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In vitro Co-culture of Mesenchymal Stem Cells and Endothelial Colony Forming Cells
间充质干细胞和内皮集落形成细胞的体外共培养   

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The FASEB Journal
Feb 2017

Abstract

The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization in the ischemic tissues. However, their engraftment after transplantation resulted to be low. Previous studies showed mesenchymal stem/stromal cells (MSCs) could improve the survival and capillary formation capacity of ECFCs in co-culture systems. In this article, we describe a protocol for in vitro co-culture of MSCs and ECFCs to prime ECFCs for better engraftment.

Keywords: Endothelial colony forming cells (内皮集落形成细胞), Endothelial progenitor cells (内皮祖细胞), Mesenchymal stem/stromal cells (间充质干细胞/基质细胞), Vascularization (血管化), Placenta (胎盘), Co-culture (共培养), in vitro (体外)

Background

Endothelial progenitor cells (EPC) are defined as a cell population capable of forming new blood vessels through a vasculogenesis process. In 2004, Ingram et al. identified a specific highly proliferative population of EPC in ex vivo culture termed ‘endothelial colony-forming cells (ECFC)’ from human umbilical cord blood (Ingram et al., 2004) and these cells have recently been declared to represent EPCs (Medina et al., 2017). A similar population can also be isolated from the human term placenta tissue with equivalent vascularization potential and at clinically relevant quantities (Patel et al., 2013; Shafiee et al., 2015). Therefore, ECFC transplantation has been proposed as a therapeutical approach for ischemic diseases such as myocardial infarction or critical leg ischemia. However, ECFCs engraftment and vasculogenic potential after transplantation are well documented to be low (Shafiee et al., 2017; Medina et al., 2017). Previous experiments have shown enhanced ECFC engraftment and function by co-transplantation of mesenchymal stem/stromal cells (MSC) with ECFC (Shafiee et al., 2017). In vitro and in the presence of MSC, ECFC showed enhanced survival in serum deprivation conditions. In normal/growth culture conditions, MSC co-culture resulted in reduced ECFC proliferation and altered appearance towards an elongated mesenchymal-like morphology. Further investigations suggested that direct contact with MSC was required for changes in ECFC morphology and proliferation rate (Shafiee et al., 2017). In addition, after being co-cultured with MSCs for 4 days, ‘primed ECFCs’ showed reduced colony forming potential but improved capacity to form tube-like structures on MatrigelTM in vitro (Shafiee et al., 2017). In this article, we describe a protocol for in vitro co-culturing of ECFCs and bone marrow-derived MSCs (BM-MSCs).

Materials and Reagents

  1. Materials
    1. 15 ml centrifuge tube (Corning, Falcon®, catalog number: 352196 )
    2. 50 ml centrifuge tube (Corning, Falcon®, catalog number: 352070 )
    3. T75 flasks
    4. 2 ml micro tubes
    5. Transwell chambers with a 0.4 µm pore size membrane (Corning, catalog number: 3397 )
    6. 0.22 μm filter (EMD Millipore, catalog number: SLGP033RS )
    7. Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3 membrane (molecular weight cut off, 3 kDa) (EMD Millipore, catalog number: UFC900308 )
    8. NuncTM 6-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
    9. NuncTM 96-well flat bottom (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167008 )
    10. 24-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140644 )
    11. Culture slides (Corning, Falcon®, catalog number: 354118 )
    12. Cell strainer (size: 40 μm) (Corning, Falcon®, catalog number: 352340 )
    13. 20 µl, 100 µl, and 1 ml pipette tips
    14. 5 ml, 10 ml, and 50 ml serological pipettes
    15. Cryogenic vials, 1.2 ml (Corning, catalog number: 430487 )
    16. 50 ml syringe (BD, catalog number: 1018841 )
    17. Fluorescence activated cell sorting (FACS) tubes (5 ml Round-Bottom Polypropylene) (Corning, Falcon®, catalog number: 352063 )
    18. FACS tubes (5 ml Round-Bottom Polystyrene, with Cell Strainer Snap Cap) (Corning, Falcon®, catalog number: 352235 )

  2. Cells
    1. Human MSCs: Adult BM-MSCs were purchased from Lonza (Lonza, USA)
    2. Human ECFCs: Human fetal placental ECFCs were isolated as reported previously (Patel et al., 2013). To distinguish between MSCs and ECFCs after co-culture, we suggest using GFP tagged ECFCs as used in the current protocol but non-GFP tagged cells can also be used

  3. Reagents
    1. Double-distilled water (ddH2O)
    2. 100% ethanol
    3. Dulbecco modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11995073 )
    4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
    5. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    6. PBS tablet (Sigma-Aldrich, catalog number: P4417 )
    7. TrypLE-express dissociation reagent (Thermo Fisher Scientific, GibcoTM, catalog number: 12605093 )
    8. Collagen, Type I solution from rat tail (concentrated stock, 100x) (Sigma-Aldrich, catalog number: C3867 )
    9. Acetic acid (Sigma-Aldrich, catalog number: ARK2183 )
    10. Endothelial basal medium-2 (EBM-2) (Lonza, catalog number: 190860 )
    11. Endothelial growth medium-2 (EGM-2) BulletKitTM (Lonza, catalog number: CC-3162 )
      Note: The components of this kit includes the following items: Hydrocortisone, GA-1000 (Gentamicin, Amphotericin-B), hEGF, VEGF, hFGF-B, R3-IGF-1, Ascorbic acid, Heparin.
    12. Growth factor reduced MatrigelTM Matrix (Corning, catalog number: 356230 )
    13. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100 )
    14. Secondary antibody goat anti-rabbit Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11034 )
    15. Secondary antibody goat anti-mouse Alexa Fluor® 568 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11004 )
    16. PE/Cy5 conjugated anti-human CD90 antibody (Thermo Fisher Scientific, eBioscienceTM, catalog number: 15-0909-42 )
    17. V450 mouse anti-human CD31 (BD, BD Biosciences, catalog number: 561653 )
    18. 7-Aminoactinomycin D (7-AAD) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A1310 )
    19. Prolong Gold reagent with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Thermo Fisher Scientific, catalog number: P36935 )
    20. Dulbecco’s phosphate buffered saline (D-PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
    21. EDTA (Merck, USA, CAS: 6381-92-6)
    22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
    23. Paraformaldehyde powder (PFA) (Sigma-Aldrich, catalog number: P6148 )
    24. 100% Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
    25. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
    26. Penicillin/streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    27. Normal goat serum
    28. Trypan blue powder
    29. 70% ethanol (see Recipes)
    30. Phosphate buffered saline (PBS) (see Recipes)
    31. Coating solution (see Recipes)
    32. Complete EGM2 medium (see Recipes)
    33. Freezing medium (see Recipes)
    34. FACS buffer (see Recipes)
    35. 4% paraformaldehyde (PFA) (see Recipes)
    36. 0.1% Triton X-100 (see Recipes)
    37. Washing solution (see Recipes)
    38. Antibodies diluting solution (see Recipes)
    39. Blocking solution (see Recipes)
    40. 0.4% Trypan blue solution (see Recipes)

