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Dec 2017

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Generation of BMEC Lines and in vitro BMEC-HSPC Co-culture Assays
建立脑微血管内皮细胞系和体外 BMEC-HSPC 共培养检测   

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Abstract

Endothelial cells (ECs) sustain the self-renewal and regeneration of adult hematopoietic stem and progenitor cells (HSPCs) via deployment of EC-derived paracrine factors, termed as angiocrine factors. Generation of durable ex vivo vascular niche that maintains EC identity and preserves the angiocrine profile of organ of origin offers platforms for in vitro dissection of the mechanism by which angiocrine factors execute their instructive function for stem cell maintenance and tissue regeneration. This protocol describes detailed methods to isolate primary bone marrow ECs (BMECs), to subsequently transduce lentiviral vector carrying myristoylated-Akt1 into primary BMECs, and to use the Akt1-BMECs to expand engraftable murine HSPCs. The BMEC-HSPC co-culture system serves as bioreactor prototype to generate scalable populations of the blood and immune systems.

Keywords: Angiocrine (血管分泌因子), BMEC (脑微血管内皮细胞), HSPCs (氢化大豆磷脂酰胆碱), Coculture (共培养), Mouse microvascular endothelial cells (小鼠微血管内皮细胞)

Background

Hematopoietic stem cells (HSCs) are multipotent adult stem cells that can self-renew to replenish themselves and differentiate into all the lineages of the blood and immune system. HSC transplantation offers the best therapeutic cure for diseases such as acute myeloid leukemia, and served as cellular platform to correct the mutation of genetic blood disease via gene targeting. There are several sources of hematopoietic stem cells, adult bone marrow-derived HSCs, cord blood-derived HSCs, and granulocyte-colony stimulating factor (GCSF)-mobilized HSCs. Compared with the bone marrow-derived HSCs, cord blood HSCs can tolerate more HLA mismatching, and have better anti-leukemia activities, and are more readily available. Unfortunately, the HSC transplantation is still a risky procedure to perform and transplant-related mortality is partially due to the leukemia relapse and/or the incidence of infections that took place during the recovery phase of HSC transplantation; all of which are attributable to the low stem cell numbers in the donor cord blood. Therefore, identifying cellular and molecular approaches that can help expand bona fide HSCs that maintain self-renewal activity is of pivotal translational significance.

Endothelial cells safeguard the self-renewal and regeneration of adult HSCs in the bone marrow via deploying endothelial-derived paracrine factors, termed as angiocrine factors, such as KitL, SDF-1, Jagged-1 and Jagged-2, etc. (Poulos et al., 2013; Mendelson and Frenette, 2014; Rafii et al., 2016, Asada et al., 2017). Rafii et al. have pioneered in the technology of isolating adult human bone marrow endothelial cells (BMECs) and performing co-culture experiments to expand HSPCs (Rafii et al., 1994). The short life span of human BMECs and human umbilical venous endothelial cells (HUVECs) were strategically (Zhang et al., 2004) overcome via overexpression of the adenoviral E4ORF1 gene. The resulting cells, termed as E4-HUVECs, maintain the angiocrine profiles of primary HUVECs and are able to support the self-renewal of long-term repopulating mouse HSPCs and human cord blood stem cells (Seandel et al., 2008; Butler et al., 2010). E4ORF1 executes such functions partially via activation of Akt1 signaling pathway. We have thus generated constitutively active Akt1 by adding a myristoylation sequence 5’ to the ORF sequence that helps target the Akt1 at the cell membrane to undergo phosphorylation. Transduction of myristoylated Akt1 into primary mouse BMECs maintains their EC identities and preserves the angiocrine profiles (Kobayashi et al., 2010). This approach is useful for in vitro assays to dissect the mechanism through which BMECs support the self-renewal and expansion of HSPCs (Poulos et al., 2013; Hadland et al., 2015; Poulos et al., 2015; Guo et al., 2017). With the concept of endothelial cell heterogeneity, Akt1-BMECs will prove to be a complementary approach for in vivo studies that highlight the instructive role of different vascular beds for the differentiation of subpopulation of the blood and immune system.
This protocol is divided into the following parts:

  1. FACSAria II sorting of primary BMECs.
  2. Dynabeads isolation of primary BMECs.
  3. Lentiviral transduction of primary BMECs.
  4. In vitro BMEC-HSPC coculture.

Materials and Reagents

  1. Falcon tube, 15 ml (Corning, catalog number: 352096)
  2. Falcon tubes, 50ml (Corning, catalog number: 352098)
  3. Falcon® 100 mm TC-treated Cell Culture Dish, 20/Pack, 200/Case, Sterile (Corning, catalog number: 353003) 
  4. Low retention tube, 1.5 ml (Fisher Scientific, catalog number, FisherbrandTM, catalog number: 02-681-320)
  5. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34120)
  6. Parafilm (Bermis, catalog number: PM996)
  7. Amicon Ultra-0.5 Centrifugal Filter Unit (Merck, catalog number: UFC503096)
  8. Sterile 40 μm nylon mesh (Corning, catalog number: 352340)
  9. 6-tube magnetic stand (Thermo Fisher Scientific, catalog number: AM10055)
  10. 24-well plate (Corning, catalog number: 353047)
  11. 12-well plate (Corning, catalog number: 353043)
  12. T75 flasks (Corning, catalog number: 353136)
  13. Bio-Spin® P-30 Gel Columns, Tris Buffer (Bio-Rad Laboratories, catalog number: 7326232)
  14. DynabeadsTM Sheep anti-rat IgG (Thermo Fisher Scientific, catalog number: 11035)
  15. DPBS, 1x without calcium and magnesium (Corning, catalog number: 21-031-CV)
  16. DMSO, dimethyl sulfoxide (Sigma-Aldrich, catalog number: D2650)
  17. Alexa FluorTM 647 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific, catalog number: A20006)
  18. Isothesia (Isoflurane) solution (Henry Schein Animal Health, catalog number: 029405)
  19. Isoflurane chamber, EZ anesthesia
  20. Oxygen (Tech Air)
  21. Potassium chloride, KCl, BioXtra, ≥ 99.0% (Sigma-Aldrich, catalog number: P9333)
  22. Calcium chloride dihydrate, CaCl2•2H2O (Sigma-Aldrich, catalog number: C3306)
  23. Magnesium chloride, MgCl2, anhydrous, ≥ 98% (Sigma-Aldrich, catalog number: M8266)
  24. Sodium bicarbonate, NaHCO3 (Sigma-Aldrich, catalog number: S5761-500G) 
  25. Bovine Serum Albumin, lyophilized powder, essentially IgG-free, low endotoxin, BioReagent, suitable for cell culture (Sigma-Aldrich, catalog number: A2058)
  26. HBSS buffer: Hank’s Balanced Salt Solution, 1x without Calcium, Magnesium and Phenol Red (Corning, catalog number: 21-022-CV)
  27. Lentiviral myristoylated-Akt1: Virus titer is measured by Lenti-X p24 Rapid Titer Kit (Takara Bio, Clontech, catalog number: 632200)
  28. Fibronectin (Sigma-Aldrich, catalog number: F1141-5MG) (working concentration is 1 μg/ml in PBS)
  29. Trypsin (Corning, catalog number: 25-052-CI)
  30. Collagenase (Roche Diagnostics, catalog number: 11088793001)
  31. Dispase (Roche Diagnostics, catalog number: 04942078001)
  32. Polybrene (Sigma-Aldrich, catalog number: H9268-5G) 
  33. Direct lineage depletion kit (Miltenyi Biotec, catalog number: 130-110-470)
  34. Accutase cell detachment solution (Corning, catalog number: 25-058-CI)
  35. StemSpan SFEM (Stem Cell Technologies, catalog number: 09650)
  36. Knockout serum replacement (Thermo Fisher Scientific, catalog number: 10828028)
  37. Recombinant human SCF, or KitL (PeproTech, catalog number: 250-03)
  38. UltraPureTM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, catalog number: 15575020)
  39. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) (Thermo Fisher Scientific, catalog number: D1306)
  40. F-12 medium (Corning, Cellgro, catalog number: 10-080-CV)
  41. DMEM low-glucose medium (Corning, Cellgro, catalog number: 10-014-CV)
  42. Heat-inactivated FBS (Denville Scientific, catalog number: FB5001)
  43. Non-essential amino acid (Corning, Cellgro, catalog number: 25-025-CI)
  44. Penicillin/streptomycin/amphotericin (Corning, Cellgro, catalog number: 30-004-CI)
  45. 1 M HEPES (Corning, Cellgro, catalog number: 25-060-CI)
  46. Endothelial cell growth supplement (Alfa Aesar, catalog number: J64516)
  47. Heparin sodium 10 mg/ml (Sigma-Aldrich, catalog number: H3149-100KU)
  48. GlutaMAX (Thermo Fisher Scientific, catalog number: 35050061)
  49. Antibodies (Table 1)
  50. 8x stock concentration of collagenase/dispase solution (see Recipes)
  51. MACS buffer (see Recipes)
  52. Polybrene stock solution (see Recipes)
  53. EC complete medium (see Recipes)

