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

Summary

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.

Abstract

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.

Introduction

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/progen....

Protocol

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

1. Isolation of VESCs from the Mouse Mammary Gland

1. Mammary gland harvest and tissue digestion.
   1. Use 8 week-old Actin-GFP mature virgin reporter mice, with weights range between 20 and 25 g, as tissue donors.
      Note: Cells originated from the donor can be easily tracked by GFP expression.
   2. Prepare tissue digestion base medium consisting of 5% fetal bovine serum (FBS),....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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