Equipment

  1. Water bath
  2. Refrigerator
  3. Portable Pipet-aid
  4. Centrifuge (Eppendorf, catalog number: 5810 R )
  5. Laminar flow work bench
  6. Centrifuge
  7. Shaker
  8. Tissue culture incubator set at 37 °C, 5% CO2 (Memmert GmbH & Co., Nurnberg, Germany)
  9. Hemocytometer (Hausser Scientific, catalog number: 3110 )
  10. IncuCyte Zoom (Essen BioScience)
  11. Zeiss Axio microscope (Carl Zeiss)
  12. Fluorescence-activated flow cytometry (FACS) Aria 11u system (BD Biosciences, FACSAriaTM)
  13. Gallios flow cytometer (Beckman Coulter, Fullerton, CA, USA)

Software

  1. Kaluza Flow Cytometry Analysis Software
  2. GraphPad Prism 7 software
  3. ZEN 2.3 (Blue edition, Carl Zeiss Microscopy GmbH)
  4. ImageJ (Fiji)

Procedure

  1. Cell culture and maintenance
    To distinguish between MSCs and ECFCs after co-culture, ECFCs were GFP-tagged using manufacturer’s protocol (https://www.systembio.com/products/imaging-and-reporter-vectors/bioluminescent-imaging-vectors/bliv101-cmv-gfp-t2a-luciferase/ System Biosciences, Mountain View, CA, USA). Alternatively, ECFCs can be identified from MSCs through their high expression level of platelet endothelial cell adhesion molecule (PECAM-1) also known as CD31.
    1. Preparation and application of coating solution
      1. Coat tissue culture flasks/plates with fresh collagen type-I coating solution (working concentration of 1x, see Recipes) for 3-4 h under a laminar flow hood at room temperature. It is recommended to prepare the coating solution fresh and decap the culture vessels during coating step for optimal performance.
      2. Then discard the coating medium thoroughly and gently wash the flasks/plates with PBS (see Recipes). Repeat washing step twice.
        Note: The coated flasks/plates can be kept at 4 °C for maximum 1 month.
    2. In vitro culture and freezing of placental ECFCs and BM-MSCs
      1. Upon defrosting, culture ECFCs (about 6.0 x 103/cm2) and BM-MSCs (about 4.0 x 103/cm2) in EGM2 (see Recipes), and DMEM + 10% FBS respectively.
      2. Change media twice per week and split the culture 1:3 when cultures reach approximately 90% of confluency. Discard the medium and gently wash the flasks with PBS. For subculturing, add TrypLE-express and incubate for 3-5 min, and then inactivate with full culture medium. Spin down the cell suspension at 330 x g for 5 min and then discard the supernatant and resuspend in fresh media.
      3. For future applications freeze the cells using the freezing media (90% FBS and 10% DMSO, see Recipes).

  2. In vitro co-culture assays
    1. Direct co-culture of MSCs and ECFCs
      To investigate the effects of MSCs co-culturing on ECFCs survival in stress conditions (here serum deprivation conditions), MSCs and ECFCs are co-cultured together in EBM-2 alone (Figure 1A).
      1. Culture 2.5 x 105 MSCs and 2.5 x 105 ECFCs together in collagen coated T75 flasks with EGM2.
      2. After 4 h and once cells attached, discard the supernatant and wash the cells using EBM-2 (without addition of serum or growth factors) to completely remove any serum leftovers and feed the cells with EBM-2. As a control, 2.5 x 105 ECFCs are cultured in collagen-coated T75 flasks containing EBM-2.
      3. After 48 h, detach cells using TrypLE-express dissociation reagent and count them.
      4. Wash the cells with PBS, and resuspend them in FACS buffer (cell concentration: 1.0 x 105 to 5.0 x 105 in 100 µl FACS buffer, see Recipes) and transfer to 2 ml microtubes and then stain with mouse PE/Cy5 conjugated anti-human CD90 antibody (1:100 dilution), CD31 antibody (1:50 dilution) and 7-AAD (1:50 dilution) at 4 °C.
      5. After 20 min, add 1 ml FACS buffer to each microtube and centrifuge cells at 400 x g for 4 min and resuspend cells in 200 µl of FACS buffer.
      6. Use the Gallios flow cytometer and measure the percentage of ECFCs (GFP+CD31+CD90-) and calculate ECFCs number using appropriate flow cytometry analysis software (Kaluza).
      7. (Optional) To assess the impact of MSCs on ECFCs in normal culture conditions, in separate experiments culture 2.5 x 105 MSCs and 2.5 x 105 ECFCs together in collagen-coated T75 flasks containing EGM2 for 48 h, and calculate the ECFC number as before (Figure 1B).
        Note: As a control, culture 2.5 x 105 ECFCs alone in collagen-coated T75 flasks containing EGM2 for 48 h.
    2. Indirect co-culture of Human Mesenchymal Stromal Cells (MSC) and ECFC
      To assess the impact of cell-cell contact on ECFC survival, indirect co-culture experiment is performed using Transwell chambers with a 0.4-mm pore size membrane (Figure 1C).
      1. Culture 0.5 x 105 MSCs on the top and 0.5 x 105 ECFCs in the bottom of Transwell chambers in separate plates with EGM2.
      2. After 4 h, transfer the upper compartment on the top of ECFC cultures wells and change the EGM2 with EBM-2 and incubate in normal culture conditions:
        5% CO2
        20% oxygen
        37 °C
        Humidity is maintained by putting a dish with sterile water
      3. After 48 h, remove the Transwell, detach ECFCs using TrypLE-express dissociation reagent and count the number of viable ECFCs by the trypan blue exclusion method using a hemocytometer.
        Note: Use ECFCs on the bottom of Transwell chambers as control culture and leave the top compartment cell-free.