    Table 1. List of antibodies used

Equipment

  1. Pipettes, Denville Ultra EZpetteTM Pipette Starter Kit (Gray/Blue) (Denville Scientific, catalog number: P3960-SK)
  2. Scissors, Straight; Sharp-Blunt; 4.5" Length (Roboz Surgical Instrument, catalog number: RS-6800)
  3. Forceps, Student Grade Thumb Dressing Forceps 4.5" Serrated (Roboz Surgical Instrument, catalog number: 65-8100)
  4. Mortar and pestles. (VWR, catalog numbers: 89038-148 and 89038-164) 
  5. 37 °C Orbital Shaker (Thermo Fisher Scientific, model: MaxQTM 4000)
  6. Centrifuge, SorvallTM LegendTM XT/XF Centrifuge Series (Thermo Fisher Scientific, model: SorvallTM LegendTM XT, catalog number: 75004505)
  7. FACS Aria II cell sorter (BD, model: FACSAria II)
  8. Laminar flow hood (The Baker Company, SterileGARD biological safety cabinets)
  9. NanoDrop Spectrometer (Thermo Fisher Scientific, model: NanoDropTM 1000, catalog number: ND-1000)

Procedure

  1. Prepare BV13-AF647 conjugated antibodies and measure the degree of labeling (DOL)
    1. Aliquot the AF647 dye into individual aliquots of 40 μg, by first resuspend 1 mg AF647 dye in 500 μl of DMSO, followed by aliquoting 20 μl into each tube and rotating desiccation.
    2. Concentrate antibodies to 1 mg/ml (100 μg total) in a 0.5 ml Amicon Ultra Filter Unit. 
    3. Collect antibody by inverting the column into a fresh tube and spinning at 1,000 x g for 3 min.
    4. Bring the volume up to 100 μl with PBS.
    5. Add 10 μl of 1 M NaHCO3.
    6. Resuspend pellet of dye with antibody solution, incubate at 37 °C for 2 h.
    7. Pipette to mix every 20 min to evenly conjugate.
    8. When there is 5 min left to the conjugation, prepare the Bio-Spin® 30 (Bio-Rad) columns for purification. 
      1. Swing tube to get slurry out the lid.
      2. Remove orange cap.
      3. Twist off bottom.
      4. Place in a 5 ml polystyrene Falcon tube to collect flow-through.
      5. Spin at 1,100 x g for 3 min in a swing bucket rotor.
    9. Load antibody/dye solution drop by drop onto the center of the resin.
    10. Place the column into a collection tube and spin for 1,100 x g for 5 min.
    11. Place into an amber tube and store at 4 °C.
    12. Calculate:



      Note that for Amax and A280 are measured using a NanoDrop under the section “proteins and labels”. AF647 dye, the Amax is measured at 651 nm. Extinction Coeffi is 239,000, Correction Factor is 0.03, and Extinction Coefficient of typical IgG is 203,000.

      For AF647, 

  2. Isolation of primary BMECs via cell sorting
    1. Prepare BV13-AF647 conjugated antibodies, at 1 mg/ml. Measure the degree of labeling (DOL). A good DOL to work with is between 4 and 8.
    2. Per mouse, prepare BV13-AF647 antibody by adding 25 μl of conjugated antibody in 75 μl of sterile PBS.
    3. Anesthetize the mouse using isofluorane and oxygen flow. Retroobitally injects the 100 μl BV13-AF647 antibody (25 μg) prepared at Step B2 into each mouse (Video 1).

      Video 1. Retroobital injection of BV13-AF647. After putting the mouse in the isoflurane chamber for 3 min, the mice become anesthetized (not shown in video). After confirming the state of anesthesia by pinching the in-between-toe area and lack of movement of mouse, the mouse was taken out from the isoflurane chamber and subjected to retro-orbital injection. Position the head of mouse closer to the needle. Firmly hold the eye area by placing the thumb and index fingers at each side of the eye ball. Using a downward motion, expose the eye ball as much as possible and meanwhile maintaining the eyeball in still position (This required placement of thumb and index fingers immediately adjacent of eye ball, but not further down below). Insert the needle from the anterior end of the eye to the area subneath the eyeball, (optional: one can inject the needle fully until the needle feels the bones behind the retroorbital plexus and retrieve the needle a little bit). Inject the 25 μg of BV13-AF647 diluted in 75 μl PBS (in total 100 μl volume) into the mouse. Quickly detach the needle and gently cover the eyes with eye lids to prevent bleeding. Mice were monitored for any signs of bleeding or other discomfort, which usually do not take place. (Study approval. All animal experiments were performed under the approval of Weill Cornell Medicine Institutional Animal Care and Use Committee, New York, NY. All experimental procedures followed the IACUC guidelines. This video was made at Weill Cornell Medical College according to the guidelines of the IACUC of Weill Cornell Medical College, New York, New York, USA under protocol # 2009-0061.)

    4. Ten minutes later, euthanize the mouse.
    5. Quickly open the mouse, dissect out the 2 femurs and 2 tibias.
    6. Clean out the muscles using Kimwipes and scissors (Video 2).

      Video 2. Dissecting femurs and tibias. Expose and peel off the skin of legs using scissors. When dissecting out the femurs from mice, make sure to cut as much as possible at the end where femur and hip joints are attached, to preserve the intactness of femurs. After the femurs and tibias are dissected out from mice, excessive muscles are removed from the leg of mice using scissors (not shown in video). The femur and tibias was then separated using a gentle twisting motion. To further clear off the muscles from femurs, Kimwipes are used to scratch off the muscles, any residual muscle that is not easily detached using Kimwipes can be cut off using scissors. To clear off muscles from tibias, hold the tibias bottom up, the toe facing upward, with the back of foot facing toward the operator. Gently cut using scissors at the wrist area to expose the skin, then the skin and muscles can all be peeled off by pulling the toe downward and simultaneously pushing the tibias bone upward (see video for detailed techniques). (Study approval. All animal experiments were performed under the approval of Weill Cornell Medicine Institutional Animal Care and Use Committee, New York, NY. All experimental procedures followed the IACUC guidelines. This video was made at Weill Cornell Medical College according to the guidelines of the IACUC of Weill Cornell Medical College, New York, New York, USA under protocol # 2009-0061.)

    7. Prepare 1x collagenase/dispase solution by diluting the 8x collagenase/dispase stock with 1x HBSS buffer.
    8. Homogenize femurs and tibias using a mortar and pestle. Use the pestle to firmly grind the bone tissues in a circular motion, clockwise for 25 times, and counter clockwise for 25 times. Make sure all the marrow tissue is released. 
    9. Add 5 ml of 1x collagenase/dispase/HBSS buffer to the mortar, and transfer all the supernatant and the bone tissues into a 15 ml Falcon tube.
    10. Seal the Falcon tube with parafilm and place the tube on an orbital shaker at 37 °C. Shake for 15 min.
    11. Add 10 ml MACS buffer to stop the enzymatic digestion.
    12. Filter cells through a sterile 40 μm nylon mesh (cell strainer) and centrifuge flow through at 500 x g for 5 min.
    13. Aspirate supernatant carefully.
    14. Carry out lineage depletion using direct lineage depletion kit (Miltenyi Biotech). Collect the Lin- cells from the flow through.
    15. Resuspend cell pellet in 50 μl of MACS buffer.
    16. Block with TruStain mouse block (FcR block, or anti-CD16/32 antibody) at 1:50 dilutions for 5 min on ice.
    17. Add 1 μl CD31 and 1 μl CD45 antibodies into the cells and stain on ice for 25 min.
    18. Add 10 ml MACS buffer to wash.
    19. Prepare resuspenstion solution (PBS + 2 mM EDTA + DAPI). After taking out the supernatant, add 0.4 ml (PBS + 2 mM EDTA + DAPI) into each tube to resuspend the pellet.
    20. Sort the BMECs gated as DAPI-CD45-CD31+VE-Cadherin+ cells at 85 μm nozzle using BD FACSAria II (Figure 1A). Alternatively, when the intravital labeling of the BV13-AF647 was not performed, BMECs can be sorted as DAPI-CD45-CD31+ cells (Figure 1B). 
    21. From one 2-month old mouse, we can obtain about 20,000-30,000 BMECs.
    22. Seed cells into one well of 24-well plate, in 0.5 ml of mouse EC complete medium.


      Figure 1. Gating of BMECs for sorting. A. The Lin- cells were further gated on CD45- and subsequently CD31+VE-Cadherin+ population. The obtained cell population are primary BMECs. B. Alternatively, BMECs can be sorted as CD31+CD45- cells.

