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 border="0" cellpadding="0" cellspacing="0" width="1265">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">0.05% Trypsin-Ethylene Diamine Tetraacetic Acid(EDTA) (1X)</td>
			<td style="width:263px;">Gibco (Life Technologies)</td>
			<td style="width:208px;">25300-062</td>
			<td style="width:387px;">0.05% Trypsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#181;m Filter Unit</td>
			<td>Merck Millipore Ltd.</td>
			<td>SLGP033RB</td>
			<td>0.22 &#181;m Filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylbutanol-2, ReagentPlus</td>
			<td>Sigma-Aldrich</td>
			<td>24, 048-6</td>
			<td>Tert Amyl Alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">222, Tribromethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25g</td>
			<td>222, Tribromethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole(DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Cell Strainer</td>
			<td>BD FAL</td>
			<td>352350</td>
			<td>70 &#181;m Cell Strainer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesion Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>188105</td>
			<td>Glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD105 APC</td>
			<td>eBioscience</td>
			<td>17-1051-82, clone MJ7/18</td>
			<td>for FACS analusis use at 1 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD201 Biotin</td>
			<td>eBioscience</td>
			<td>13-2012-82</td>
			<td>for FACS analusis use at 2.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD31 PE-Cyanine 7</td>
			<td>eBioscience</td>
			<td>25-0311-82, clone 390</td>
			<td>for FACS analusis use at 1 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSJazz Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>655489</td>
			<td>FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>100190332</td>
			<td>BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 3</td>
			<td>Worthington-Biochem</td>
			<td>#LS004183</td>
			<td>Collagenase III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscopy</td>
			<td>Leica</td>
			<td>sp8</td>
			<td>Confocal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D2463-5VL</td>
			<td>Dnase I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-Rat Cy3</td>
			<td>Jackson ImmunResearch</td>
			<td>712-165-150</td>
			<td>Secondary antibody, use at 0.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps</td>
			<td>WPI</td>
			<td>500342</td>
			<td>Froceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps - Micro-Blunted Tips</td>
			<td>FST</td>
			<td>11253-20</td>
			<td style="width:387px;">Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 &#181;l per test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma</td>
			<td>G6279</td>
			<td>Glycerol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscov&#39;s Modified Dulbecco&#39;s Medium</td>
			<td>Gibco (Life Technologies)</td>
			<td>12440-053</td>
			<td>IMDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolectin GS-IB4 from Griffonia Simplicifolia, Alexa Fluora&#160; 647</td>
			<td>Invitrogen</td>
			<td>Z32450</td>
			<td>Isolectin-647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel&#160; Matrix (growth factor reduced)</td>
			<td>BD</td>
			<td>356231</td>
			<td>Matrigel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain (Actin-GFP)</td>
			<td>Jax Laboratories</td>
			<td>3773</td>
			<td>Actin-GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain (C57BL/6)</td>
			<td>SLAC</td>
			<td>C57BL/6</td>
			<td>C57BL/6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain&#160; (BALB/c nude)</td>
			<td>SLAC</td>
			<td>BALB/c nude</td>
			<td>Nude mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>PFA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilin Streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>5140-122</td>
			<td>Pen/Strep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (pH7.2) 1X</td>
			<td>Gibco (Life Technologies)</td>
			<td>c20012500BT</td>
			<td>PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRIM1640</td>
			<td>Gibco (Life Technologies)</td>
			<td>c11875500CP</td>
			<td>PRIM1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td>Life Technologies</td>
			<td>P36934</td>
			<td>Mounting Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Cell Lysis Buffer</td>
			<td>Sigma</td>
			<td>R7767-100ML</td>
			<td>Red blood cell lysing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight/Sharp-Blunt/10cm</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Fine Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin eFluor 450</td>
			<td>eBioscience</td>
			<td>48-4317-82</td>
			<td>for FACS analysis use at 0.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tamoxifen</td>
			<td>Sigma</td>
			<td>101551374</td>
			<td>TAM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-250ML</td>
			<td>TritonX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Disposable Scaplels</td>
			<td>Swann Morton</td>
			<td>#10</td>
			<td>Scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td>Yifeng Medical</td>
			<td align="right">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

9.1K 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

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...