洗所述板。收集的冲洗和结合的细胞进入的同一管中。
确定细胞数,将细胞悬液以24 0 x g 离心,室温离心5分钟,然后吸出上清液。
将细胞重悬在EGM-2培养基中,然后以5,000细胞/ cm 2 (稀疏)或10,000细胞/ cm 2 的细胞密度将细胞铺板在经过FN涂层的组织培养物处理过的培养皿上。
HemSC ,GLUT1阳性的HemEC 和HemPericyte 通常以5,000细胞/ cm 2 接种。90%融合的p100板具有5 x 10 6 -6 x 10 6个细胞。
GLUT1阴性的HemEC 通常以10,000细胞/ cm 2 接种。90%融合的p100板具有3 x 10 6 -4 x 10 6个细胞。
 


冷冻IH来源的细胞
当细胞是汇合时,冲洗所述单层两次用PBS。
胰蛋白酶化的细胞,收集的细胞悬浮液和确定细胞数。
于240 x g 离心细胞悬浮液,室温离心5分钟。
用通过次数(+1),单元格编号和日期标记一个冷冻管。
暂停在细胞沉淀1 X 10 6 - 5×10 6 细胞在1ml冷冻介质和等分0.5-1 毫升每冷冻管。
将冷冻管到的“先生。冰霜” 并在-80 °C下放置过夜。
在接下来的一天,移动冷冻管到所述液体氮罐用于长期存储。
 


解冻IH衍生的细胞
准备FN涂层的,经组织培养处理的板(0.1 µg / cm 2 ),预热的EGM2培养基和淬灭/融化培养基。
准备15 毫升Falcon 管。向每个管中添加5 ml 淬灭/解冻介质。
用PBS 洗涤FN包被的板两次。
迅速解冻冷冻的细胞直接后除去从液体氮通过放置在一个37 ℃的水浴中为1-2 分钟。轻轻晃动的管,以方便解冻。
将细胞转移到预装有淬灭/解冻介质的15 ml 管中(避免剧烈吸液)。确定单元格编号。
于240 x g 离心细胞悬浮液,室温离心5分钟。
抽吸淬火/解冻介质。将细胞重悬于EGM-2培养基中,并平板接种在FN包被的组织培养物处理过的培养皿中(3 ml / p60; 10 ml / p100; 25 ml / p150)。
标记的涂板用细胞类型,通道号,日期,和缩写。
细胞应在接种后4-6 小时内附着。每隔2-3 天添加新鲜的媒体。
 


资料分析


 


在第2-3代通过流式细胞仪,免疫荧光和qPCR分析细胞表型和纯度。HemSC 对于CD90和VEGFR1 是阳性的,而对于CD31是阴性的(Khan 等,2008)。GLUT1 + HemECs 最初通过qPCR对CD31,VE-钙粘着蛋白和VEGFR2呈阳性,但随着培养时间的推移,不再检测到这些内皮标志物,而是细胞表达间充质标志物CD90(参见Huang 等人,2015)。GLUT1- 阴性/ CD31 + HemEC 表达CD31,VE-钙黏着蛋白和VEGFR2,不表达CD90 (Huang 等,2015 ),半绒毛细胞表达PDGFRβ,NG2,钙蛋白,α-平滑肌肌动蛋白,NOTCH3但不表达CD31 (Boscololo 等人,2013)。根据需要对细胞进行重新分析,例如在体内实验之前,以验证表型。我们通常使用的细胞通路小号4和10 。


 


笔记


 


CD133 +细胞的百分比在不同的IH标本中有所不同(Yu 等,2004)。
超过1岁患者的IH标本中GLUT1 + 内皮细胞的百分比降低(Huang 等,2015 )。
PBS中的1%明胶可以代替FN,用于覆盖培养板。
始终使用新鲜制备的EGM2培养基,以确保单因子(VEGF-A,碱性FGF,EGF和IGF1)中生长因子的活性完全一致。制备好介质后,可以将其等份保存在-20°C下直至使用。准备或解冻后的3-4天内应使用2-8 °C 储存的培养基。
 


菜谱


 


热灭活FBS(hiFBS )
将500 ml的HyClone FBS 瓶在温水中解冻(持续数小时)或在4°C下过夜。
将解冻的FBS放入56°C的预热水浴中30分钟(确保水覆盖瓶中的所有FBS。每隔10分钟摇动/倒置一次,将FBS混合在瓶中,以使整个过程中温度均匀) 500毫升瓶。
让FBS冷却到室温。
等分试样45毫升到无菌的50ml管并储存于- 20℃的非除霜冰箱中。
在使用前,将37 ml热灭活(hi)-FBS的45 ml冷冻等分试样解冻约20分钟或4°C过夜。
Liberase TM 储备液(0.5mg / ml)
5 mg 冻干的Liberase TM             