  3. Isolation of primary BMECs via CD31-Dynabeads
    1. Day 1: Coat sheep anti-rat Dynabeads with rat anti-mouse CD31 (Clone 13.3) antibody
      1. Use 10 μl beads for femurs and tibias from one mouse.
      2. Wash beads three times each with 1 ml MACS buffer on magnetic rack.
      3. Resuspend beads in 200 μl MACS buffer.
      4. Add 4.8 μl of rat anti-mouse CD31 antibody. 
      5. When working with several mice’s bone marrow samples, scale up the beads volume at Step B1a and the antibody volume at Step B1d, but keep the MACS buffer volume at 200 μl.
      6. Incubate beads and antibody with gentle mixing for 1 h at room temperature and then keep mixing at 4 °C overnight.
    2. Day 2: Isolate primary BMECs
      1. Euthanize the mouse.
      2. Quickly open the mouse, dissect out the 2 femurs and 2 tibias.
      3. Clean out the muscles using Kimwipes and scissors.
      4. Prepare 1x collagenase/dispase solution by diluting the 8x collagenase/dispase stock with 1x HBSS buffer.
      5. Homogenize femurs and tibias using a mortar and pestle. Use the pestle to firmly grind the bone tissues in a circular motion, clockwise for 25 times, and counter clockwise for 25 times. Make sure all the marrow tissue is released.
      6. Add 5 ml of 1x collagenase/dispase/HBSS buffer to the mortar, and transfer all the supernatant and the bone tissues into a 15 ml Falcon tube.
      7. Cap the Falcon tube, and then seal the Falcon tube with parafilm. Place the tube on an orbital shaker at 37 °C. Shake for 15 min.
      8. Add 10 ml MACS buffer to stop the digestion. 
      9. Filter cells through a sterile 40 μm nylon mesh (cell strainer) and centrifuge flow through at 500 x g for 5 min.
      10. Aspirate supernatant carefully.
      11. Resuspend the cell pellet in 0.5 ml MACS buffer in a low retention tube.
      12. Wash beads 3 times in MACS buffer to remove excessive antibody. Resuspend beads in 50 μl MACS buffer per 10 μl beads volume at Step B1a of Day 1. 
      13. Add CD31-coated beads and incubate for 45 min at 4 °C, with gentle shaking.
      14. Collect the bead-bound cells using a magnet and wash 5 times in MACS buffer.
      15. Precoat one well of 12-well plate using fibronectin at 1 μg/ml in PBS.
      16. Resuspend the beads using 1 ml mouse EC complete medium and transfer to a 12-well plate for culture. After Dynabeads-CD31 enrichment, we put all the CD31+ cells obtained from one mouse’s 2 femurs and 2 tibias into one well of 12-well plate pre-coated with fibronectin.
    Notes:
    1. It is recommended to use an appropriate amount of Dynabeads to enrich CD31+ cells. Excessive beads tend to accumulate at the bottom of the well plate and prevent the cells from attaching, decreasing the yield (Figure 2).
    2. The Dynabeads can be removed by enzymatic digestion using trypsin or accutase cell detachment medium. Alternatively, as cells divide or during cell passages, Dynabeads will gradually disappear. 
    3. Comparison between the cell sorting techniques and the Dynabeads enrichment approach: Pure population of BMECs will be obtained from FACS sorting, albeit at low cell number. For generation of stable lines of Akt1-BMECs, Dynabeads approach yields more cells and better viability for seeding. Any impurities at the initial step can be taken care of at the later steps via FACS sorting.


      Figure 2. Representative images of primary mouse CD31+ cells post Dynabeads enrichment. A-B. Bright field image of Dynabeads enriched C31+ lung ECs. C-D. Bright field image of Dynabeads enriched C31+ liver ECs. There are few free Dynabeads floating in the cell culture dish, making it easier for cells to attach. Scale bars =200 μm.

  4. Lentiviral transduction of primary BMECs and cell passages
    1. Day 1
      1. Precoat the plate with fibronectin/PBS solution (1:1,000 dilution of fibronectin in sterile PBS) for 30 min at room temperature in the laminar flow hood.
      2. Aspirate off the fibronectin coating.
      3. Seed the primary BMECs in mouse EC complete medium in the precoated wells. Transform the number of sorted events and divide by 3 to come up with the actual cell number, and seed within smaller well plates, such as 24-well plate. From one mouse’s 2 femurs and 2 tibias, we put the Dynabeads enriched CD31+ cells into one well of 12-well plate. We put the FACS purified BMECs into one well of 24-well plate.
    2. Day 2
      1. One day after plating, some of the cells should have attached. Cells tend to attach at the center and the very edge of the plate.
      2. Add polybrene solution into the medium to a final concentration of 4 μg/ml.
      3. Let sit for 5 min at 37 °C in the cell incubator.
      4. Add 2,500 pg of virus for one well of 24-well plate. Scale up or down as needed.
    3. Day 3
      Add 0.5 ml of fresh medium into the transduced wells (24-well plate).
    4. Days 4-6
      1. Small colonies should be growing out, at the center and at the very edge of the plate.
      2. Gently change the medium after emergence of such colonies.
    5. Days 7-20
      1. Change the medium every 3 days, until it reaches more than 80% confluence.
      2. Around Day 20, passage the cells into a bigger well place, at 1:2. This first-time passage is tricky and should be done very carefully. It is normal to lose cells after replating them into a new well. Trypsin is better than accutase. The primary mouse ECs have been sitting in the same well for 3-4 weeks, and are very firmly attached to the bottom of the plate. Using accutase takes at least 30 min to detach the cells, and thus very harmful. 0.05% trypsin detaches the cells faster and preserves the cells significantly better than accutase. Add 1ml of 0.05% trypsin into 1 well of a 12-well plate, and incubate at 37 °C for 7 min, or until the cells round up when observed under a microscope. Gently detach the cells using pipettes.
    6. Day 20 and beyond
      1. Change the medium every 3-4 days until the cells reach more than 80% confluence.
      2. Passage 1:2 into one well of a 6-well plate.
      3. Repeat Steps C6a-C6b, until the cells reach confluence in T75 flasks. Confirm the purity of cultured Akt1-mouse ECs and purity by sorting if necessary.
      4. Cryopreserve aliquots of early passage Akt1-mouse ECs for future usage.
    Notes:
    1. Timeline: After CD31-Dynabeads isolation of primary ECs, the cells were seeded into 12-well plate. After lentiviral transduction and subsequent culture, it takes about 3-4 weeks to reach one confluent well in a 12-well plate. The subsequent cultures and passages are easier than the first cell passage. It usually takes 2 months to reach confluence in one T75 flask of cells.
    2. Use trypsin, not accutase for cell passaging of murine ECs, especially the first time cell passage.
    3. Dilute 1:2 when doing the initial rounds of cell passage.
    4. Due to intrinsic endothelial cell heterogeneity, it is recommended to generate multiple lines of ECs and compare their functions.
    5. Especially for the earlier passages of ECs, there are multiple colonies in the same well, and the cells look heterogenous (Figure 3A). After passaging for more than 10 times, the cells tend to become more and more homogenous (Figure 3B).
    6. Most of the times, I used 20% oxygen culture conditions when generating the murine ECs lines. 5% oxygen condition should be explored for better yield.
    7. Using the above protocols, mouse EC lines from lung, liver, and brain etc. have been successfully generated.
    8. Key events: If you see cells attach after 2-3 days post seeding, and especially observe a few colonies growing out and persistently gets larger after lentiviral transduction around Days 6-7 post seeding, most likely the cells are going to grow fine. A small number of colonies go a long way, and they proliferate remarkably to generate a murine EC line with scalable cell numbers.


      Figure 3. Representative images of Akt1-BMECs at different stages. A. At day 10 post Akt1 transduction of the Dynabeads enriched CD31+ primary BMECs. Note that several colonies of ECs exist in the well (Scale bar = 500 μm). B. After cell passaging for more than 10 times, the resulting Akt1-BMECs become homogenous (Scale bar = 500 μm). 

  5. In vitro Akt1-BMEC and HSPC coculture assays
    1. Seed BMECs into a 12-well plate and let it grow into confluence in mouse EC complete medium. 
    2. Euthanize Mice. Dissect femurs and tibias out. 
    3. Add 5 ml of MACS buffer into the mortar and then homogenize femurs and tibias (Video 3).
    4. Filter the supernatant through a 40 μm cell strainer into a 50 ml Falcon tube (Video 3).

