The overall goal of this experiment is to use an aorta-gonad-mesonephros explant culture to investigate the critical regulators of hematopoietic stem cell development. This method can help answer key questions in HSC biology and function fields about how to identify the factors involved in the regulation of HSC development in mammals. The main advantage of this technique is that these regulators can be verified through DNA expression analysis and the colony formation ability and the reconstitution capacity of the HSC.
Generally individuals new to this method will struggle because the precise separation of AGM from the surrounding tissues in the trunk region requires practice to master. Before beginning the procedure place stainless steel meshes into individual wells of a six-well plate containing two milliliters of freshly prepared fluoxetine-supplemented medium per well. Place one UV-sterilized 0.65 microliter filter per experimental well into a glass cell culture dish containing fresh boiling water for two three-minute washes, immersing the filters in a container of room temperature PBS for two minutes after each wash.
Then place one filter onto each steel mesh at the air-liquid interface of each well. Next use the dissecting scissors to strip the uterus from an embryonic day-11 pregnant mouse and harvest the embryos from the uterus. Using dissecting scissors remove the yolk sac, umbilical cord and viscera from the embryo body.
And carefully remove the dorsal nerve tissues and surrounding muscular tissues. Using forceps remove the tissues from the anterior and hind limbs. And separate the AGM region from the embryo body.
Then place each AGM onto a single filter at the air-liquid interface of each well. Place the cultures in a 37 degree Celsius and 5%CO2 incubator. After two to three days, transfer the AGM samples into individual 1.5-milliliter microcentrifuge tubes containing 200 microliters of collagenase for their dissociation at 37 degrees Celsius for about 20 minutes with shaking every five minutes.
At the end of the digestion stop the reactions with 200 microlilters of serum per explant. Transfer the entire contents of each tube into individual wells of a 24-well ultra-low attachment plate containing 500 to 600 microliters of M3434 medium per well. Mix the cells in each well with a one milliliter syringe without a needle.
Place the plate in the cell culture incubator. The number of colony-forming units within each well can then be scored after seven to 10 days of culture. For in vivo AGM explant transplantation after two to three days of AGM culture as just demonstrated, lethally eradiate eight to 10-week-old male C57 black six mice with nine grays of radiation.
Four to five hours later dissociate the harvested explants in 200 microliters of collagenase per samples as demonstrated. Load of 0.5 to one embryo equivalent of the resulting single-cell suspensions into one one-milliliter syringe per recipient animal. Place the first irradiated mouse into a restrainer and wipe the tail with a 75%ethanol-soaked cotton swab to promote tail vein vasodilation.
Intravenously inject the entire contents of one syringe into a dilated tail vein. Use an antiseptic swab to stop the bleeding after removal of the needle. Then return the animal to its cage, and inject the next animal.
11 days after the adoptive transfer the spleens can be fixed in Bouin's solution for macroscopic enumeration of the visible spleen colonies. Fluoxetine-supplemented AGM cultures demonstrate an increased expression of Runx1, a pivotal gene for HSC development at the mRNA and protein expression levels. Fluoxetine can also significantly increase the colony formation ability of HSC in the AGM including burst-forming unit erythroids, colony-forming unit granular monocytes and colony-forming unit granulocytes, erythrocytes, macrophages and megakaryocytes.
Further in vivo transplantation of fluoxetine-treated HSC isolated from AGM explants results in increased numbers of colonies in the spleens of irradiated adult recipient animals. After watching this video, you should have a good understanding of how to isolate the AGM and to perform an AGM explant culture for the identification of the critical regulators of HSC development within the AGM.