  3. Preparation and application of MSC-derived conditioned medium (MSC-CM)
    To investigate the contribution of MSC-secreted factors on ECFCs, ECFCs are cultured in the presence of MSC-CM (Figure 1D).
    1. Culture 5 x 105 MSCs in a T75 flask with EBM-2 and incubate in normal cell culture conditions.
    2. After 48 h, collect the supernatant and centrifuge at 330 x g for 5 min to remove any dead cells.
    3. Filter the supernatant through a 0.22 µm filter to remove any remaining cell debris. The MSC-CM can immediately be used.
    4. In the meantime, culture 5.0 x 105 ECFCs in EGM2 in a T75 tissue culture flask. After 4 h, remove the medium and wash the cultures with PBS and culture ECFCs with MSC-CM and incubate in normal culture conditions.
    5. After 48 h, remove the supernatant and then count the number of viable ECFCs by the trypan blue exclusion method using a hemocytometer. As control culture, use ECFCs in EBM-2 and EGM2.
      Note: MSC-CM can be stored at -20 °C prior to use.
    6. The MSC-CM can be concentrated using a centricon column. Under the laminar hood assemble the Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3 membrane column according to manufacturer’s instructions.
    7. Add 15 ml MSC-CM to the loading chamber (top) of the column. Centrifuge at 4 °C at 4,000 x g for 30 min. Concentrated MSC-CM can immediately be transferred on top of the ECFCs or can be stored at -20 °C after collection. 50 folds of concentrating are recommended.
    8. In the meantime, culture ECFCs in collagen-coated 6-well plates (5 x 104/well) with EGM2 and place in the incubator. After 4 h, remove the medium and wash the plates with PBS and deliver concentrated MSC-CM on top of ECFCs.
    9. After 48 h, count the number of viable ECFCs by the trypan blue exclusion method using a hemocytometer. As a control, culture ECFCs in EBM-2 and EGM2.


      Figure 1. Protocols for investigating the effects of mesenchymal stem cells (MSC) co-cultured with endothelial colony forming cells (ECFC) in vitro. A. Direct co-culture of MSC and ECFC in serum-free culture medium; B. Direct co-culture in growth medium; C. Indirect co-culturing MSC and ECFC; D. Harvesting the MSC conditioned medium (MSC-CM) and deliver on ECFC cultures.

  4. Immunostaining analysis
    1. Co-culture 5.0 x 103 of MSCs and 5.0 x 103 of ECFCs in collagen-coated culture slides with EGM2 and incubate in normal condition. Change the media every second day.
    2. After 4 days, discard the media, wash the cultures using DPBS and fix using 4% paraformaldehyde (PFA) solution (see Recipes) in PBS (the homemade PBS is used for immunostaining experiments) for 20 min at 4 °C.
      Note: For CD31 immunostaining, either use 2% PFA for 15 min at 4 °C or apply freezer cold acetone for 10 min at 4 °C.
    3. Permeabilize cells with PBS/Triton X-100 (0.1%) (see Recipes) for 10 min at room temperature (RT).
      Note: Excess incubation with PBS/Triton X-100 (0.1%) can damage cell morphology.
    4. To block nonspecific protein-binding sites, wash the slides with BSA blocking solution (see Recipes) for 20 min at RT.
    5. Then, incubate slides for 2 h at RT with primary antibodies against CD31 (1:50), CD144 (1:100) and CD90 (1:100) diluted in antibody diluting solution (PBS/BSA [3%]/Tween [0.1%], see Recipes).
    6. Wash the slides with 300 µl of PBS/Tween (0.1%) solution for 5 min. Repeat three times.
    7. Then, incubate slides for 45 min at RT with 250 µl secondary antibodies diluted in antibody diluting solution (PBS/BSA [3%]/Tween [0.1%]).
    8. Wash the slides with 300 µl of PBS/Tween (0.1%) solution for 5 min. Repeat three times.
    9. Take off the upper compartment of culture slide according to manufacturer’s instruction.
    10. Put one drop of Prolong Gold reagent with DAPI in each well of the 8-well plate, and cover the 8 wells with coverslip and observe under an Axio microscope.

  5. Preparation of primed-ECFCs
    1. Co-culture 2.5 x 105 of MSCs and 2.5 x 105 of ECFCs in collagen-coated T75 tissue culture flasks with EGM2 and incubate in normal condition. Change the media every second day (Figure 2A). Figure 3 shows representative images of ECFC alone or co-cultured with MSC in EGM2 for 4 days.


      Figure 2. Preparation (A) and characterization (B) of primed-endothelial colony forming cells (ECFC). MSC: mesenchymal stem/stromal cells.


      Figure 3. Representative images of GFP tagged-endothelial colony forming cells (ECFC) alone or co-cultured with mesenchymal stem/stromal cells (MSC) in EGM2 for 4 days. The top panels: merged phase contrast and fluorescent images; the down panels: phase contrast images. Scale bars = 100 µm.

    2. After 4 days discard the media, wash the cultures using DPBS and detach cells using TrypLE-express dissociation reagent and count them. Spin down cells at 330 x g for 5 min, and resuspend them in 100 µl ice-cold FACS buffer and stain with mouse PE/Cy5 conjugated anti-human CD90 antibody (1:100 dilution) and 7-Aminoactinomycin D (7-AAD, 1:50 dilution) and V450 conjugated anti-human CD31 antibody (1:50 dilution) at 4 °C.
    3. After 20 min, centrifuge cells at 330 x g for 4 min and resuspend cells in 100 µl of ice-cold FACS buffer.
      Note: When performing flow cytometry, single stained samples are important to determine the levels of staining and compensation, so staining of MSCs alone for CD90 antibody, and ECFCs for CD31 is recommended. Dead cells can result in false positives results due to autofluorescence and increased level of non-specific binding. So, application of live/dead markers to eliminate dead cells is also recommended.
    4. Filter the cells through a 40 μm cell strainer to reduce cell aggregates before running samples through a FACS machine. Sort the GFP+CD90-CD31+ cells (primed-ECFCs) in ice-cold FBS in Falcon round-bottom polypropylene tubes.
      Note: Sorted cells in PBS (or PBS with BSA/FBS) have limited viability, so sorting in FBS is recommended.
    5. These cells can be then subjected to tube-formation assay, limiting dilution assay and gene expression analysis.
    6. In separate experiments, and as control culture ECFCs alone in a T75 flask with EGM2 for 4 days and sort the GFP+CD90-CD31+ cells.
      Note: Change the medium at day 2.

  6. Tube-formation assay
    1. Thaw MatrigelTM one night before use at 4 °C.
    2. Pre-chill the 24-well plates and the pipet tips at 4 °C for 2-3 h.
    3. Aliquot 100-150 μl of MatrigelTM per well of the pre-chilled 24-well plate on ice (Figure 2B).
      Note: Make sure the MatrigelTM is evenly distributed over the wells and try to avoid air bubble formation inside the gel. MatrigelTM must not dry.
    4. Leave the coated plates in an incubator (humidified 37 °C incubator) and allow gel to polymerize for 30 to 60 min.
    5. Transfer the ECFCs harvested from sort (primed-ECFCs) to 15 ml tubes and spin down at 330 x g for 5 min. Remove the supernatant and resuspend cells in fresh EGM2. Count the cells and dispense 400 μl of primed-ECFCs (1.0 x 104) into the MatrigelTM-coated wells. Use 1.0 x 104 non-primed ECFCs as a control.
    6. Place the plates in the IncuCyte® live-cell analysis system, and capture phase contrast and fluorescence images every 2 h for 48 h. Count capillary-like structures. Figure 4 shows representative images of primed ECFCs seeded on MatrigelTM.