      Video 3. Homogenizing femurs and tibias to obtain bone marrow cells. Put the 2 femurs and 2 tibias dissected from one mouse into the mortar, and add 5 ml of cold, sterile MACS buffer. Firmly hold the pestle and apply downward force to grind up the femurs and tibias. After the femurs and tibias are broken into pieces, use circular motion to fully release the bone marrow cells within the bone cavities. Apply circular grinding motion for 25 times clockwise and 25 times counter clockwise. For the isolation of BMECs as we discussed in this protocol, both the white bony tissues and the cell suspension are collected into the 15 ml Falcon tube, followed by enzymatic digestion. For collecting hematopoietic cells to retrieve Lin- cells for coculture, collect 5 ml of supernatant containing the cell suspension and immediately filter through a 40 μm cell strainer into a 50 ml Falcon tube. Add 5 ml MACS buffer into the white bony remains and apply circular grinding motion. Collect the supernatant until the red bone marrows have been completely rinsed out, leaving only white bone tissues. Usually, it takes 3 rounds of 5 ml MACS buffer and circular grinding to complete the homogenization processes. No enzymatic digestion (collagenase and dispase) is needed. In total, for collecting the hematopoietic stem cells, we have about 15 ml cell suspension in MACS buffer, ready for downstream processing.

    5. Repeat Steps D3-D4 two more times until all the pieces of bone marrow appear white.
    6. Enrich lineage-negative cells (Lin-) cells using direct lineage depletion kit.
    7. To prepare the Akt1-BMECs for co-culture, first aspirate off the mouse EC complete medium from the wells.
    8. Wash the Akt1-BMECs once with 1x PBS without calcium/magnesium.
    9. Prepare the mouse HSPC culture medium using StemSpan supplemented with 20 ng/ml sKitL, knockout serum replacement, penicillin/streptomycin/amphotericin and glutaMAX.
    10. Resuspend the Lin- cells in 0.1 million/ml StemSpan medium. For example, if 3 wells of 12-well plate of coculture are needed, resuspend 0.3 million of Lin- cells in 3 ml of StemSpan medium.
    11. Day 0: Add 0.1 million of Lin- cells into one well of a 12-well plate. This is considered as Day 0.
    12. Day 2: Add 1 ml of StemSpan medium supplemented with sKitL into each well.
    13. Prepare a second 12-well plate of Akt1-BMECs and let it become confluence by Day 4.
    14. Day 4: Gently collect the floating hematopoietic cells and spin them down at 500 x g for 5 min. Resuspend into 1ml of StemSpan medium and distribute into 1 well of 12-well plate as on Day 0. Add 1 ml each of fresh medium into the old wells of BMECs with Lin- cell coculture. Keep both the old wells and the new wells for culture.
    15. Day 6: Add 1 ml of fresh medium to both the new well and old wells. 
    16. Day 7 and onward: Cell collection, data analysis and downstream functional assays.
      1. On Day 7, collect the floating hematopoietic cells. Add 0.3 ml of accutase into each well of the 12-well plate to detach both the attached HSPCs and the BMECs.
      2. For calculation of total expanded HSPCS: the total hematopoietic cell number are counted. 
        1. Enrich Lin- cells using direct lineage depletion kit and count the resulting Lin- cell numbers. 
        2. And then stain the Lin- cells with CD45, c-Kit, and Sca1 antibodies to obtain the frequency of cKit+Sca1+Lin- HSPCs among the Lin- cells. 
        3. Calculate the total numbers of expanded HSPCs using the Lin- cell number and the percentage of HSPC among Lin- cells.
      3. Phenotyping the lineage cells after 7 days of co-culture
        1. Take out approximately 1 million total expanded cells and wash with PBS once. After decanting the supernatant, resuspend the cells in 50 μl of MACS buffer. Block the cells with mouse FcR block at 1:50 dilution for 10 min on ice. Add lineage antibodies (1 μl of each undiluted antibody) including CD45, Gr1, CD11b, B220, CD3, CD41 into the cell suspension. Stain the antibodies and the cells on ice for 25 min. 
        2. Wash off the excessive and unspecific bound antibodies using 1 ml of MACS buffer. Finally, resuspend the cells in 0.4 ml (PBS + 2 mM EDTA + DAPI) solution for flow cytometry analysis. 
        3. If the cells are not analyzed on the same day when the staining is done, after washing off the excessive antibodies, fix the cells in 200 μl of 1% PFA/PBS solution at room temperature for 3 min. Then wash the PFA using 1 ml of MACS buffer. Finally, resuspend the cells in 0.4 ml of (PBS + 2 mM) buffer. The stained cells can be stored for 2 days before flow cytometry analysis.
        4. The myeloid cells are defined as CD45+Gr1+ or CD45+CD11b+, T cells are defined as CD45+CD3+ cells, and B cells are defined as CD45+B220+ cells. Megakaryocyte lineages are defined as CD45+CD41+ cells.
      4. Functional analysis of the HSPCs can be performed using in vitro methylcellulose assays and competitive transplantation assays. For the methylcellulose assays, sort out 350 cKit+Sca1+Lin- cells (events) in 300 μl of StemSpan medium. Then add the cells into 3 ml of thawed methylcult, and split into 2 low-attachment Petri dish. On Day 8 post culture, analyze the resulting colony number and colony type.
      5. For competitive transplantation assays, irradiate CD45.1 mice on Day 6 of coculture at 9 Gy. On Day 7, count 0.5 million of CD45.2 co-cultured hematopoietic cells and mix with 0.5 million of CD45.1 bone marrow mononucleated cells (BMMNCs), and next retroorbitally inject into the CD45.1 mice. The BMECs are not sorted out when performing such competitive transplantation assays, and will not engraft into the CD45.1 mice. At 4, 8, 12 and 16 weeks post-transplantation, analyze the peripheral chimerism and lineage differentiation potential for CD45.2 cells.

Data analysis

Please refer to the methods sections named “In vitro BMEC-HSPC coculture assays’ in the article (Guo et al., 2017) and Figures 2A-2K for the data analysis.

Recipes

  1. 8x stock concentration of collagenase/dispase solution
    1. Prepare buffer to resuspend the enzymes in: 500 ml PBS, with 5 mM KCl, 10 mM HEPES, 2 mM CaCl2, 1.3 mM MgCl2. Filter to sterilize
    2. Take a sterile bottle, add 2.5 g collagenase A, and 1 g Dispase. Add 125 ml of buffer prepared in step a; Mix to resuspend. Do not filter. The solution is too sticky to be filtered
  2. MACS buffer
    PBS
    2 mM EDTA
    0.1% BSA
    Penicillin/streptomycin/amphotericin (Final concentration: 1x. The stock is 100x, dilute 1:100 when making MACS buffer)
    Filter to sterilize
  3. Polybrene stock solution
    Prepare stock concentration using ddH2O at 4 mg/ml
    Filter to sterilize in a laminar flow hood.
    Final working concentration: 4 μg/ml-8 μg/ml depending on the cell types
  4. Mouse EC complete medium

    Note: Prepare heparin sodium powder to a stock concentration of 10 mg/ml using F-12 medium. Filter to sterilize.

Acknowledgments

SR is supported by the Ansary Stem Cell Institute, the Starr Foundation Tri-Institutional Stem Cell core project, the Tri-Institutional Stem Cell Initiative (TRI-SCI 2013-032, 2014-023, 2016-013), the Empire State Stem Cell Board, and New York State Department of Health grants, and by grants from the NIH R01 (DK095039, HL119872, HL128158, HL115128, HL099997) and U54 CA163167.

Competing interests

The authors declare no conflict of interest.

Ethics

All animal experiments were performed under the approval of Weill Cornell Medicine Institutional Animal Care and Use Committee, New York, NY. All experimental procedures followed the IACUC guidelines. The videos were made at Weill Cornell Medical College according to the guidelines of the IACUC of Weill Cornell Medical College, New York, New York, USA under protocol # 2009-0061).

