The overall goal of this experiment is to assess the proliferation, differentiation, and vessel generation potential of vascular endothelial stem cells through a mammary fat pad transplantation technique followed by whole-mount tissue preparation for microscopic observation. This method provides a new way to optimally evaluate the stemness properties of vascular endothelial stem cells in vivo. The main advantage of this technique is that during puberty, the mammary gland provides an ideal angiogenic environment for the generation and integration of newly-formed vessels.
This enables the functional evaluation of transplanted endothelium cells. The implications of this technique aren't restricted to testing endothelial function. They can extend toward evaluating the effect of angiogenic factors on localized vessel remodeling.
These approaches are efficient tools that can be applied to multiple disciplines including studies of endothelium population in tumor environment and of alterations in cell potency. Demonstrating the procedure will be Wenqian Song, a grad student from our laboratory. After preparing tissue digestion based medium and sacrificing eight-week-old Actin-GFP mature virgin reporter mice according to the text protocol, use a scalpel to dissect out the fourth pair of inguinal mammary glands by first making a vertical skin incision at the lower abdominal midline.
Weigh the minced tissue and place it in a 50 milliliter tube. To constitute the digestion buffer, add 300 units of collagenase type three per 10 milliliters of digestion based medium. Mix evenly and add it to the minced tissue.
Horizontally, place the 50 milliliter centrifuge tube on a rocking platform and set the speed to 100 rpm and the temperature to 37 degrees Celsius. Agitate the mammary tissue for two hours. After digestion, top up the 50 milliliter centrifuge tube with pre-warmed sterile PBS and invert the tube to mix evenly.
Then centrifuge the tube at 200 times g for five minutes. Remove the supernatant and flick the bottom of the tube to loosen up the cell pellet. Then add three milliliters of red blood cell lysing buffer and incubate the tube at room temperature for five minutes to eliminate the red blood cells.
Add 10 milliliters of sterile PBS and invert the tube to evenly mix. Then after centrifuging the tissue and discarding the supernatant, add three milliliters of pre-warmed 0.05%trypsin and incubate the tube at 37 degrees Celsius for five minutes to further digest the remaining tissue. Next, add three milliliters of pre-warmed IMDM and then add 100 microliters of DNase I solution.
Incubate the tube at 37 degrees Celsius to break up the DNA filaments. Then pass the tissue mixture through a 70 micron cell strainer to obtain the cell solution. Use two milliliters of PBS plus 5%FBS to rinse the strainer twice to neutralize the enzymatic digestion.
Centrifuge the cell mixture at 200 times g for five minutes. Discard the supernatant and resuspend the cell pellet in one milliliter of PBS plus 5%FBS for antibody staining. After antibody staining, top up the cells with PBS plus 5%FBS and then centrifuge the sample at 200 times g for five minutes.
Then after discarding the supernatant, use one milliliter of PBS plus 5%FBS to resuspend the cells. Filter the suspension through a 40 micron cell strainer. Then carry out FACS-based sorting to isolate protein C receptor positive vascular endothelial stem cells according to the text protocol.
Top up the FASC-sorted cells with 10 milliliters of PBS plus 5%FBS and then centrifuge the samples at 200 times g for five minutes to settle the cells at the bottom of the tube. After carefully aspirating the liquid, use a hemocytometer to count the cell number and resuspend the cell pellet with basement membrane matrix mixture to 15, 000 cells per 15 microliters. Serially dilute the sorted and counted cells to reach the required input concentrations in a 1.5 milliliter microcentrifuge tube making sure that the final injection volumes do not exceed 15 microliters.
Then add 0.1%trypan blue to each tube for better content visualization during transplantation. Pipette thoroughly to mix and store the tubes on ice until use. To transplant primary endothelial cells or ECs into the mammary fat pad, lay each mouse in a supine position which its back on the operating table.
Then use adhesive tape to immobilize the limbs. Dip a cotton swab into iodine-based antiseptic solution. Thoroughly wipe the entire abdomen area of the mouse to sterilize the skin surface before surgery.
