A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Experimental procedures were approved by the Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences under the approved protocol SIBCB-NAF-15-002-S335-003.

1. Isolation of VESCs from the Mouse Mammary Gland

1. Mammary gland harvest and tissue digestion.
   1. Use 8 week-old Actin-GFP mature virgin reporter mice, with weights range between 20 and 25 g, as tissue donors.
      Note: Cells originated from the donor can be easily tracked by GFP expression.
   2. Prepare tissue digestion base medium consisting of 5% fetal bovine serum (FBS), 1% pen/strep, and 25 mM HEPES in RPMI1640. Filter the mixture through a 0.22 µm filter to sterilize and pre-warm it to 37 °C in a water bath.
   3. Sacrifice the mice in a CO₂ chamber. Dissect out the fourth pair of inguinal mammary glands by first making a vertical skin incision at the lower abdominal midline using a scalpel (or surgical scissors). Gently peel the skin to one side to expose the inguinal mammary gland using forceps. Obtain the fourth inguinal mammary glands from both sides and cut them into fine pieces in a 6 cm Petri dish using scissors.
      1. Measure the total weight of minced tissue and place it in a 50 mL tube.
         Note: Make sure the resultant tissue mince is smaller than 1 mm³ in size for each piece. A typical yield is approximately 5,000 VESCs per animal.
   4. Prepare digestion base medium in a 50-mL capped centrifuge tube at the volume of 10 mL per 1 g of tissue mince. Add collagenase Type 3 at the concentration of 300 units per 10 mL of digestion base medium to constitute the digestion buffer. Mix evenly and add it to the tissue mince.
   5. Horizontally place the 50-mL centrifuge tube on a rocking platform and set the speed to 100 rpm and the temperature to 37 °C. Agitate the mammary tissue for 2 h. Check and manually shake the tube every 20 min to ensure proper digestion.
      Note: The purpose of tissue digestion prior to FACS is to prepare a single-cell solution from solid tissue. Therefore, by the end of digestion, the content should be a thick solution without visible tissue pieces.
2. Single-cell solution preparation.
   1. After digestion, top-up the 50 mL centrifuge tube with pre-warmed, sterile PBS and invert the tube to mix evenly. Centrifuge the tube at 200 x g for 5 min to obtain a cell pellet.
   2. Remove the supernatant and flick the bottom of the tube to loosen up the cell pellet. Add in 3 mL of red blood cell lysing buffer (see the Table of Materials) and incubate in a tissue culture hood at room temperature (RT) for 5 min to eliminate the red blood cells.
   3. Add 10 mL of sterile PBS and invert the tube to mix evenly. Centrifuge the tissue content at 200 x g for 5 min and discard the supernatant. Add in 3 mL of pre-warmed 0.05% trypsin and store the tube at 37 °C for 5 min to further digest the remaining tissue.
   4. Add in 3 mL of pre-warmed IMDM and then add 100 µL of DNase I solution (0.25 mg of DNase I diluted in 1 mL of PBS). Store the tube at 37 °C to break up the DNA filaments. Pass the tissue mixture through a 70-µm cell strainer to obtain the cell solution. Rinse the strainer twice with 2 mL of PBS + 5% FBS to neutralize the enzymatic digestion.
   5. Centrifuge the cell mixture at 200 x g for 5 min to pellet the cells. Discard the supernatant and resuspend the cell pellet in 1 mL of PBS + 5% FBS for antibody staining.
3. FACS-based Procr⁺ VESC isolation.
   1. Subject the cell mixture obtained from donor mammary tissue to antibody staining following published routine protocols⁸.
      Note: The following antibodies were used: anti-mouse blood lineage-FITC, anti-mouse CD31(PECAM1)-PECY7, anti-mouse CD105-APC (all used at a concentration of 1 µg/mL), anti-mouse CD201 (Procr)-biotin (used at a concentration of 2.5 µg/mL), and streptavidin-v450 (used at a concentration of 0.5 µg/mL).
      1. After antibody staining, top-up the cells with PBS + 5% FBS and then centrifuge at 200 g for 5 min. Discard the supernatant and resuspend the cells with 1 mL of PBS + 5% FBS. Filter through a 40-µm cell strainer. Place on ice until FACS sorting.
   2. Proceed to perform FACS-based sorting to isolate Procr⁺ VESCs.
      1. Analyze the unstained sample and each single-color control to determine the voltage for each color. Calculate the compensation parameters to avoid auto-fluorescence or background staining. Set up the template for proper gating and sorting for the desired cell populations.
      2. To establish the hierarchy of FACS sorting, first eliminate debris from all events and then discard doublets or adhesive cell clusters by triggering with width. Gate-out blood lineage-cells and then use CD31+ and CD105+ to define the endothelial populations.
         Note: Procr+ cells were isolated from CD31+ CD105+ ECs compared to an FMO control. A representative FACS analysis plot is shown in Figure 1. A FACS-isolated VESC population should be suspended in IMDM + 50% FBS and stored on ice until further processing.

