This work was supported by the National Natural Science Foundation of China (31530045 and 31371500 to Y.A.Z., 31401245 to Q.C.Y.), the Ministry of Science and Technology of China (2014CB964800), The Chinese Academy of Sciences (XDB19000000 to Y.A.Z.), and the Chinese Society of Cell Biology (Early Career Fellowship to Q.C.Y.).

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
<ol>
	<li>Mammary gland harvest and tissue digestion.
	<ol>
		<li>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.</li>
		<li>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 &#181;m filter to sterilize and pre-warm it to 37 &#176;C in a water bath.</li>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>Horizontally place the 50-mL centrifuge tube on a rocking platform and set the speed to 100 rpm and the temperature to 37 &#176;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.</li>
	</ol>
	</li>
	<li>Single-cell solution preparation.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#176;C for 5 min to further digest the remaining tissue.</li>
		<li>Add in 3 mL of pre-warmed IMDM and then add 100 &#181;L of DNase I solution (0.25 mg of DNase I diluted in 1 mL of PBS). Store the tube at 37 &#176;C to break up the DNA filaments. Pass the tissue mixture through a 70-&#181;m cell strainer to obtain the cell solution. Rinse the strainer twice with 2 mL of PBS + 5% FBS to neutralize the enzymatic digestion.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>FACS-based Procr+ VESC isolation.
	<ol>
		<li>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 &#181;g/mL), anti-mouse CD201 (Procr)-biotin (used at a concentration of 2.5 &#181;g/mL), and streptavidin-v450 (used at a concentration of 0.5 &#181;g/mL).
		<ol>
			<li>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-&#181;m cell strainer. Place on ice until FACS sorting.</li>
		</ol>
		</li>
		<li>Proceed to perform FACS-based sorting to isolate Procr+ VESCs.
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. In Vivo VESC Vessel Formation Assay Using the Mammary Fat Pad
<ol>
	<li>Prepare primary ECs for transplantation.
	<ol>
		<li>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.</li>
		<li>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 &#181;L. 
		Note: The basement membrane matrix mixture should always be stored on ice.</li>
		<li>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 &#181;L 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.</li>
	</ol>
	</li>
	<li>Transplant primary ECs to the mammary fat pad.
	<ol>
		<li>Disinfect the surgical instruments and benchtop. If using the same instruments on multiple animals, a bead sterilzer can be used for disinfection.</li>
		<li>Anesthetize 3-week-old BALB/c nude mice by using the anesthetic protocol recommended by your institution&#39;s veterinarian and IACUC. Lay each mouse in supine position, with its back on the operation table. Immobilize its limbs using adhesive tape.</li>
		<li>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.</li>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Place a 27G 1/2&#34; 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 &#181;L of mixed solution onto the cap of the microcentrifuge tube and then aspirate the entire volume into the primed syringe.</li>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Close the skin flap with absorbable sutures.&#160;Suture the skin flap using size 5-0 non-reactive monofilament suture&#160;and close the wounds with a surgeon&#39;s-style knots, each knot approximately 0.3 cm apart. Alternatively, close the skin with wound staples.</li>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Whole-mount Tissue Preparation for Microscopic Observation
<ol>
	<li>Tissue harvest
	<ol>
		<li>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.</li>
		<li>Label the recipient systemic circulation with a tail-vein injection of isolectin-647 dye at a dosage of 50 &#181;g/25 g of bodyweight, resuspended in 50 &#181;L of sterile PBS. Allow a resting period of 5 - 10 min. Sacrifice the mice using CO2.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Tissue digestion and preparation for antibody staining.
	<ol>
		<li>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).</li>
		<li>Place the 15 mL centrifuge tube horizontally on a rocking platform with the speed set to 100 rpm and the temperature set to 37 &#176;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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Whole-mount tissue antibody staining.
	<ol>
		<li>Dilute the primary antibody in antibody staining buffer in a 2 mL centrifuge tube. Incubate the tissue with primary antibody overnight at 4 &#176;C. 
		Note: Rat anti-CD31 (concentration: 10 &#181;g/mL) or rat anti-VE-Cadherin (CD144/Cdh5; concentration: 2.5 &#181;g/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.</li>
		<li>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 &#176;C. 
		Note: Use DAPI (4&#39;,6-diamidino-2-phenylindole) at a final concentration of 5 &#181;g/mL to visualize the nuclei.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;m. 
		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.</li>
	</ol>
	</li>
</ol>
4. Lineage Tracing Using a Genetically Modified Mouse Model
<ol>
	<li>Detect Procr+ VESCs using the mouse model.
	<ol>
		<li>Use the ProcrCreERT2-IRES-tdTomato/+ (referred to as ProcrCreERT2) knock-in mouse strain. 
		Note: In this mouse model, a CreERT2-IRES-tdTomato cassette10 is inserted after the first ATG codon of Procr11.
		<ol>
			<li>Cross ProcrCreERT2 mice with the Rosa26mTtomatomGFP (referred to as R26mTmG) reporter strain to trace the fate of endogenous Procr+ ECs in vivo11. Identify all labeled endogenous Procr+ ECs and their descendent using GFP expression.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Angiogenesis assays represent a good experimental approach to study vascular dynamics. Mouse retinal vasculature, which develops postnatally, has proven to be an attractive model to study angiogenesis12. Despite it being relatively accessible, actual manipulation within the retina vascular bed is rather difficult. So far, the best-described in vivo transplantation has been the plug assay, which encloses cells inside a basement membrane matrix mass and surgically implants/injects subcutane...