      Figure 4. Representative images of primed-endothelial colony forming cells seeded on MatrigelTM after 24 h (left: phase contrast, right: phase contrast merged with fluorescent). Scale bars = 100 µm.

  7. Single cell assay
    1. Coat desired number of 96-well plate with 50 μl coating solution for 3-4 h under the laminar flow hood and make sure the coating solution is evenly distributed over the wells (Figure 2B). It is recommended to prepare the coating solution fresh and decap the plates during coating step for optimal performance.
    2. Discard the coating medium thoroughly and gently wash the flasks with PBS. Repeat washing step twice.
      Note: The coated 96-well plate can be kept at 4 °C for maximum 1 month.
    3. Prior to cell culture, aliquot 100-150 μl of EGM2 into each well of 96-well plate.
    4. Seed one single primed or non-primed ECFCs (GFP+CD90-CD31+) in each well by using the BD FACSAria sort machine. Incubate plates in normal cell culture conditions and change the media with fresh EGM2 twice a week by removing half of the medium and replacing it with fresh EGM2.
    5. After 2 weeks, visualize the cells under a light microscope and count/score colonies. Endothelial colonies can be scored as endothelial clusters (ECs; < 50 cells), low proliferative potential EPCs (LPP; 50-2,000 cells), and high proliferative potential EPCs (HPP; > 2,000 cells) (Figure 5).


      Figure 5. Representative images of primed-endothelial colony forming cells subjected to single cell assay after 10 days. Endothelial colonies can be scored as endothelial clusters (EC), low proliferative potential endothelial progenitor cells (EPCs) (LPP), and high proliferative potential EPCs (HPP). Scale bars = 100 µm.

Data analysis

  1. Kaluza software can be used to analyze the flow data. For direct and indirect co-culture assays at least six biological replicates should be used. Limiting dilution and tube-formation assays are performed in triplicate and quadruplicate respectively for at least 3 independent experiments. For the tube-formation assay, we counted capillary-like structures in at least 20 fields of view.
  2. All Statistical analysis can be performed using GraphPad Prism 7 software. Paired or unpaired Student’s t-test can be used to determine statistical differences among different groups.

Notes

  1. For better functionality and reproducibility of the results, we recommend using stem/progenitor cells at passages 3-5 for all the experiments.
  2. FACS solutions, culture media, immunostaining solutions need to be prepared on the day of use for best results.
  3. All cell culture reagents should be warmed at 37 °C before use.
  4. For highest viability during the FACS sorting, cells should be kept on ice.
  5. We recommend to keep collagen type-I solution at 2-8 °C. After coating, completely remove coating solution by aspirating, then gently wash with PBS and keep at 2-8 °C before use. The coated flasks/plates should be stored at 2-8 °C. We recommend using the coated flask within 1 month.

Recipes

  1. 70% ethanol (1 L)
    700 ml 100% ethanol
    300 ml deionized water
  2. Phosphate buffer saline (PBS, 1 L)
    Note: This is homemade PBS and only used for immunostaining experiments.
    10 PBS tablets
    1,000 ml MilliQ water
  3. Collagen coating solution (100 ml)
    1,000 µl collagen solution (concentrated stock, 100x)
    560 µl glacial acetic acid
    99 ml sterile PBS
  4. Complete EGM2 medium (EBMTM-2 plus SingleQuotsTM of Growth Supplements) (500 ml)
    500 ml EBMTM-2
    10 ml fetal bovine serum (FBS)
    0.5 ml hEGF
    0.2 ml hydrocortisone
    0.5 ml GA-1000 (Gentamicin, Amphotericin-B)
    0.5 ml VEGF
    2.0 ml hFGF-B
    0.5 ml R3-IGF-1
    0.5 ml ascorbic acid
    0.5 ml Heparin
  5. Freezing medium
    90% FBS
    10% DMSO
  6. FACS buffer
    1x D-PBS
    2.5 mM EDTA
    0.5% BSA
  7. 4% paraformaldehyde (PFA) (1 L)
    40 g of paraformaldehyde powder
    1,000 ml PBS
  8. 0.1% Triton X-100 (1 L)
    1 ml of Triton solution
    1,000 ml PBS
  9. Washing solution for immunofluorescent staining (1 L)
    1 ml Tween 20
    1,000 ml PBS
  10. Antibodies diluting solution for immunofluorescent staining
    DPBS, pH 7.2-7.4
    3% BSA
    0.05% Tween 20
  11. Blocking solution for immunofluorescent staining (100 ml)
    20 ml of normal goat serum
    80 ml immunofluorescent washing solution
  12. 0.4% trypan blue solution (RT)
    4 mg of trypan blue powder
    1 ml of 1x PBS
    Note: For counting the cells, dilute the cell suspension in a 1:1 dilution with 0.4% trypan blue solution and incubate for 1-2 min.

Acknowledgments

This protocol has been adapted and slightly modified from the previously published article (Shafiee et al., 2017). This study was supported by National Health and Medical Research Council Project Grant.

References

  1. Ingram, D. A., Mead, L. E., Tanaka, H., Meade, V., Fenoglio, A., Mortell, K., Pollok, K., Ferkowicz, M. J., Gilley, D. and Yoder, M. C. (2004). Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104(9): 2752-2760.
  2. Medina, R. J., Barber, C. L., Sabatier, F., Dignat-George, F., Melero-Martin, J. M., Khosrotehrani, K., Ohneda, O., Randi, A. M., Chan, J. K. Y., Yamaguchi, T., Van Hinsbergh, V. W. M., Yoder, M. C. and Stitt, A. W. (2017). Endothelial progenitors: A consensus statement on nomenclature. Stem Cells Transl Med 6(5): 1316-1320.
  3. Patel, J., Seppanen, E., Chong, M. S., Yeo, J. S., Teo, E. Y., Chan, J. K., Fisk, N. M. and Khosrotehrani, K. (2013). Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta. Stem Cells Transl Med 2(11): 839-847.
  4. Shafiee, A., Fisk, N. M., Hutmacher, D. W., Khosrotehrani, K. and Patel, J. (2015). Fetal endothelial and mesenchymal progenitors from the human term placenta: potency and clinical potential. Stem Cells Transl Med 4(5): 419-423.
  5. Shafiee, A., Patel, J., Wong, H. Y., Donovan, P., Hutmacher, D. W., Fisk, N. M. and Khosrotehrani, K. (2017). Priming of endothelial colony-forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling. FASEB J 31(2): 610-624.