Next, use forceps to hold and lift the abdomen skin of the mouse. Then with a surgical scalpel, make a small one centimeter vertical incision. Make two cuts about 0.5 to one centimeters in length each toward the hind leg to achieve an upside down Y-shaped incision.
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. Then place the mouse under a stereoscope and set the magnification to 2X. Place a 27 gauge 1/2 inch needle on ice to pre-chill.
After priming the syringe, pipette 15 microliters of basement membrane matrix solution onto the cap of the microcentrifuge tube and then aspirate the entire volume into the primed syringe. To ensure the complete injection of the cell content, make sure no air is trapped in the needle after priming and the taking of cells. Using fine tweezers, hold the mammary fat pad in front of the inguinal lymph node and lift it slightly for easy injection.
Then gently insert about 1/4 of the syringe needle into the fat pad. Release the tweezers from the fat pad to grip and stabilize the syringe needle. Slowly inject the solution to prevent liquid leak.
Then hold the needle in place for a few seconds to make sure that the cell mixture is solidified to minimize leakage when pulling out the needle. Use 5-0 gut absorbable sutures to close and suture the skin flap and close the wounds with surgeon-style knots, each knot approximately 0.3 centimeters apart. To harvest the tissue, label the recipient systemic circulation with a tail vein injection of Isolectin 647 dye in PBS.
Allow a resting period of five to 10 minutes. After sacrificing the mice according to the text protocol, use surgical scissors to dissect out the area of mammary tissue where the cells were initially injected. In a six centimeter Petri dish, use a surgical blade to chop the tissue into roughly three to four cubic millimeter cubes.
Then to digest the tissue pieces, transfer them to a 15 milliliter tube filled with 10 milliliters of pre-warmed digestion medium supplemented with collagenase three. Place the 15 milliliter centrifuge tube horizontally on a rocking platform at 100 rpm and 37 degrees Celsius for approximately one hour to loosen the tissue structure and to remove excess adipocyte tissue. Next, use PBS to wash the tissue at room temperature for 10 minutes.
Then to preserve the fluorescence, fix the whole-mount tissue by carefully placing the pieces into a six centimeter Petri dish half filled with 4%PFA. Place the Petri dish on a rocking platform and gently agitate the tissue for 30 minutes at room temperature. After washing the tissue and incubating it in the diluted primary antibody according to the text protocol, remove the stain and use wash buffer to wash the tissue three times for 10 minutes each.
Then incubate the tissue with secondary antibody plus DAPI at room temperature for four hours on a rocking platform or overnight at four degrees Celsius. After washing the sample according to the text protocol, transfer the tissue to 80%glycerol or 80%sucrose overnight to dehydrate it for better preservation. Mount the tissue and carry out confocal microscopy according to the text protocol.
A representative result of cell isolation using FACS is shown here. Blood lineages were gated out and CD31 positive, CD105 positive were used to define endothelial cells followed by enrichment of protein C receptor positive endothelial stem cells. This confocal image represents a recipient mammary fat pad at the site of transplantation.
Blood vessels formed from transplanted protein C receptor positive endothelial cells have integrated and contributed toward host mammary vasculature. The recipient mammary vasculature is presented in this figure. Intravenous injection of Isolectin in recipient mice revealed that protein C receptor positive endothelial cells formed lumenized functional vessels.
Once mastered, the transplantation procedure can be done in under five minutes if it is performed properly. While attempting the procedure, it is important that you remember to closely monitor the recipient animals after surgery. Appropriate care procedures should be provided if signs of distress and infection are observed.
Following the procedure, other methods like cell lineage tracing can be performed in order to examine the behavior of targeted cells during various biological processes in vivo. After its development, this technique paved the way for the researchers in the field of vascular biology to explore the potency of endothelium subpopulations under both physiological and pathological conditions. After watching this video, you should have a good understanding of how to use mammary fat pad transplantation and whole-mount tissue preparation to investigate the vessel-forming ability of endothelium populations and also to evaluate the functionality of formed vessels.