During development and homeostasis, vascular growth and remodeling faithfully take place in accordance with organ growth and repair. Angiogenesis describes the generation of new vessels from pre-existing blood vessels and is deemed a major force mediating these dynamic vascular changes. Each blood vessel is inner-lined with a layer of endothelial cells (ECs), and they appear to be the foundation of vessel architecture. For a long time, the mechanism through which the EC pool is replenished during homeostasis remained unclear, and arguments were raised over whether vascular turnover is the result of mature EC proliferation or is the contribution of vascular stem/progenitor cell activities. Due to the lack of direct physiological evidence, the existence and cellular identity of vascular endothelial stem cells (VESCs) also remained controversial.
One of the most common approaches used to verify stem cell behavior is through the transplantation of putative stem cells into recipient mice. This method measures the stemness potential of candidate stem cells in vivo. Transplantation was first applied to the study of bone marrow stem cells1, which contributed to the establishment of the hierarchical characteristics of the hematopoietic system2. In the endothelial field, a basement membrane matrix (e.g., matrigel) plug inserted subcutaneously under the flank skin has been a standard in vivo angiogenesis assay used to address the vessel formation capabilities of transplanted ECs. Multiple experimental methods, including colony formation in 3D culture systems and transplantation, have suggested potential EC progenitor/VESC populations3,4,5,6. However, since ECs embedded in basement membrane matrix are relatively separate from the surrounding tissue, this does not provide the optimal niche environment required to fully explore the angiogenic potential of transplanted cells. As a result, vessels formed within the matrix plug are predominantly capillary-like and are functionally unmeasurable.
The mammary gland develops postnatally, with the most robust growth occurring during puberty and pregnancy. At the pubertal stage, the mammary epithelium undergoes rapid expansion, to occupy the whole mammary fat pad, accompanied by the efficient remodeling of the surrounding vascular structures. Thus, the mammary gland offers an excellent model for the study of angiogenesis. It provides space, matrix, and rich angiogenic stimuli from the growing mammary epithelium and therefore is an ideal grafting site for assessing the angiogenic potential of exogenous cells. In addition, the mammary fat pad allows the formed exogenous vessels to integrate with the host circulation system, enabling further functional evaluation and representing an advantage over subcutaneous transplantation.
Although in vitro culturing and transplantation assays are as effective way to investigate the regeneration properties of a cell population, it is known that such assays may stimulate plasticity as cells are taken away from their native habitats, and changes might be induced when cells are disconnected from their physiological surroundings7. Therefore, obtaining direct in vivo evidence of cell fate is the key approach to advancing the current understanding of the behavior of endothelial populations.
Genetic fate mapping (i.e., in vivo lineage tracing) is imperative for the identification of VESCs and for the investigation of their properties in the body system, as it can reveal in vivo stem cell behavior in its physiological context, and the actual stemness can be assessed. Lineage tracing provides direct evidence of the long-term persistence (i.e., self-renewal) of candidate VESCs and their ability to produce cell types for the tissue of origin (i.e., differentiation potency).
This protocol describes a novel mammary fat pad transplantation technique and a lineage tracing method to observe the vessel generation capability of VESCs. These techniques overcome shortcomings of currently available assays and provide a new way to optimally evaluate the stem cell properties of VESCs. These approaches are efficient tools that can be used to assess the behavior and vessel-forming properties of endothelial populations, as well as to determine vascular cell potency alteration within a pathological environment.