简介

发现具有强大自我更新和从头血管形成潜力的内皮细胞集落形成细胞(ECFCs)表明,ECFC可以通过改善缺血组织的新生血管形成,成为心血管疾病治疗的优良细胞来源。 然而,移植后的移植导致了低位移植。 以前的研究显示间充质干/基质细胞(MSC)可以改善共培养系统中ECFCs的存活和毛细管形成能力。 在这篇文章中,我们描述了体外协调MSCs和ECFCs共同培养ECFC以实现更好的移植。
【背景】内皮祖细胞(EPC)被定义为能够通过血管发生过程形成新血管的细胞群。 2004年,Ingram等人鉴定了来自人脐带血的称为“内皮细胞集落形成细胞(ECFC)”的离体培养物中的特异性高度增殖的EPC群体Ingram等人,2004),并且这些细胞最近被宣布代表EPCs(Medina等人,2017)。类似的群体也可以从具有等效血管化潜力和临床相关数量的人类胎盘组织中分离(Patel等人,2013; Shafiee等人,2015) )。因此,ECFC移植已被提出作为缺血性疾病如心肌梗塞或关键性腿部缺血的治疗方法。然而,移植后的ECFCs植入物和血管生成潜力被证明是低的(Shafiee等人,2017; Medina等人,2017)。以前的实验已经显示通过与ECFC(Shafiee等人,2017)共同移植间充质干/基质细胞(MSC)来增强ECFC植入和功能。在体外,在MSC的存在下,ECFC在血清剥夺条件下显示增强的存活。在正常/生长培养条件下,MSC共培养导致ECFC增殖减少并且改变了外观,达到细长的间质样形态。进一步的研究表明,ECFC形态和增殖率的变化需要与MSC直接接触(Shafiee et al。,2017)。此外,在与MSC共培养4天后,“引发的ECFC”显示降低的集落形成潜力,但是改善了在Matrigel TM体外形成管状结构的能力[/ sup] (Shafiee等人,2017)。在本文中,我们描述了一种在体外共培养ECFC和骨髓来源的MSC(BM-MSC)的方案。

关键字:内皮集落形成细胞, 内皮祖细胞, 间充质干细胞/基质细胞, 血管化, 胎盘, 共培养, 体外

材料和试剂

  1. 物料
    1. 15ml离心管(Corning,Falcon ®,目录号:352196)
    2. 50ml离心管(Corning,Falcon ®,目录号:352070)
    3. T75烧瓶
    4. 2毫升微管
    5. 具有0.4μm孔径膜的Transwell腔室(Corning,目录号:3397)
    6. 0.22μm过滤器(EMD Millipore,目录号:SLGP033RS)
    7. 具有Ultracel-3膜(分子量截止,3kDa)的Amicon Ultra-15离心过滤器单元(EMD Millipore,目录号:UFC900308)
    8. Nunc TM 6孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
    9. Nunc TM 96孔平底(Thermo Fisher Scientific,Thermo Scientific TM,目录号:167008)
    10. 24孔板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:140644)
    11. 文化幻灯片(Corning,Falcon ®,目录号:354118)
    12. 细胞过滤器(尺寸:40μm)(Corning,Falcon ®,目录号:352340)
    13. 20μl,100μl和1 ml移液管吸头
    14. 5ml,10ml,和50ml血清移液管
    15. 低温小瓶1.2ml(Corning,目录号:430487)
    16. 50ml注射器(BD,目录号:1018841)
    17. 荧光活化细胞分选(FACS)管(5ml圆底聚丙烯)(Corning,Falcon,目录号:352063)
    18. FACS管(5ml圆底聚苯乙烯,带有细胞过滤器捕捉帽)(Corning,Falcon,目录号:352235)

  2. 细胞
    1. 人类MSC:成人BM-MSC购自Lonza(Lonza,USA)
    2. 人类ECFCs:如先前报道的(Patel等人,2013)分离人胎盘胎盘ECFCs。为了在共培养后区分MSC和ECFC,我们建议使用目前协议中使用的GFP标记的ECFC,但也可以使用非GFP标记的细胞。
  3. 试剂
    1. 双蒸水(ddH 2 O)
    2. 100%乙醇
    3. Dulbecco改性Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:11995073)
    4. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
    5. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
    6. PBS片剂(Sigma-Aldrich,目录号:P4417)
    7. TrypLE表达离解试剂(Thermo Fisher Scientific,Gibco TM,目录号:12605093)
    8. 胶原,大鼠尾巴(浓缩原料,100x)的I型溶液(Sigma-Aldrich,目录号:C3867)
    9. 乙酸(Sigma-Aldrich,目录号:ARK2183)
    10. 内皮基底培养基-2(EBM-2)(Lonza,目录号:190860)
    11. 内皮生长培养基-2(EGM-2)BulletKit TM(Lonza,目录号:CC-3162)
      注意:该试剂盒的组分包括以下物质:氢化可的松,GA-1000(庆大霉素,两性霉素-B),hEGF,VEGF,hFGF-B,R3-IGF-1,抗坏血酸,肝素。 >
    12. 生长因子减少Matrigel TM Matrix(Corning,目录号:356230)
    13. 二甲基亚砜(DMSO)(Fisher Scientific,目录号:BP231-100)
    14. 第二抗体山羊抗兔Alexa Fluor 488缀合物(Thermo Fisher Scientific,Invitrogen TM,目录号:A-11034)
    15. 第二抗体山羊抗小鼠Alexa Fluor 568缀合物(Thermo Fisher Scientific,Invitrogen,目录号:A-11004)
    16. PE / Cy5缀合的抗人CD90抗体(Thermo Fisher Scientific,eBioscience TM,目录号:15-0909-42)
    17. V450小鼠抗人CD31(BD,BD Biosciences,目录号:561653)
    18. 7-氨基生物霉素D(7-AAD)(Thermo Fisher Scientific,Invitrogen TM,目录号:A1310)
    19. 具有4',6-二脒基-2-苯基吲哚二氢氯化物(DAPI)的延长金试剂(Thermo Fisher Scientific,目录号:P36935)
    20. Dulbecco的磷酸盐缓冲盐水(D-PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190250)
    21. EDTA(Merck,USA,CAS:6381-92-6)
    22. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
    23. 多聚甲醛粉末(PFA)(Sigma-Aldrich,目录号:P6148)
    24. 100%Triton X-100(Fisher Scientific,目录号:BP151-500)
    25. 吐温20(Sigma-Aldrich,目录号:P9416)
    26. 青霉素/链霉素10,000U / ml(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    27. 正常山羊血清
    28. 台盼蓝色粉末
    29. 70%乙醇(见食谱)
    30. 磷酸盐缓冲盐水(PBS)(见食谱)
    31. 涂层溶液(见配方)
    32. 完成EGM2培养基(见食谱)
    33. 冷冻介质(参见食谱)
    34. FACS缓冲区(见配方)
    35. 4%多聚甲醛(PFA)(见配方)
    36. 0.1%Triton X-100(参见食谱)
    37. 洗涤液(参见食谱)
    38. 抗体稀释液(见配方)
    39. 阻塞解决方案(见配方)
    40. 0.4%台盼蓝溶液(见配方)

