Name: a novel mammary fat pad transplantation technique to visualize the vessel generation of vascular endothelial stem cells
Uploaded: 2017-08-03T13:00:00
Description: 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

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
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		<li>Use 8 week-old Actin-GFP mature virgin reporter mice, with weights range between 20 and 25 g, as tissue donors. 
		Note: Cells origina...

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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.

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		<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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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 ...

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 ...

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 ...

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

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 ...

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 ...

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 ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

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

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 ...

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. ...