<table><tbody><tr><td>0.05% Trypsin-Ethylene Diamine Tetraacetic Acid(EDTA) (1x)</td><td>Gibco (Life Technologies)</td><td>25300-062</td><td>0.05% Trypsin</td></tr><tr><td>0.22 &amp;micro;m Filter Unit</td><td>Merck Millipore Ltd.</td><td>SLGP033RB</td><td>0.22 &amp;micro;m Filter</td></tr><tr><td>2-Methylbutanol-2, ReagentPlus</td><td>Sigma-Aldrich</td><td>24, 048-6</td><td>Tert Amyl Alcohol</td></tr><tr><td>222, Tribromethanol</td><td>Sigma-Aldrich</td><td>T48402-25g</td><td>222, Tribromethanol</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole(DAPI)</td><td>Invitrogen</td><td>D1306</td><td>DAPI</td></tr><tr><td>70 &amp;micro;m Cell Strainer</td><td>BD FAL</td><td>352350</td><td>70 &amp;micro;m Cell Strainer</td></tr><tr><td>Adhesion Microscope Slides</td><td>CITOGLAS</td><td>188105</td><td>Glass slides</td></tr><tr><td>Anti-mouse CD105 APC</td><td>eBioscience</td><td>17-1051-82, clone MJ7/18</td><td>for FACS analusis use at 1 &amp;micro;g/mL</td></tr><tr><td>Anti-mouse CD201 Biotin</td><td>eBioscience</td><td>13-2012-82</td><td>for FACS analusis use at 2.5 &amp;micro;g/mL</td></tr><tr><td>Anti-mouse CD31 PE-Cyanine 7</td><td>eBioscience</td><td>25-0311-82, clone 390</td><td>for FACS analusis use at 1 &amp;micro;g/mL</td></tr><tr><td>BD FACSJazz Cell Sorter</td><td>BD Biosciences</td><td>655489</td><td>FACS</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>100190332</td><td>BSA</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Centrifuge</td></tr><tr><td>Collagenase type 3</td><td>Worthington-Biochem</td><td>#LS004183</td><td>Collagenase III</td></tr><tr><td>Confocal microscopy</td><td>Leica</td><td>sp8</td><td>Confocal</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D2463-5VL</td><td>Dnase I</td></tr><tr><td>Donkey anti-Rat Cy3</td><td>Jackson ImmunResearch</td><td>712-165-150</td><td>Secondary antibody, use at 0.5 &amp;micro;g/mL</td></tr><tr><td>Dumont Forceps</td><td>WPI</td><td>500342</td><td>Froceps</td></tr><tr><td>Dumont Forceps - Micro-Blunted Tips</td><td>FST</td><td>11253-20</td><td>Forceps</td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>FBS</td></tr><tr><td>FITC Ms CD3/Gr1/CD11b/CD45R(B220)/Ter-119</td><td>BioLegend</td><td>78022</td><td>Blood lineage cocktail for FACS analysis, use at 25 &amp;micro;L per test</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G6279</td><td>Glycerol</td></tr><tr><td>Iscov&#039;s Modified Dulbecco&#039;s Medium</td><td>Gibco (Life Technologies)</td><td>12440-053</td><td>IMDM</td></tr><tr><td>Isolectin GS-IB4 from Griffonia Simplicifolia, Alexa Fluora&amp;nbsp; 647</td><td>Invitrogen</td><td>Z32450</td><td>Isolectin-647</td></tr><tr><td>Matrigel&amp;nbsp; Matrix (growth factor reduced)</td><td>BD</td><td>356231</td><td>Matrigel</td></tr><tr><td>Mouse strain (Actin-GFP)</td><td>Jax Laboratories</td><td>3773</td><td>Actin-GFP</td></tr><tr><td>Mouse strain (C57BL/6)</td><td>SLAC</td><td>C57BL/6</td><td>C57BL/6</td></tr><tr><td>Mouse strain&amp;nbsp; (BALB/c nude)</td><td>SLAC</td><td>BALB/c nude</td><td>Nude mice</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>PFA</td></tr><tr><td>Penicilin Streptomycin</td><td>Gibco (Life Technologies)</td><td>5140-122</td><td>Pen/Strep</td></tr><tr><td>Phosphate Buffered Saline (pH7.2) 1x</td><td>Gibco (Life Technologies)</td><td>c20012500BT</td><td>PBS</td></tr><tr><td>PRIM1640</td><td>Gibco (Life Technologies)</td><td>c11875500CP</td><td>PRIM1640</td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36934</td><td>Mounting