References

  1. Asada, N., Takeishi, S. and Frenette, P. S. (2017). Complexity of bone marrow hematopoietic stem cell niche. Int J Hematol 106(1): 45-54.
  2. Butler, J. M., Nolan, D. J., Vertes, E. L., Varnum-Finney, B., Kobayashi, H., Hooper, A. T., Seandel, M., Shido, K., White, I. A., Kobayashi, M., Witte, L., May, C., Shawber, C., Kimura, Y., Kitajewski, J., Rosenwaks, Z., Bernstein, I. D. and Rafii, S. (2010). Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6(3): 251-264. 
  3. Guo, P., Poulos, M. G., Palikuqi, B., Badwe, C. R., Lis, R., Kunar, B., Ding, B. S., Rabbany, S. Y., Shido, K., Butler, J. M. and Rafii, S. (2017). Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression. J Clin Invest 127(12): 4242-4256.
  4. Hadland, B. K., Varnum-Finney, B., Poulos, M. G., Moon, R. T., Butler, J. M., Rafii, S. and Bernstein, I. D. (2015). Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells. J Clin Invest 125(5): 2032-2045.
  5. Kobayashi, H., Butler, J. M., O'Donnell, R., Kobayashi, M., Ding, B. S., Bonner, B., Chiu, V. K., Nolan, D. J., Shido, K., Benjamin, L. and Rafii, S. (2010). Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol 12(11): 1046-1056.
  6. Mendelson, A. and Frenette, P. S. (2014). Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat Med 20(8): 833-846.
  7. Poulos, M. G., Guo, P., Kofler, N. M., Pinho, S., Gutkin, M. C., Tikhonova, A., Aifantis, I., Frenette, P. S., Kitajewski, J., Rafii, S. and Butler, J. M. (2013). Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis. Cell Rep 4(5): 1022-1034. 
  8. Poulos, M. G., Crowley, M. J. P., Gutkin, M. C., Ramalingam, P., Schachterle, W., Thomas, J. L., Elemento, O. and Butler, J. M. (2015). Vascular platform to define hematopoietic stem cell factors and enhance regenerative hematopoiesis. Stem Cell Rep 5(5): 881-894.
  9. Rafii, S., Butler, J. M. and Ding, B. S. (2016). Angiocrine functions of organ-specific endothelial cells. Nature 529(7586): 316-325.
  10. Rafii, S., Shapiro, F., Rimarachin, J., Nachman, R. L., Ferris, B., Weksler, B., Moore, M. A. and Asch, A. S. (1994). Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 84(1): 10-19.
  11. Seandel, M., Butler, J. M., Kobayashi, H., Hooper, A. T., White, I. A., Zhang, F., Vertes, E. L., Kobayashi, M., Zhang, Y., Shmelkov, S. V., Hackett, N. R., Rabbany, S., Boyer, J. L. and Rafii, S. (2008). Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene. Proc Natl Acad Sci U S A 105(49): 19288-19293.
  12. Zhang, F., Cheng, J., Hackett, N. R., Lam, G., Shido, K., Pergolizzi, R., Jin, D. K., Crystal, R. G. and Rafii, S. (2004). Adenovirus E4 gene promotes selective endothelial cell survival and angiogenesis via activation of the vascular endothelial-cadherin/Akt signaling pathway. J Biol Chem 279(12): 11760-11766.

简介

内皮细胞(EC)通过部署EC衍生的旁分泌因子(称为血管分泌因子)维持成人造血干细胞和祖细胞(HSPCs)的自我更新和再生。 产生持久的离体血管生态位,维持EC同一性并保留血管器官的血管分泌谱,为体外解剖血管分泌因子执行其指导的机制提供平台。 干细胞维持和组织再生的功能。 该方案描述了分离原代骨髓EC(BMEC),随后将携带豆蔻酰化-Akt1的慢病毒载体转导入原代BMEC的详细方法,并使用Akt1-BMEC扩展可雕刻的鼠HSPC。 BMEC-HSPC共培养系统用作生物反应器原型,以产生可扩展的血液和免疫系统群体。

【背景】 造血干细胞(HSCs)是多能成体干细胞,可以自我更新以补充自身并分化成血液和免疫系统的所有谱系。 HSC移植为急性髓性白血病等疾病提供了最佳治疗方法,并作为通过基因靶向纠正遗传性血液病突变的细胞平台。有几种来源的造血干细胞,成人骨髓来源的HSC,脐带血来源的HSC和粒细胞集落刺激因子(GCSF) - 复合的HSC。与骨髓来源的HSC相比,脐带血HSC可以耐受更多的HLA错配,并且具有更好的抗白血病活性,并且更容易获得。不幸的是,HSC移植仍然是一个危险的手术程序,移植相关的死亡率部分是由于白血病复发和/或HSC移植恢复期间发生的感染发生率;所有这些都归因于供体脐带血中干细胞数量低。因此,鉴定能够帮助扩展保持自我更新活性的真正HSC的细胞和分子方法具有关键的转化意义。

内皮细胞通过部署内皮源性旁分泌因子(称为血管分泌因子,如KitL,SDF-1,Jagged-1和Jagged-2等)来保护骨髓中成体HSCs的自我更新和再生。 (Poulos et al。,2013; Mendelson and Frenette,2014; Rafii et al。,2016,Asada et al。 ,2017)。 Rafii 等人在分离成人人骨髓内皮细胞(BMECs)和进行共培养实验以扩展HSPCs的技术方面处于领先地位(Rafii et al。,1994 )。人类BMECs和人类脐静脉内皮细胞(HUVECs)的短寿命在策略上(Zhang et al。,2004)通过过表达腺病毒E4ORF1基因克服。得到的细胞称为E4-HUVEC,维持原代HUVEC的血管分布,并能够支持长期重建小鼠HSPCs和人脐带血干细胞的自我更新(Seandel et al。,2008; Butler et al。,2010)。 E4ORF1通过激活Akt1信号通路部分地执行这些功能。因此,我们通过向ORF序列添加5'的豆蔻酰化序列产生组成型活性Akt1,其有助于靶细胞膜上的Akt1进行磷酸化。将豆蔻酰化Akt1转导至原代小鼠BMEC中维持其EC同一性并保留血管分泌谱(Kobayashi et al。,2010)。这种方法可用于体外检测,以剖析BMEC支持HSPCs自我更新和扩增的机制(Poulos et al。,2013; Hadland et al。,2015; Poulos et al。,2015; Guo et al。,2017)。利用内皮细胞异质性的概念,Akt1-BMECs将被证明是体内研究的补充方法,突出了不同血管床对血液和免疫系统亚群分化的指导作用。
该协议分为以下几个部分:

  1. FACSAria II分选原代BMECs。
  2. Dynabeads分离原代BMECs。
  3. 慢病毒转导原代BMECs。
  4. 体外 BMEC-HSPC共培养。

  5. 关键字:血管分泌因子, 脑微血管内皮细胞, 氢化大豆磷脂酰胆碱, 共培养, 小鼠微血管内皮细胞

    材料和试剂

    1. Falcon管,15 ml(Corning,目录号:352096)
    2. Falcon管,50ml(Corning,目录号:352098)
    3. Falcon ® 100 mm TC处理的细胞培养皿,20个/包,200个/箱,无菌(Corning,目录号:353003) 
    4. 低保留管,1.5 ml(Fisher Scientific,目录号,Fisherbrand TM ,目录号:02-681-320)
    5. Kimwipes(KCWW,Kimberly-Clark,目录号:34120)
    6. Parafilm(Bermis,目录号:PM996)
    7. Amicon Ultra-0.5离心过滤装置(默克,产品目录号:UFC503096)
    8. 无菌40微米尼龙网(康宁,目录号:352340)
    9. 6管磁力架(Thermo Fisher Scientific,目录号:AM10055)
    10. 24孔板(康宁,目录号:353047)
    11. 12孔板(康宁,目录号:353043)
    12. T75烧瓶(康宁,目录号:353136)
    13. Bio-Spin ® P-30凝胶柱,Tris缓冲液(Bio-Rad Laboratories,目录号:7326232)
    14. Dynabeads TM 绵羊抗大鼠IgG(赛默飞世尔科技,目录号:11035)
    15. DPBS,1x不含钙和镁(Corning,目录号:21-031-CV)
    16. DMSO,二甲基亚砜(Sigma-Aldrich,目录号:D2650)
    17. Alexa Fluor TM 647 NHS酯(琥珀酰亚胺酯)(Thermo Fisher Scientific,目录号:A20006)
    18. Isothesia(Isoflurane)解决方案(Henry Schein Animal Health,目录号:029405)
    19. 异氟醚腔,EZ麻醉
    20. 氧气(Tech Air)
    21. 氯化钾,KCl,BioXtra,≥99.0%(Sigma-Aldrich,目录号:P9333)
    22. 氯化钙二水合物,CaCl 2 •2H 2 O(Sigma-Aldrich,目录号:C3306)
    23. 氯化镁,MgCl 2 ,无水,≥98%(Sigma-Aldrich,目录号:M8266)
    24. 碳酸氢钠,NaHCO 3 (Sigma-Aldrich,目录号:S5761-500G) 
    25. 牛血清白蛋白,冻干粉,基本上不含IgG,低内毒素,BioReagent,适合细胞培养(Sigma-Aldrich,目录号:A2058)
    26. HBSS缓冲液:Hank's平衡盐溶液,1x不含钙,镁和酚红(Corning,目录号:21-022-CV)
    27. 慢病毒myristoylated-Akt1:通过Lenti-X p24 Rapid Titer Kit(Takara Bio,Clontech,目录号:632200)测量病毒滴度
    28. 纤连蛋白(Sigma-Aldrich,目录号:F1141-5MG)(PBS中的工作浓度为1μg/ ml)
    29. 胰蛋白酶(Corning,目录号:25-052-CI)
    30. 胶原酶(罗氏诊断,目录号:11088793001)
    31. Dispase(Roche Diagnostics,目录号:04942078001)
    32. Polybrene(Sigma-Aldrich,目录号:H9268-5G) 
    33. 直接谱系消耗试剂盒(Miltenyi Biotec,目录号:130-110-470)
    34. Accutase细胞分离解决方案(Corning,目录号:25-058-CI)
    35. StemSpan SFEM(干细胞技术,目录号:09650)
    36. Knockout血清替代品(Thermo Fisher Scientific,目录号:10828028)
    37. 重组人SCF,或KitL(PeproTech,目录号:250-03)
    38. UltraPure TM 0.5 M EDTA,pH 8.0(Thermo Fisher Scientific,目录号:15575020)
    39. DAPI(4',6-二脒基-2-苯基吲哚,二盐酸盐)(Thermo Fisher Scientific,目录号:D1306)
    40. F-12 medium(Corning,Cellgro,目录号:10-080-CV)
    41. DMEM低葡萄糖培养基(Corning,Cellgro,目录号:10-014-CV)
    42. 热灭活FBS(Denville Scientific,目录号:FB5001)
    43. 非必需氨基酸(Corning,Cellgro,目录号:25-025-CI)
    44. 青霉素/链霉素/两性霉素(Corning,Cellgro,目录号:30-004-CI)
    45. 1 M HEPES(Corning,Cellgro,目录号:25-060-CI)
    46. 内皮细胞生长补充剂(Alfa Aesar,目录号:J64516)
    47. 肝素钠10 mg / ml(Sigma-Aldrich,目录号:H3149-100KU)
    48. GlutaMAX(赛默飞世尔科技,目录号:35050061)
    49. 抗体(表1)
    50. 胶原酶/分散酶溶液的8倍浓度(参见食谱)
    51. MACS缓冲区(参见食谱)
    52. Polybrene原液(见食谱)
    53. EC完全中等(见食谱)