设备

  1. 水浴
  2. 冰箱
  3. 便携式吸管
  4. 离心机(Eppendorf,目录号:5810 R)
  5. 层流工作台
  6. 离心机
  7. 振动器
  8. 组织培养箱设置在37℃,5%CO 2(Memmert GmbH&amp; Co.,Nurnberg,Germany)上
  9. 血细胞计数器(Hausser Scientific,目录号:3110)
  10. IncuCyte Zoom(埃森生物科学)
  11. 蔡司Axio显微镜(Carl Zeiss)
  12. 荧光活化流式细胞术(FACS)Aria 11u系统(BD Biosciences,FACSAria TM)
  13. Gallios流式细胞仪(Beckman Coulter,Fullerton,CA,USA)

软件

  1. Kaluza流式细胞分析软件
  2. GraphPad Prism 7软件
  3. ZEN 2.3(Blue edition,Carl Zeiss Microscopy GmbH)
  4. ImageJ(斐济)

程序

  1. 细胞培养和维护
    为了在共培养后区分MSC和ECFCs,使用制造商的方案对ECFC进行GFP标记( https://www.systembio.com/products/imaging-and-reporter-vectors/bioluminescent-imaging-vectors/bliv101-cmv-gfp- t2a-luciferase / System Biosciences,Mountain View,CA,USA)。或者,可以通过其也称为CD31的血小板内皮细胞粘附分子(PECAM-1)的高表达水平从MSC鉴定ECFC。
    1. 涂层溶液的制备与应用
      1. 在室温下,在层流罩下,用新鲜胶原I型涂层溶液(1x的工作浓度,参见食谱)涂覆组织培养瓶/板3-4小时。建议在涂布步骤中将新鲜的涂层溶液制备成分解培养容器,以达到最佳性能。
      2. 然后彻底丢弃涂层介质,并用PBS轻轻洗涤烧瓶/板(参见食谱)。重复洗涤步骤两次。
        注意:涂覆的烧瓶/板可以保持在4℃最多1个月。
    2. 体外培养和冷冻胎盘ECFCs和BM-MSC
      1. 在除霜后,培养ECFC(约6.0×10 3 / sup 2 / cm 2)和BM-MSC(约4.0×10 3 / sup / cm 3 > 2 )(见配方),DMEM + 10%FBS。
      2. 每周更换培养基两次,分离培养物1:3,当培养物达到约90%的融合时。弃去培养基,用PBS轻轻洗涤烧瓶。对于传代培养,添加TrypLE表达并孵育3-5分钟,然后用完全培养基灭活。将细胞悬浮液在330℃下旋转5分钟,然后弃去上清液并重新悬浮于新鲜培养基中。
      3. 对于未来的应用,使用冷冻介质(90%FBS和10%DMSO,见食谱)冷冻细胞。

  2. 共培养试验
    1. MSC和ECFC的直接共同文化
      为了研究MSCs共培养对应激条件(这里为血清剥夺条件)的ECFCs存活的影响,将MSC和ECFCs单独共同培养在EBM-2中(图1A)。
      1. 将具有EGM2的胶原包被的T75烧瓶中的2.5×10 5个MSCs和2.5×10 5个ECFC合并在一起。
      2. 4小时后,一旦细胞连接,丢弃上清液并使用EBM-2(不加血清或生长因子)洗涤细胞,以完全除去任何血清残留物,并用EBM-2喂养细胞。作为对照,将2.5×10 5个ECFCs在含有EBM-2的胶原包被的T75烧瓶中培养。
      3. 48小时后,用TrypLE-表达解离试剂分离细胞并计数
      4. 用PBS洗涤细胞,并将其重新悬浮于100μlFACS缓冲液中的FACS缓冲液(细胞浓度:1.0×10 5至5.0×10 5)中,参见Recipes)并转移至2ml微管,然后在4℃下用小鼠PE / Cy5缀合的抗人CD90抗体(1:100稀释),CD31抗体(1:50稀释)和7-AAD(1:50稀释))染色。 />
      5. 20分钟后,向每个微管中加入1ml FACS缓冲液,并以400×g离心细胞4分钟,并将细胞悬浮于200μlFACS缓冲液中。
      6. 使用Gallios流式细胞仪测量ECFCs的百分比(GFP + CD31 + CD90 - ),并使用适当的流式细胞分析软件计算ECFCs数(卡鲁扎)。
      7. (可选)为了评估MSC在正常培养条件下对ECFCs的影响,在单独的实验中,将2.5×10 5个MSCs和2.5×10 5个ECFC共同在胶原包被将含有EGM2的T75烧瓶放置48小时,并计算如前所述的ECFC编号(图1B)。
        注意:作为对照,在含有EGM2的胶原包被的T75烧瓶中单独培养2.5×10 5个ECFCs 48小时。
    2. 间接共培养人类间充质基质细胞(MSC)和ECFC
      为了评估细胞接触对ECFC存活的影响,使用具有0.4mm孔径膜的Transwell腔进行间接共培养实验(图1C)。
      1. 培养在具有EGM2的分离板中的Transwell室底部的顶部的MSCs和0.5×10 5个ECFC的培养物。
      2. 4小时后,转移ECFC培养孔顶部的上隔室,并用EBM-2转化EGM2,并在正常培养条件下孵育:
        5%CO 2
        20%氧气
        37°C
        通过放入无菌水的碟子保持湿度
      3. 48小时后,取出Transwell,使用TrypLE-表达离解试剂分离ECFCs,并使用血细胞计数器通过台盼蓝排除法计数可行的ECFCs数量。
        注意:使用Transwell腔室底部的ECFC作为对照培养物,并保留顶部隔室无细胞。