2. In Vivo VESC Vessel Formation Assay Using the Mammary Fat Pad

1. Prepare primary ECs for transplantation.
   1. Top-up the FACS-sorted cells with 10 mL of PBS + 5% FBS and then centrifuge at 200 x g for 5 min to settle the cells down to the bottom.
   2. Aspirate the liquid carefully, count the cell number using a hemocytometer, and resuspend the cell pellet with basement membrane matrix mixture (consisting of 50% basement membrane matrix and PBS with 20% FBS, supplemented with 100 ng/mL rmVEGF and 60 U/mL heparin) to reach a working concentration of 15,000 cells/15 µL.
      Note: The basement membrane matrix mixture should always be stored on ice.
   3. Serially dilute the sorted and counted cells to reach the required input concentrations in a 1.5-mL microtube, making sure that the final injection volumes do not exceed 15 µL in total. Add 0.1% Trypan blue to each tube for better content visualization during transplantation. Pipette thoroughly to mix. Store the tubes on ice until use.
      Note: Successful vessel generation could be achieved with a minimum of 100 VESCs.
2. Transplant primary ECs to the mammary fat pad.
   1. Disinfect the surgical instruments and benchtop. If using the same instruments on multiple animals, a bead sterilzer can be used for disinfection.
   2. Anesthetize 3-week-old BALB/c nude mice by using the anesthetic protocol recommended by your institution's veterinarian and IACUC. Lay each mouse in supine position, with its back on the operation table. Immobilize its limbs using adhesive tape.
   3. Dip a cotton swab into iodine-based antiseptic solution. Wipe the entire abdomen area of the mouse thoroughly to sterilize the skin surface before surgery.
   4. Hold and lift the abdomen skin of the mouse with forceps. Use a surgical scalpel to make a small, 1 cm vertical incision. Make two cuts about 0.5 – 1 cm in length, each towards the hind leg, to achieve an upside-down, Y-shaped incision.
      1. Use forceps and a cotton swab to gently separate the skin from the abdomen and pin the skin sideways to expose the inguinal mammary glands. Place the mouse under a stereoscope and set the magnification to 2.0X. Make sure to adjust the focus so that the mammary fat pad can be clearly seen and operated on.
   5. Place a 27G 1/2" needle on ice to pre-chill. Prime a 1-mL syringe with basement membrane matrix mixture in advance to remove possible air bubbles trapped in the needle-attaching region. Pipette out 15 µL of mixed solution onto the cap of the microcentrifuge tube and then aspirate the entire volume into the primed syringe.
   6. Hold the mammary fat pad in front of the inguinal lymph node, using fine tweezers, and lift it slightly for easy injection. Insert about 1/4 of the syringe needle gently into the fat pad.
      1. Release the tweezers from the fad pad to grip and stabilize the syringe needle. Inject the solution slowly to prevent liquid leak. Hold for a few seconds to make sure that the cell mixture is solidified to minimize the leakage when pulling out the needle.
   7. Close the skin flap with absorbable sutures. Suture the skin flap using size 5-0 non-reactive monofilament suture and close the wounds with a surgeon's-style knots, each knot approximately 0.3 cm apart. Alternatively, close the skin with wound staples.
   8. Place operated mice in a separate cage for recovery. Place a heating lamp over the cage to prevent the anesthetized mice from suffering from hypothermia. Place soft paper and cotton at the bottom of the cage to keep the mice warm post-surgery.
      1. Observe the mice carefully until all recipients are fully awake. For the next few days, apply post-surgical analgesics pre-emptively to all animals or as directed by the facility veterinarian. Apply antibiotics according to the approved animal protocol if wound infection occurs. If the surgical wound was closed with wound staples, remove the wound clips 10 days after surgery, when the wound has fully closed.