      表1.使用的抗体列表

    设备

    1. 移液器,Denville Ultra EZpette TM 移液器入门套件(灰色/蓝色)(Denville Scientific,目录号:P3960-SK)
    2. 剪刀,直;夏普钝; 4.5“长度(Roboz手术器械,目录号:RS-6800)
    3. 镊子,学生等级拇指敷料钳4.5“锯齿(Roboz手术器械,目录号:65-8100)
    4. 砂浆和杵。 (VWR,目录号:89038-148和89038-164) 
    5. 37°C轨道振荡器(Thermo Fisher Scientific,型号:MaxQ TM 4000)
    6. 离心机,Sorvall TM Legend TM XT / XF离心机系列(Thermo Fisher Scientific,型号:Sorvall TM Legend TM XT,目录号:75004505)
    7. FACS Aria II细胞分选仪(BD,型号:FACSAria II)
    8. 层流罩(The Baker Company,SterileGARD生物安全柜)
    9. NanoDrop光谱仪(Thermo Fisher Scientific,型号:NanoDrop TM 1000,目录号:ND-1000)

    程序

    1. 准备BV13-AF647偶联抗体并测量标记程度(DOL)
      1. 将AF647染料等分成40μg的单个等分试样,首先将1mg AF647染料重悬于500μlDMSO中,然后将20μl等分至每个管中并旋转干燥。
      2. 在0.5 ml Amicon Ultra Filter Unit中将抗体浓缩至1 mg / ml(总共100μg)。 
      3. 通过将柱倒置到新管中并在1,000 x g 下旋转3分钟来收集抗体。
      4. 用PBS将体积增加至100μl。
      5. 加入10μl1MNaHCO 3, 3 。
      6. 用抗体溶液重悬沉淀染料,在37℃下孵育2小时。
      7. 移液管每20分钟混合均匀结合。
      8. 当共轭离开5分钟时,准备Bio-Spin ® 30(Bio-Rad)柱进行纯化。 
        1. 旋转管从盖子中取出浆液。
        2. 取下橙色帽子。
        3. 从底部扭转。
        4. 置于5毫升聚苯乙烯Falcon管中以收集流过液。
        5. 在摆式转子转子中以1,100 x g 旋转3分钟。
      9. 将抗体/染料溶液逐滴加载到树脂的中心。
      10. 将柱放入收集管中并旋转1,100 x g 5分钟。
      11. 放入琥珀色管中,在4°C下储存。
      12. 计算:



        注意,对于Amax和A280,使用NanoDrop在“蛋白质和标签”部分下测量。 AF647染料,Amax在651nm处测量。灭绝Coeffi为239,000,校正因子为0.03,典型IgG的消光系数为203,000。



    2. 通过细胞分选分离原代BMEC
      1. 以1mg / ml制备BV13-AF647缀合的抗体。测量标记程度(DOL)。一个好的DOL可以使用4到8个。
      2. 每只小鼠,通过在75μl无菌PBS中加入25μl缀合的抗体制备BV13-AF647抗体。
      3. 使用异氟醚和氧气流麻醉小鼠。将在步骤B2中制备的100μlBV13-AF647抗体(25μg)逐渐倒入每只小鼠(视频1)。


        视频1.BV13-AF647的逆向注射。将小鼠放入异氟醚室中3分钟后,将小鼠麻醉(未在视频中显示)。在通过捏住脚趾间区域和缺乏小鼠运动确认麻醉状态后,将小鼠从异氟醚室中取出并进行眼眶后注射。将鼠标头靠近针头。将拇指和食指放在眼球的两侧,牢牢抓住眼部区域。使用向下运动,尽可能地暴露眼球,同时将眼球保持在静止位置(这需要将拇指和食指紧靠眼球放置,但不要在下方进一步向下)。将针头从眼睛的前端插入眼球下方的区域(可选:可以完全注射针头,直到针头感觉到眶后丛后面的骨骼并稍微取回针头)。将在75μlPBS(总共100μl体积)中稀释的25μgBV13-AF647注射到小鼠中。快速取下针头,用眼睑轻轻盖住眼睛,以防止出血。监测小鼠是否有任何出血或其他不适的迹象,这些迹象通常不会发生。 (研究批准。所有动物实验均在纽约州纽约市威尔康奈尔医学院动物护理和使用委员会的批准下进行。所有实验程序均遵循IACUC指南。该视频是在威尔康奈尔医学院根据指南制作的。根据#2009-0061号协议,美国纽约威尔康奈尔医学院的IACUC。)

      4. 十分钟后,安乐死鼠标。
      5. 快速打开鼠标,剖开2个股骨和2个胫骨。
      6. 使用Kimwipes和剪刀清洁肌肉(视频2)。


        视频2.解剖股骨和胫骨。用剪刀揭开并剥掉腿部皮肤。当从小鼠中解剖股骨时,确保尽可能在股骨和髋关节附着的末端切开,以保持股骨的完整性。在从小鼠中解剖出股骨和胫骨之后,使用剪刀从小鼠腿部移除过多的肌肉(未在视频中示出)。然后使用轻微的扭转运动分离股骨和胫骨。为了进一步清除股骨肌肉,Kimwipes用于刮除肌肉,任何使用Kimwipes不易分离的残留肌肉都可以用剪刀剪掉。为了清除胫骨肌肉,将胫骨向下保持,脚趾朝上,脚背朝向操作者。在手腕区域使用剪刀轻轻切割以露出皮肤,然后通过向下拉动脚趾并同时向上推动胫骨骨骼来剥离皮肤和肌肉(有关详细技术,请参阅视频)。 (研究批准。所有动物实验均在纽约州纽约市威尔康奈尔医学院动物护理和使用委员会的批准下进行。所有实验程序均遵循IACUC指南。该视频是在威尔康奈尔医学院根据指南制作的。根据#2009-0061号协议,美国纽约威尔康奈尔医学院的IACUC。)

      7. 通过用1x HBSS缓冲液稀释8x胶原酶/分散酶原液制备1x胶原酶/分散酶溶液。
      8. 使用研钵和研杵使股骨和胫骨均匀化。使用研杵以圆周方式将骨组织牢固地研磨,顺时针旋转25次,逆时针旋转25次。确保释放所有骨髓组织。 
      9. 向研钵中加入5ml 1x胶原酶/分散酶/ HBSS缓冲液,将所有上清液和骨组织转移到15ml Falcon管中。
      10. 用封口膜密封Falcon管并将管置于37°C的轨道振荡器上。摇动15分钟。
      11. 加入10 ml MACS缓冲液以停止酶消化。
      12. 通过无菌的40μm尼龙网(细胞过滤器)过滤细胞,并以500 x g 离心5分钟。
      13. 小心地吸出上清液。
      14. 使用直接谱系消除试剂盒(Miltenyi Biotech)进行谱系消耗。收集流过的Lin-细胞。
      15. 将细胞沉淀重悬于50μlMACS缓冲液中。
      16. 用1:50稀释度用TruStain小鼠阻断(FcR阻断或抗CD16 / 32抗体)在冰上封闭5分钟。
      17. 将1μlCD31和1μlCD45抗体加入细胞中并在冰上染色25分钟。
      18. 加入10 ml MACS缓冲液进行清洗。
      19. 准备重新溶解溶液(PBS + 2 mM EDTA + DAPI)。取出上清液后,在每个试管中加入0.4ml(PBS + 2mM EDTA + DAPI)以重悬沉淀。
      20. 使用BD对85nm喷嘴处的DAPI - CD45 - CD31 + VE-钙粘蛋白 + 细胞的BMEC进行分类FACSAria II(图1A)。或者,当不进行BV13-AF647的活体标记时,可以将BMEC分类为DAPI - CD45 - CD31 + 细胞(图1B)群组。
      21. 从一只2个月大的小鼠中,我们可以获得大约20,000-30,000个BMEC。
      22. 将细胞接种到24孔板的一个孔中,在0.5ml小鼠EC完全培养基中。