Medium</td></tr><tr><td>Rat anti CD144</td><td>BD Biosciences</td><td>550548</td><td>for whole-mount analysis, anti-VE-Cadherin / Cdh5 antibdy for endothelial tight junction, use at 2.5 &amp;micro;g/mL</td></tr><tr><td>Rat anti CD31-biotin</td><td>BD Biosciences</td><td>553371</td><td>for whole-mount analusis, anti-CD31/PECAM1 antibody for endothelial surface adhesion molecule, use at 10 &amp;micro;g/mL</td></tr><tr><td>Red Cell Lysis Buffer</td><td>Sigma</td><td>R7767-100ML</td><td>Red blood cell lysing buffer</td></tr><tr><td>Straight/Sharp-Blunt/10cm</td><td>FST</td><td>14028-10</td><td>Fine Scissors</td></tr><tr><td>Streptavidin eFluor 450</td><td>eBioscience</td><td>48-4317-82</td><td>for FACS analysis use at 0.5 &amp;micro;g/mL</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>101551374</td><td>TAM</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-250ML</td><td>TritonX</td></tr><tr><td>Wax Coated Braided Silk (Size 5-0 USP (1 Metric), 18 inches (45 cm) BLACK on C-1 Needle)</td><td>COVIDIEN</td><td>S1173</td><td>Suture&amp;nbsp;</td></tr><tr><td>Sterile Disposable Scaplels</td><td>Swann Morton</td><td>#10</td><td>Scalpel</td></tr><tr><td>Betadine</td><td>Yifeng Medical</td><td>20160101</td><td>Antiseptic solution</td></tr></tbody></table>

a novel mammary fat pad transplantation technique to visualize the vessel generation of vascular endothelial stem cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

Mammary VESCs Isolation:
To isolate the endothelial population for the consequent transplantation assay, mature (8 week-old), virgin mouse mammary glands were harvested as donor material. Endothelial cells were isolated using an antibody-based cell sorting technique. Representative plots of the FACS analysis of the VESC population in 8-week-old C57BL/6 mammary gland ECs are shown in Figure 1 (Figure ad...

Watch this Scientific Journal Video about A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells at JoVE.com

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

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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Research

JoVE Journal

Developmental Biology

9.2K Views.  Chinese Academy of Sciences. This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

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

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Cancer Research

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Bioengineering

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Biology

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Medicine

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models

Source: Flesken-Nikitin, A. et al. <a href="https://www.jove.com/t/54444">Transplantation Into the Mouse Ovarian Fat Pad.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an assay for transplantation of experimental cells and tissue fragments into the mouse ovarian fat pad. This method can be used to monitor cell regeneration and tumor transformation via non-invasive techniques.

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

Summary

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Chapters in this video

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Mammary Epithelial Transplant Procedure

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Transplantation Into the Mouse Ovarian Fat Pad

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models