3. Whole-mount Tissue Preparation for Microscopic Observation

1. Tissue harvest
   1. At 2 – 4 weeks post-implantation, anesthetize the recipient BALB/c nude mice with an intra-peritoneal injection of 2.5% anesthetizing agent at a dosage of 0.12 mL per 20 g of bodyweight, as in step 2.2.2.
      Note: Positive vessel growth can be detected 2 – 4 weeks after cell transplantation.
   2. Label the recipient systemic circulation with a tail-vein injection of isolectin-647 dye at a dosage of 50 µg/25 g of bodyweight, resuspended in 50 µL of sterile PBS. Allow a resting period of 5 – 10 min. Sacrifice the mice using CO₂.
   3. Dissect out the area of mammary tissue where the cells were initially injected following step 2.2.4; use surgical scissors. Using a surgical blade, chop the tissue into roughly 3- to 4-mm³ cubes in a 6-cm Petri dish.
2. Tissue digestion and preparation for antibody staining.
   1. Digest the tissue pieces in a 15 mL centrifuge tube filled with 10 mL of pre-warmed digestion base medium (step 1.1.2) supplemented with collagenase III (300 U per 10 mL).
   2. Place the 15 mL centrifuge tube horizontally on a rocking platform with the speed set to 100 rpm and the temperature set to 37 °C for approximately 1 h to loosen up the tissue structure and to remove excessive adipocyte tissue.
      Note: Adipocytes tend to produce auto-fluorescence which could result in a noisy fluorescent background. By the end of the digestion, the tissue pieces should have a soft, cloudy appearance. The optimal digestion period depends upon the size of chopped tissue pieces and should therefore be adjusted accordingly to obtain the best results. Since the tissue vasculature has already been labeled with fluorescence-tagged Isolectin, all following procedures should be carried out protected from light.
   3. Wash the tissue pieces with PBS at RT for 10 min. Fix the whole-mount tissue by carefully placing the pieces into a 6-cm Petri dish half-full with 4% PFA (pH 7.2 – 7.4) to preserve the fluorescence. Place the Petri dish on a rocking platform and gently agitate the tissue for 30 min at RT.
      Note: PFA is carcinogen and should be handled with caution.
   4. Remove the remaining PFA and wash the fixed tissue three times with whole-mount wash buffer (0.1% tween in PBS), with a 10-min interval between each wash.
3. Whole-mount tissue antibody staining.
   1. Dilute the primary antibody in antibody staining buffer in a 2 mL centrifuge tube. Incubate the tissue with primary antibody overnight at 4 °C.
      Note: Rat anti-CD31 (concentration: 10 µg/mL) or rat anti-VE-Cadherin (CD144/Cdh5; concentration: 2.5 µg/mL) were used as the primary antibodies. Both CD31 and VE-Cadherin are cell surface markers used to recognize endothelium. 5x antibody staining buffer is prepared with 500 mM maleic acid, pH 7.5; 750 mM NaCl; and 0.5% (v/v) tween dissolve in PBS. Only add in an appropriate serum volume (5%) when preparing fresh 1x working solution⁹.
   2. Remove the antibody staining buffer and wash the whole-mount tissue three times with wash buffer, with a 10-min interval. Incubate the tissue with secondary antibody (see the Table of Materials) plus DAPI at RT for 4 h on a rocking platform or overnight at 4 °C.
      Note: Use DAPI (4',6-diamidino-2-phenylindole) at a final concentration of 5 µg/mL to visualize the nuclei.
   3. Wash the whole-mount tissue with wash buffer 3 times for 10 min each. Incubate the tissue pieces in 80% glycerol or 80% sucrose overnight to dehydrate the tissue for better preservation.
   4. Place the individual tissue pieces onto a glass slide and remove the excessive dehydration buffer. Cover the tissue with mounting medium, place a coverslip, and gently squeeze the tissue to set it.
   5. Perform confocal microscopic image acquisition. For whole-mount mammary glands, acquire images using a 40X with N.A. = 1.3 oil objective. Acquire tile scans of Z-stacks at an optical section resolution of 1,024 x 1,024 and a layer separation of 1.0 – 1.2 µm.
      Note: To capture the correct fluorescence light signal, the filter beam splitter should be set to triple dichroic (TD) 488/553/638, with the gain setting at PMT Gain 600-800. The following excitation/emission sources should be used: mGFP, with excitation/emission maxima of 488/509 nm; mTomato with excitation/emission maxima of 532/588 nm; Alexa-647, with excitation/emission maxima of 594/665; and DAPI, with excitation/emission maxima of 350/470.

4. Lineage Tracing Using a Genetically Modified Mouse Model

1. Detect Procr⁺ VESCs using the mouse model.
   1. Use the Procr^{CreERT2-IRES-tdTomato/+} (referred to as Procr^CreERT2) knock-in mouse strain.
      Note: In this mouse model, a CreERT2-IRES-tdTomato cassette¹⁰ is inserted after the first ATG codon of Procr¹¹.
      1. Cross Procr^CreERT2 mice with the Rosa26^mTtomatomGFP (referred to as R26^mTmG) reporter strain to trace the fate of endogenous Procr+ ECs in vivo¹¹. Identify all labeled endogenous Procr⁺ ECs and their descendent using GFP expression.
   2. Administer tamoxifen solution¹¹ (10 mg of tamoxifen dissolved in 1 mL of peanut oil + 10% ethanol to make up a stock solution of 10 mg/mL) intraperitoneally at a dose of 4 mg/25 g bodyweight during the pubertal stage (5 weeks old) to track the developmental fate of Procr+ ECs.
   3. Analyze the clone-forming efficiency of labeled cells by whole-mount immunostaining following the preparation steps outlined in step 3. Obtain mammary fat pads from treated mice both after short-term (2 days) and extended time periods (up to 10 months). See Figure 3.