        图1.用于分选的BMEC门控。 A. Lin - 细胞在CD45 - 上进一步门控,随后CD31 + < / sup> VE-钙粘着蛋白 + 群体。获得的细胞群是主要BMEC。 B.或者,BMEC可以分类为CD31 + CD45 - 细胞。

    3. 通过CD31-Dynabeads分离原代BMEC
      1. 第1天:用大鼠抗小鼠CD31(克隆13.3)抗体涂羊抗大鼠Dynabeads
        1. 使用10μl珠子从一只小鼠的股骨和胫骨。
        2. 在磁架上用1ml MACS缓冲液洗涤珠子三次。
        3. 将珠子重悬于200μlMACS缓冲液中。
        4. 加入4.8μl大鼠抗小鼠CD31抗体。&nbsp;
        5. 当使用几只小鼠的骨髓样品时,在步骤B1a放大珠子体积并在步骤B1d放大抗体体积,但保持MACS缓冲液体积为200μl。
        6. 在室温下温和混合孵育珠子和抗体1小时,然后在4℃下保持混合过夜。
      2. 第2天:分离主要BMEC
        1. 安乐死鼠标。
        2. 快速打开鼠标,剖开2个股骨和2个胫骨。
        3. 使用Kimwipes和剪刀清洁肌肉。
        4. 通过用1x HBSS缓冲液稀释8x胶原酶/分散酶原液制备1x胶原酶/分散酶溶液。
        5. 使用研钵和研杵使股骨和胫骨均匀化。使用研杵以圆周方式将骨组织牢固地研磨,顺时针旋转25次,逆时针旋转25次。确保释放所有骨髓组织。
        6. 向研钵中加入5ml 1x胶原酶/分散酶/ HBSS缓冲液,将所有上清液和骨组织转移到15ml Falcon管中。
        7. 盖上Falcon管,然后用封口膜密封Falcon管。将管置于37°C的轨道振荡器上。摇动15分钟。
        8. 加入10毫升MACS缓冲液以阻止消化。&nbsp;
        9. 通过无菌的40μm尼龙网(细胞过滤器)过滤细胞,并以500 x g 离心5分钟。
        10. 小心地吸出上清液。
        11. 将细胞沉淀重悬于0.5ml MACS缓冲液中的低保留管中。
        12. 在MACS缓冲液中洗涤珠子3次以除去过量的抗体。在第1天的步骤B1a,每10μl珠子体积在50μlMACS缓冲液中重悬珠子。&nbsp;
        13. 加入CD31包被的珠子,在4℃下温育45分钟,轻轻摇动。
        14. 使用磁铁收集珠子结合的细胞并在MACS缓冲液中洗涤5次。
        15. 使用PBS中1μg/ ml的纤连蛋白预涂覆12孔板的一个孔。
        16. 使用1ml小鼠EC完全培养基重悬珠子,并转移到12孔板中进行培养。在Dynabeads-CD31富集后,我们将从一只小鼠的2个股骨和2个胫骨获得的所有CD31 + 细胞放入预先涂有纤连蛋白的12孔板的一个孔中。
      注意:
      1. 建议使用适量的Dynabeads来富集CD31 + 细胞。过多的珠子倾向于积聚在孔板底部并阻止细胞附着,从而降低产量(图2)。
      2. 可以使用胰蛋白酶或accutase细胞分离培养基通过酶消化除去Dynabeads。或者,当细胞分裂或在细胞传代期间,Dynabeads将逐渐消失。
      3. 细胞分选技术和Dynabeads富集方法之间的比较:BMEC的纯种群将从FACS分选获得,尽管细胞数量低。为了产生稳定的Akt1-BMEC系,Dynabeads方法产生更多的细胞和更好的接种活力。初始步骤中的任何杂质可以通过FACS分选在后面的步骤中处理。


        图2. Dynabeads富集后的原代小鼠CD31 + 细胞的代表性图像。 A-B。 Dynabeads的明场图像富含C31 +肺EC。光盘。 Dynabeads的明场图像富含C31 +肝脏ECs。很少有免费的Dynabeads漂浮在细胞培养皿中,使细胞更容易附着。比例尺=200μm。

    4. 原发性BMEC和细胞传代的慢病毒转导
      1. 第1天
        1. 在室温下在层流罩中用纤连蛋白/ PBS溶液(在无菌PBS中1:1,000稀释纤连蛋白)预涂覆板30分钟。
        2. 吸出纤连蛋白涂层。
        3. 在预涂孔中使小鼠EC完全培养基中的初级BMEC接种。转换排序事件的数量并除以3以得出实际的细胞数,并在较小的孔板内种子,例如24孔板。从一只小鼠的2只股骨和2只胫骨,我们将Dynabeads富集的CD31 + 细胞放入12孔板的一个孔中。我们将FACS纯化的BMEC放入24孔板的一个孔中。
      2. 第2天
        1. 接种后一天,一些细胞应附着。细胞倾向于附着在板的中心和边缘。
        2. 将聚凝胺溶液加入培养基中至终浓度为4μg/ ml。
        3. 在37℃下在细胞培养箱中静置5分钟。
        4. 在24孔板的一个孔中加入2,500pg病毒。根据需要放大或缩小。
      3. 第3天
        将0.5ml新鲜培养基加入转导孔(24孔板)中。
      4. 第4-6天
        1. 小菌落应该在板的中心和边缘生长出来。
        2. 在这些菌落出现后轻轻更换培养基。
      5. 第7-20天
        1. 每3天更换一次培养基,直至达到80%以上的汇合。
        2. 在第20天左右,将细胞以1:2的比例传送到更大的孔中。这个第一次通过很棘手,应该非常小心。将它们重新放入新井后丢失细胞是正常的。胰蛋白酶比accutase更好。原代小鼠EC已经在同一个孔中放置3-4周,并且非常牢固地附着在板的底部。使用accutase需要至少30分钟来分离细胞,因此非常有害。 0.05%胰蛋白酶可以更快地分离细胞,并且比accutase显着更好地保留细胞。将1ml 0.05%胰蛋白酶加入12孔板的1孔中,并在37℃下孵育7分钟,或直到在显微镜下观察时细胞向上翻转。使用移液器轻轻地分离细胞。
      6. 第20天及以后
        1. 每3-4天更换培养基,直至细胞达到80%以上汇合。
        2. 将1:2通道插入6孔板的一个孔中。
        3. 重复步骤C6a-C6b,直至细胞在T75烧瓶中达到汇合。如有必要,通过分选确认培养的Akt1-小鼠EC的纯度和纯度。
        4. 冷冻保存早期传代Akt1-小鼠ECs的等分试样以备将来使用。
      注意:
      1. 时间线:在CD31-Dynabeads分离初级EC后,将细胞接种到12孔板中。在慢病毒转导和随后的培养后,在12孔板中到达一个汇合孔需要约3-4周。随后的培养和传代比第一次细胞传代更容易。在一个T75培养瓶中通常需要2个月才能达到融合。
      2. 使用胰蛋白酶,而不是用于小鼠ECs的细胞传代,特别是第一次细胞传代。
      3. 在进行第一轮细胞传代时稀释1:2。
      4. 由于内在的内皮细胞异质性,建议生成多行EC并比较它们的功能。
      5. 特别是对于EC的早期传代,在同一个孔中存在多个菌落,并且细胞看起来是异质的(图3A)。传代超过10次后,细胞趋于变得越来越均匀(图3B)。
      6. 大多数时候,我在产生小鼠ECs细胞系时使用了20%的氧培养条件。应探索5%的氧气条件以获得更好的产量。
      7. 使用上述方案,已成功生成肺,肝和脑等小鼠EC系列。
      8. 关键事件:如果您在播种后2-3天看到细胞附着,特别是观察到一些菌落长出并且在接种后第6-7天的慢病毒转导后持续变大,很可能细胞将会生长精细。少数菌落长期存在,并且它们显着增殖以产生具有可扩展细胞数的小鼠EC系列。