溶于10毫升无菌ddH 2 O                           
取0.5 毫升等分试样并储存在-20 °C
分配储备液(50 U / ml,100 ml)
制作5毫升等分试样,并在-20°C下储存


10x Ca 2+ / Mg 2+ 溶液500 ml
927毫克氯化钙2· 2H 2 O (最终浓度:1.26 mM)                           


1.0 g MgSO 4 ·7H 2 O (最终浓度:0.8 mM)                                         


溶于无菌ddH 2 O ,最终体积为500 ml
过滤器(0.2 微米),并储存在室温下
收集介质100毫升
                            87 ml DMEM(高葡萄糖,GlutaMAX TM 补充剂)


10 ml 10x Ca 2+ / Mg 2+ 溶液


2 ml高FBS                                                       


1毫升100x GPS                                         


混合和过滤器(0.2 微米)
分装至5或10 ml /管,并在-20°C下保存长达3个月
消化缓冲液6毫升(新鲜配制)             
解冻5毫升收集培养基
加入0.5 ml Liberase TM (0.5 mg / ml)(1:10;工作浓度:50 µg / ml)                                                        
加入0.5 ml Dispase(50 U / ml)(1:10; 工作浓度:5 U / ml)                                                                                                 
6%ACD-A溶液,1 L
22.3克葡萄糖                           


22.0克柠檬酸钠             


8.0克柠檬酸


溶解ddH 2 O至终体积1 L                                                                                    
过滤器(0.2 微米),并存储在4℃下
缓冲液A (PBS / 0.6%ACD-A / 0.5%BSA)500毫升
2.5克BSA


50 ml 6%ACD-A溶液


450毫升PBS                           


溶解,过滤器(0.2 微米),并存储在4℃下


缓冲液B (PBS / 0.6%ACD-A / 0.1%BSA),500毫升
0.5克BSA


50 ml 6%ACD-A溶液


450毫升PBS                           


溶解,过滤器(0.2 微米),并存储在4℃下


EGM-2培养基500毫升                           
445毫升EBM-2                                         


50毫升hiFBS             


5毫升100x GPS                                         


EGM-2单Q UOT 补充剂(除了氢化可的松)


过滤器(0.2 微米),等分试样(45 毫升/管),并储存在-20 ℃下直到需要或在4个商店℃,并使用在3-4 天


纤连蛋白(FN)包被缓冲液(0.1 M Na 2 CO 3 ),500毫升
5.3克Na 2 CO 3


溶于ddH 2 O,最终体积为500 ml
用12 N HCl将pH调节至9.4
过滤(0.2 µm)并在室温下保存
用纤连蛋白(FN)包被培养板
计算所需的FN量;库存为1 µg / µl
使用1 微克/厘米2 为新鲜分离的细胞和0.1 微克/厘米2 为EXP 原代培养细胞的安森
将FN稀释到恰好足以覆盖板的FN涂层缓冲液中
充分混合并用无菌移液器添加板
在37 °C的培养箱中孵育30分钟或最多过夜(如果蒸发,可以添加更多的包被缓冲液过夜孵育)
淬火/解冻介质(500毫升)
445 ml DMEM(高葡萄糖,GlutaMAX TM 补充剂)


50毫升hiFBS (最终10%)


5毫升100x GPS


过滤(0.2 µm)并储存在4°C


EGM-2介质也可以用于淬灭,但比DMEM贵


冷冻介质(使新鲜)
95%hiFBS


5%二甲基亚砜


过滤(0.2 µm)并储存在4 °C


 


致谢


 


在发展中的方法描述在这个手稿是支持通过的NHLBI 的的国家研究院的健康下授予数量R01 HL096384 到J.B. 的内容是完全的责任的的作者和它不必然代表了官方观点的对国家机构的健康。


 


利益竞争


 


作者没有要披露的金融和非金融竞争利益。


 


伦理


 


用于这些隔离程序的IH组织是根据IRB批准的规程从波士顿儿童医院临床研究委员会(04-12-175R; 11/11/2010 – 6/6/2020)获得的。从受试者的父母或监护人那里获得了知情同意。


 


参考文献


 


Boscolo ,E.和Bischoff,J.(2009)。婴儿血管瘤的血管生成。血管生成12(2):197-207。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huang, L. and Bischoff, J. (2020). Isolation of Stem Cells, Endothelial Cells and Pericytes from Human Infantile Hemangioma. Bio-protocol 10(2): e3487. DOI: 10.21769/BioProtoc.3487.
  2. Overman, J., Fontaine, F., Wylie-Sears, J., Moustaqil, M., Huang, L., Meurer, M., Chiang, I. K., Lesieur, E., Patel, J., Zuegg, J., Pasquier, E., Sierecki, E., Gambin, Y., Hamdan, M., Khosrotehrani, K., Andelfinger, G., Bischoff, J. and Francois, M. (2019). R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma. eLife 8: e43026.
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