  3. MSC衍生条件培养基(MSC-CM)的制备和应用
    为了研究MSC分泌因子对ECFCs的贡献,在MSC-CM存在下培养ECFC(图1D)。
    1. 培养5×10 5个具有EBM-2的T75烧瓶中的MSC,并在正常细胞培养条件下孵育。
    2. 48小时后,收集上清液并以330×g离心5分钟以除去任何死细胞。
    3. 通过0.22μm过滤器过滤上清液,以除去任何剩余的细胞碎片。可立即使用MSC-CM。
    4. 同时,在T75组织培养瓶中EGM2中培养5.0×10 5个ECFC。 4小时后,取出培养基并用PBS洗涤培养物,并用MSC-CM培养ECFC并在正常培养条件下孵育。
    5. 48小时后,取出上清液,然后使用血细胞计数器通过台盼蓝排除法计数活的ECFCs数。作为对照培养,在EBM-2和EGM2中使用ECFCs。
      注意:MSC-CM可以在-20°C使用前储存。
    6. MSC-CM可以使用中心柱进行浓缩。在层流罩下,根据制造商的说明书,使用Ultracel-3膜柱组装Amicon Ultra-15离心过滤器。
    7. 将15ml MSC-CM加入柱的装载室(顶部)。在4℃下以4,000xg离心30分钟。浓缩的MSC-CM可以立即转移到ECFC的顶部,或者可以在-20℃下储存。推荐使用50倍的浓缩液。
    8. 同时,用含有EGM2的胶原蛋白包被的6孔板(5×10 4 /孔)培养ECFC并置于培养箱中。 4小时后,取出培养基,用PBS洗板,并将浓缩的MSC-CM放在ECFC的顶部
    9. 48小时后,使用血细胞计数器通过台盼蓝排除法计数可行的ECFCs数。作为对照,EBM-2和EGM2培育ECFCs。


      图1.用于研究与体内内皮细胞集落形成细胞(ECFC)共培养的间充质干细胞(MSC)的作用的方案。 A.直接共培养MSC和ECFC在无血清培养基中; B.生长培养基中直接共培养; C.间接共培养MSC和ECFC; D.收集MSC条件培养基(MSC-CM)并提供ECFC培养物。

  4. 免疫染色分析
    1. 在含EGM2的胶原包被的培养载玻片中共培养5.0×10 3个MSCs和5.0×10 3个ECFCs,并在正常条件下孵育。每隔一天更换一次媒体。
    2. 4天后,丢弃培养基,使用DPBS洗涤培养物,并在4℃下用PBS中的4%多聚甲醛(PFA)溶液(参见食谱)固定20分钟(自制PBS用于免疫染色实验)。
      注意:对于CD31免疫染色,在4℃下使用2%PFA 15分钟,或在4℃下使用冷冻冷丙酮10分钟。
    3. 在室温(RT)下,用PBS / Triton X-100(0.1%)(参见食谱)将细胞渗透10分钟。
      注意:用PBS / Triton X-100(0.1%)过量培养可能会破坏细胞形态。
    4. 为阻断非特异性蛋白质结合位点,用PBS封闭溶液洗涤载玻片(见食谱)20分钟。
    5. 然后,在抗体稀释溶液(PBS / BSA [3%] /吐温[0.1])中稀释的CD31(1:50),CD144(1:100)和CD90(1:100) %],请参阅食谱)。
    6. 用300μlPBS /吐温(0.1%)溶液洗涤载玻片5分钟。重复三次。
    7. 然后,用PBS稀释溶液(PBS / BSA [3%] /吐温[0.1%])的250μl二次抗体在室温孵育载玻片45分钟。
    8. 用300μlPBS /吐温(0.1%)溶液洗涤载玻片5分钟。重复三次。
    9. 根据制造商的说明,将培养皿的上部隔离开。
    10. 在8孔板的每个孔中放入一滴具有DAPI的Prolong Gold试剂,并用盖玻片覆盖8个孔,并在Axio显微镜下观察。

  5. 预处理ECFCs的准备
    1. 在具有EGM2的胶原包被的T75组织培养烧瓶中共培养2.5×10 5个MSCs和2.5×10 5个ECFC,并在正常条件下孵育。每隔一天更换一次媒体(图2A)。图3显示了ECFC单独或与MSC在EGM2中共培养4天的代表性图像

      图2.引发内皮细胞集落形成细胞(ECFC)的制备(A)和表征(B)。 MSC:间充质干/基质细胞

      图3.单独的GFP标记内皮集落形成细胞(ECFC)的代表性图像或与EGM2中的间充质干/基质细胞(MSC)共培养4天。顶部面板:合并相位对比和荧光图像;下面板:相位对比图像。比例尺=100μm
    2. 4天后丢弃培养基,使用DPBS洗涤培养物,并使用TrypLE-express解离试剂分离细胞并计数。在330℃下将细胞分解5分钟,并将其重悬于100μl冰冷的FACS缓冲液中,并用小鼠PE / Cy5缀合的抗人CD90抗体(1:100稀释)和7- 4℃下的氨基生殖霉素D(7-AAD,1:50稀释)和V450缀合的抗人CD31抗体(1:50稀释)。
    3. 20分钟后,以330×g离心细胞4分钟,并将细胞重悬于100μl冰冷的FACS缓冲液中。
      注意:当进行流式细胞术时,单个染色的样品对于确定染色和补偿的水平很重要,因此建议单独用于CD90抗体的MSC和CD31的ECFC染色。死细胞可能导致由于自体荧光引起的假阳性结果,并增加非特异性结合的水平。因此,也推荐使用活/死标记来消除死细胞。
    4. 在通过FACS机器运行样品之前,通过40μm的细胞过滤器过滤细胞以减少细胞聚集。在Falcon圆底聚丙烯管中的冰冷FBS中将GFP + CD90 - CD31 + 细胞(底漆-ECFCs)排序。
      注意:PBS中的分选细胞(或含有BSA / FBS的PBS)的活力有限,因此建议在FBS中进行分选。
    5. 然后可以对这些细胞进行管形成测定,限制稀释测定和基因表达分析。
    6. 在单独的实验中,以及作为对照培养ECFC,在具有EGM2的T75烧瓶中单独培养4天,并分选GFP + CD90 CD31 + 细胞。
      注意:在第2天更换培养基。

  6. 管形成试验
    1. 解冻Matrigel TM 在4°C使用前一天。
    2. 预先将24孔板和吸头在4℃预冷2-3小时
    3. 将预先冷冻的24孔板在冰上每孔100-150μlMatrigel TM(图2B)。
      注意:确保Matrigel TM 均匀地分布在孔中,并尝试避免凝胶内形成气泡。 Matrigel TM 不能干。
    4. 将涂布板放在培养箱中(加湿37℃的培养箱),使凝胶聚合30至60分钟。
    5. 将从排序(底漆ECFCs)收获的ECFC转移到15 ml管中,并以330 x g旋转5分钟。去除上清液并将细胞重新悬浮在新鲜的EGM2中。对细胞进行计数,并将400μl的底漆ECFC(1.0×10 4 / sup>)分配到Matrigel TM 涂层的孔中。使用1.0 x 10 4 非底漆ECFC作为对照。
    6. 将板放在IncuCyte ®活细胞分析系统中,每2小时捕获相位对比度和荧光图像48小时。计数毛细管样结构。图4显示了在Matrigel TM上种植的已引发ECFCs的代表性图像。