        图3.不同阶段的Akt1-BMEC的代表性图像。 A.在Akt1转导后第10天,对Dynabeads富集的CD31 +原代BMEC进行转导。注意,孔中存在几个EC菌落(比例尺=500μm)。 B.细胞传代超过10次后,得到的Akt1-BMEC变得均匀(比例尺=500μm)。&nbsp;

    5. 体外 Akt1-BMEC和HSPC共培养试验
      1. 将BMEC培养到12孔板中,使其在小鼠EC完全培养基中生长融合。&nbsp;
      2. 安乐死小鼠。解剖股骨和胫骨。&nbsp;
      3. 在研钵中加入5ml MACS缓冲液,然后均匀化股骨和胫骨(视频3)。
      4. 将上清液通过40μm细胞过滤器过滤到50ml Falcon管中(视频3)。


        视频3.均匀化股骨和胫骨以获得骨髓细胞。将从一只小鼠中解剖出的2个股骨和2个胫骨放入研钵中,并加入5ml冷的无菌MACS缓冲液。牢牢握住杵并施加向下的力来磨碎股骨和胫骨。在股骨和胫骨碎成碎片后,使用圆周运动完全释放骨腔内的骨髓细胞。顺时针旋转25次,逆时针旋转25次。对于我们在该方案中讨论的BMEC的分离,将白色骨组织和细胞悬浮液收集到15ml Falcon管中,然后进行酶消化。为了收集造血细胞以回收用于共培养的Lin-细胞,收集5ml含有细胞悬浮液的上清液,并立即通过40μm细胞过滤器过滤到50ml Falcon管中。将5ml MACS缓冲液加入白色骨质残留物中并进行圆形研磨运动。收集上清液直至红色骨髓完全冲洗掉,只留下白色骨组织。通常,需要3轮5毫升MACS缓冲液和圆形研磨来完成均质化过程。不需要酶消化(胶原酶和分散酶)。总的来说,为了收集造血干细胞,我们在MACS缓冲液中含有约15 ml细胞悬液,可以进行下游处理。

      5. 再重复步骤D3-D4两次,直到所有骨髓都呈现白色。
      6. 使用直接谱系消除试剂盒富集谱系阴性细胞(Lin - )细胞。
      7. 为了制备用于共培养的Akt1-BMEC,首先从孔中吸出小鼠EC完全培养基。
      8. 用不含钙/镁的1x PBS洗涤Akt1-BMECs一次。
      9. 使用补充有20ng / ml sKitL,敲除血清替代物,青霉素/链霉素/两性霉素和glutaMAX的StemSpan制备小鼠HSPC培养基。
      10. 将Lin - 细胞重悬于0.1百万/ ml StemSpan培养基中。例如,如果需要共培养12孔板的3个孔,则在3ml StemSpan培养基中重悬30万个Lin - 细胞。
      11. 第0天:将10百万个Lin - 细胞加入12孔板的一个孔中。这被视为第0天。
      12. 第2天:向每个孔中加入1ml补充有sKitL的StemSpan培养基。
      13. 准备第二个12孔板的Akt1-BMECs,让它在第4天变得融合。
      14. 第4天:轻轻收集漂浮的造血细胞并在500 x g 下旋转5分钟。重悬于1ml StemSpan培养基中,并在第0天分配到12孔板的1孔中。将每种新鲜培养基1ml加入到具有Lin - 细胞共培养物的BMEC的旧孔中。保留旧井和新井进行培养。
      15. 第6天:在新井和旧井中加入1毫升新鲜培养基。&nbsp;
      16. 第7天及以后:细胞收集,数据分析和下游功能分析。
        1. 在第7天,收集漂浮的造血细胞。向12孔板的每个孔中加入0.3ml accutase以分离附着的HSPC和BMEC。
        2. 计算总扩张HSPCS:计算总造血细胞数。&nbsp;
          1. 使用直接谱系消除试剂盒富集Lin - 细胞并计算得到的Lin - 细胞数。&nbsp;
          2. 然后用CD45,c-Kit和Sca1抗体染色Lin - 细胞以获得cKit + Sca1 + Lin Lin - 细胞中的HSPCs。&nbsp;
          3. 使用Lin - 细胞数和Lin - 细胞中HSPC的百分比计算扩增的HSPC的总数。
        3. 共培养7天后对谱系细胞进行表型分析
          1. 取出约100万个扩增的细胞,用PBS洗一次。倾析上清液后,将细胞重悬于50μlMACS缓冲液中。用1:50稀释的小鼠FcR阻断细胞在冰上封闭细胞10分钟。将谱系抗体(每种未稀释的抗体1μl)(包括CD45,Gr1,CD11b,B220,CD3,CD41)添加到细胞悬浮液中。将抗体和细胞在冰上染色25分钟。&nbsp;
          2. 使用1ml MACS缓冲液洗掉过量和非特异性结合的抗体。最后,将细胞重悬于0.4 ml(PBS + 2 mM EDTA + DAPI)溶液中进行流式细胞分析。&nbsp;
          3. 如果在染色完成的同一天未对细胞进行分析,则在洗去过量抗体后,将细胞在室温下固定在200μl的1%PFA / PBS溶液中3分钟。然后使用1ml MACS缓冲液洗涤PFA。最后,将细胞重悬于0.4ml(PBS + 2mM)缓冲液中。在流式细胞术分析之前,染色的细胞可以储存2天。
          4. 髓样细胞定义为CD45 + Gr1 + 或CD45 + CD11b + ,T细胞定义为CD45 + CD3 + 细胞,B细胞定义为CD45 + B220 + 细胞。巨核细胞谱系定义为CD45 + CD41 + 细胞。
        4. 可以使用体外甲基纤维素测定和竞争性移植测定来进行HSPC的功能分析。对于甲基纤维素测定,在300μlStemSpan培养基中挑选350 cKit + Sca1 + Lin - 细胞(事件)。然后将细胞加入3ml解冻的甲基培养物中,并分成2个低附着的培养皿。在培养后第8天,分析所得的菌落数和菌落类型。
        5. 对于竞争性移植测定,在共培养的第6天以9Gy照射CD45.1小鼠。在第7天,计数50万CD45.2共培养的造血细胞并与0.5百万CD45.1骨髓单核细胞(BMMNC)混合,然后将其后向后注射到CD45.1小鼠中。当进行这种竞争性移植测定时,BMEC没有被分类,并且不会植入CD45.1小鼠。在移植后第4,8,12和16周,分析CD45.2细胞的外周嵌合和谱系分化潜能。

    数据分析

    请参阅文章(Guo et al。,2017)和图2A-2K中名为“体外 BMEC-HSPC共培养测定”的方法部分进行数据分析。

    食谱

    1. 胶原酶/分散酶溶液的8倍浓度
      1. 制备缓冲液以将酶重悬于:500ml PBS,含有5mM KCl,10mM HEPES,2mM CaCl 2,,1.3mM MgCl 2 。过滤消毒
      2. 取无菌瓶,加入2.5克胶原酶A和1克Dispase。加入步骤a中制备的125ml缓冲液;混合重新悬浮。不要过滤。该溶液太粘,无法过滤
    2. MACS缓冲区
      PBS
      2 mM EDTA
      0.1%BSA
      青霉素/链霉素/两性霉素(最终浓度:1x。原料为100x,制成MACS缓冲液时稀释1:100)
      过滤消毒
    3. Polybrene库存解决方案
      使用4 mg / ml的ddH 2 O制备原液浓度
      过滤器在层流罩中消毒。
      最终工作浓度:4μg/ ml-8μg/ ml,具体取决于细胞类型
    4. 小鼠EC完全培养基

      注意:使用F-12培养基制备浓度为10 mg / ml的肝素钠粉末。过滤消毒。

    致谢

    SR由Ansary干细胞研究所,Starr基金会三机制干细胞核心项目,三机制干细胞计划(TRI-SCI 2013-032,2014-023,2016-013),帝国状态干细胞支持理事会和纽约州卫生部授予,并通过NIH R01(DK095039,HL119872,HL128158,HL115128,HL099997)和U54 CA163167的拨款。

    利益争夺

    作者宣称没有利益冲突。

    伦理

    所有动物实验均在纽约州纽约的Weill Cornell Medicine Institutional Animal Care and Use Committee的批准下进行。所有实验程序均遵循IACUC指南。这些视频是根据美国纽约州纽约市威尔康奈尔医学院IACUC指南,根据协议#2009-0061)在威尔康奈尔医学院制作的。

    参考

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引用:Guo, P. and Rafii, S. (2018). Generation of BMEC Lines and in vitro BMEC-HSPC Co-culture Assays. Bio-protocol 8(21): e3079. DOI: 10.21769/BioProtoc.3079.
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