      图4. 24小时后接种于Matrigel TM上的起始内皮细胞集落形成细胞的代表性图像(左:相位对比度,右:与荧光相结合的相位对比)。 比例尺=100μm。

  7. 单细胞测定
    1. 在层流罩下将所需数量的96孔板用50μl涂层溶液涂覆3-4小时,并确保涂层溶液均匀分布在孔上(图2B)。建议在涂层步骤中新鲜涂布涂层溶液并对其进行脱膜,以获得最佳性能
    2. 彻底丢弃涂层介质,并用PBS轻轻洗涤烧瓶。重复洗涤步骤两次。
      注意:涂层的96孔板可以在4℃保存最多1个月。
    3. 在细胞培养之前,将100-150μlEGM2等分至96孔板的每个孔中
    4. 通过使用BD FACSAria分选机在每个孔中种上一个单一的底漆或非底色ECFC(GFP + CD90 - CD31 + )。在正常的细胞培养条件下孵育平板,并用新鲜的EGM2每周更换培养基,通过除去一半的培养基并用新鲜的EGM2代替。
    5. 2周后,在光学显微镜下观察细胞,计数/评分菌落。内皮集落可以作为内皮簇(ECs; <50细胞),低增殖潜能EPCs(LPP; 50-2,000个细胞)和高增殖潜能EPCs(HPP;> 2,000个细胞)评分(图5)。 />

      图5.在10天后进行单细胞测定的起始内皮细胞集落形成细胞的代表性图像。内皮细胞集合可以作为内皮簇(EC),低增殖潜能内皮祖细胞(EPCs) LPP)和高增殖潜能EPCs(HPP)。比例尺=100μm

数据分析

  1. Kaluza软件可用于分析流量数据。对于直接和间接共培养测定,应使用至少六个生物重复。对于至少3次独立实验,限制性稀释和管形成测定分别进行一式三份和一式四份。对于管形成测定,我们在至少20个视野中计数毛细管样结构
  2. 所有统计分析都可以使用GraphPad Prism 7软件进行。配对或不配对的学生测试可用于确定不同组之间的统计差异。

笔记

  1. 为了更好的功能和重复性的结果,我们建议在所有实验的第3-5节使用干细胞/祖细胞。
  2. 需要在使用当天准备FACS解决方案,培养基,免疫染色解决方案以获得最佳效果
  3. 所有细胞培养试剂应在37°C使用前加温。
  4. 为了在FACS分选过程中获得最高的生存力,细胞应保持在冰上
  5. 我们建议将胶原I型溶液保持在2-8°C。涂布后,通过抽吸完全除去涂层溶液,然后用PBS轻轻洗涤,并保持在2-8°C使用前。涂覆的烧瓶/板应储存在2-8°C。我们建议在1个月内使用涂层烧瓶。

食谱

  1. 70%乙醇(1升)
    700 ml 100%乙醇 300毫升去离子水
  2. 磷酸盐缓冲盐水(PBS,1L)
    注意:这是自制PBS,仅用于免疫染色实验。
    10个PBS片剂
    1000毫升MilliQ水
  3. 胶原涂层溶液(100ml)
    1000μl胶原溶液(浓缩原液,100x)
    560μl冰醋酸
    99 ml无菌PBS
  4. 完成EGM2培养基(EBM TM -2加上生长补充剂的SingleQuots TM / sup>)(500ml)
    500 ml EBM TM -2
    10ml胎牛血清(FBS)
    0.5 ml hEGF
    0.2 ml氢化可的松
    0.5 ml GA-1000(庆大霉素,两性霉素B)
    0.5 ml VEGF
    2.0 ml hFGF-B
    0.5ml R3-IGF-1
    0.5毫升抗坏血酸
    0.5毫升肝素
  5. 冻结介质
    90%FBS
    10%DMSO
  6. FACS缓冲区
    1x D-PBS
    2.5 mM EDTA
    0.5%BSA
  7. 4%多聚甲醛(PFA)(1升)
    40克多聚甲醛粉末 1000毫升PBS
  8. 0.1%Triton X-100(1L)
    1毫升Triton溶液
    1000毫升PBS
  9. 免疫荧光染色洗涤液(1升) 1毫升吐温20
    1000毫升PBS
  10. 抗体稀释溶液免疫荧光染色
    DPBS,pH 7.2-7.4
    3%BSA
    0.05%吐温20
  11. 免疫荧光染色的阻断溶液(100 ml)
    20毫升正常山羊血清
    80 ml免疫荧光清洗液
  12. 0.4%台盼蓝溶液(RT)
    4毫克台盼蓝粉末 1 ml 1x PBS
    注意:对于细胞计数,用0.4%台盼蓝溶液以1:1的稀释液稀释细胞悬液,孵育1-2分钟。

致谢

该协议已经从以前发表的文章(Shafiee等人,2017年)进行了修改和略微修改。本研究得到国家卫生和医学研究理事会项目资助。

参考

  1. Ingram,D.A.,Mead,L.E.,Tanaka,H.,Meade,V.,Fenoglio,A.,Mortell,K.,Pollok,K.,Ferkowicz,M.J.,Gilley,D.and Yoder,M.C。(2004)。 使用人类外周血和脐带血鉴定内皮祖细胞的新颖等级。 血液 104(9):2752-2760。
  2. Medina,RJ,Barber,CL,Sabatier,F.,Dignat-George,F.,Melero-Martin,JM,Khosrotehrani,K.,Ohneda,O.,Randi,AM,Chan,JKY,Yamaguchi, Hinsbergh,VWM,Yoder,MC和Stitt,AW(2017)。 内皮祖细胞:关于术语的共识声明。 干细胞翻译Med < 6(5):1316-1320。
  3. Patel,J.,Seppanen,E.,Chong,M.S.Yeo,J.S.Teo,E.Y.,Chan,J.K.,Fisk,N.M。和Khosrotehrani,K。(2013)。 来自人类胎盘的胎儿内皮细胞集落形成细胞的前瞻性表面标记物分离和扩增。 / a> Stem Cells Transl Med 2(11):839-847。
  4. Shafiee,A.,Fisk,N.M.,Hutmacher,D.W.,Khosrotehrani,K.and Patel,J。(2015)。 来自人类胎盘的胎儿内皮和间充质祖细胞:潜力和临床潜力。 Stem Cells Transl Med 4(5):419-423。
  5. Shafiee,A.,Patel,J.,Wong,H.Y.,Donovan,P.,Hutmacher,D.W.,Fisk,N.M.和Khosrotehrani,K.(2017)。 间充质小窝中内皮细胞集落形成细胞的引发通过协调开发间充质转化来改善移植和血管发生的潜力通过NOTCH信令。 FASEB J 31(2):610-624。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shafiee, A. and Khosrotehrani, K. (2017). In vitro Co-culture of Mesenchymal Stem Cells and Endothelial Colony Forming Cells. Bio-protocol 7(20): e2587. DOI: 10.21769/BioProtoc.2587.
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