In the mouse embryo. Early hematopoiesis occurs simultaneously in multiple organs, which includes a yolk sac, an aorta goad, meris region. More importantly, early hematopoietic stem cells can be isolated from these organs through micro dissection of the embryo, followed by flow cytometric sorting to obtain a more pure population.
In this video, we demonstrate the microdissection procedure we commonly use and also show the results of a typical fax plot. Hi, I am Kelly Morgan from the laboratory of Dr.Gary Gilland at the Howard Hughes Medical Institute and the Department of Medicine at Brigham and Women's Hospital and formally trained by Elaine Dza at Rasmus University Medical Center in Rotterdam. Today I'll show you procedure for embryonic dissection to obtain early hematopoietic stem cells from the yolk sac and the aorta goad meph region or a GM.And I'm Michael Care.
Also from the Go Lab. I'll be showing you how to make single cell suspensions of a GM and yolk cells for HSCs using facts. This procedure involves dissection of the early hematopoietic organs from E 10 and a half mirroring embryos, dissociation of the tissues and facts of single cell suspensions to isolate hematopoietic stem cells.
So let's get started. Before starting, it is important to have all the reagents and tools necessary for the procedure. Scissors, forceps, size four, surgical grade bent tipped forceps size five 40 fifths, 30 gauge needles with syringe, 35 millimeter and 100 millimeter Petri dishes PBS with 10%and 20%fetal bovine serum and collagenase.
To begin the dissection, first we need to harvest the uterine hordes from a pregnant female. At 10 and a half days post concept, the female's abdomen is wet with ethanol and then the skin is opened with scissors holding the skin with forceps. Additional cuts are made to open the peritoneum.
Now the abdomen is opened and the uterine horns can be visualized. Using forceps to hold the uterine horns cut each distal end and the center to excise both horns. Uterine horns are then placed in a Petri dish containing cold PBS and 10%FBS.
To remove uterine tissue using size four forceps and working under the microscope, gently remove the endometrial tissue from around each embryo. Conceptus being careful not to puncture the placenta. Once removed, the embryos are placed in a fresh dish with cold PBS and 10%FDS carefully remove the placental tissue to reveal the embryo encased in the dilk sack.
This is done by using the forceps to cut the embryo, encased the yolk sac away from the placenta. At their juncture, the yolk sac containing vilin arteries and a portion of umbilical cord can be gently teased away from the embryo and set aside on ice. For dissociation, the embryo is moved to a small Petri dish with minimal levels of PBS with 10%FBS just enough to wet the embryo, but not too much.
Optimal levels would keep the embryo stationary while the plate was slowly agitated. This will keep the embryo securely in place during dissection. Switch to the 30 gauge needles to remove the upper and lower portions of the embryo by cutting above the for LIMS and below hind limbs.
Becoming familiar with using the needles as cutting instruments may take some practice. Repeat small cuts by crossing the needles to dissect the tissue. Use one needle as a guide as the other performs the cuts.
Next, remove the dorsal portion of the embryo. This is a tissue that contains the somites. This is accomplished by gently cutting away the dorsal MOS tissue while remaining a sufficient distance from the aorta to prevent any nicks.
Cutting the aorta would result in loss of blood in the inability to easily visualize its location. Again, making small cuts with the needles as needed to maintain a precise cutting location. Push the extra tissue out of you and turn the embryo around to cut the ventral tissue.
Again, cutting as close to the aorta as possible without nicking it. Cuts made too far from the optimal location would result in abdominal tissues being present, which can be removed once the A GM is isolated. Now position the embryo with dors side down, or its back against the plate.
Gently splay the embryo open by pushing the sides down and visualizing the location of the gonadal ridges. These are located slightly off center as tube-like structures on either side of the aorta. Complete the dissection by removing the excess side tissues, using the same cutting techniques as before.
Once the side tissue with the embryo is removed, then the embryo is turned upside down to remove the remaining side tissue. At this point, the A GM is inspected for any excess tissue that can be easily removed with the needles without injury to the aorta or goad. Removing as much excess as possible results in better isolation of the stem cell population.
Since we rely on only a few cell surface markers for purification. Using the bent tip forceps, gently scoop up each A GM and placing cold PBS with 10%FBS until ready for dissociation. Now to dissociate the tissues, first place yolk sack and AG GMs in separate tubes, spin briefly to pellet tissues and discard PBS with 10%FBS resuspend tissues and 0.25%collagenase in PBS with 20%FBS with enough volume to adequately cover tissues.
Incubate the tissues at 37 degrees, 25 to 30 minutes for AGMs and 35 to 40 minutes for yolk sacks. At the end of the incubation, gently pipette tissues up and down to dissociate into single cell suspensions. Wash the tissues with excess PBS with 10%FPS pellet, the cells and resus suspend in fresh sterile PBS with 10%FBS cells are now ready for staining.
And for this, I'll hand them off to Mike. Thanks Kelly. For fax analysis, cells are stain according to manufacturer's recommendations.
We use a fax aria from BD bios to perform flow cytometry. Due to the size and fragility of embryonic cells, the sorting aperture is adjusted for a large diameter. The nozzle is adjusted to 100 microns and the sort pressure is 30 PSI.
For stem cell analysis, triple positive self expressing the markers C kit C 34 and CD 41 are isolated and sorted from the yolk sac and double positives. C kit CD 34 are isolated from the A-G-M-V-F-X. A typical plot would look like this.
C kit positive cells are analyzed for CD 34 and CD 41 simultaneously or individually. Usually cells display gradation of positivity for each marker. With the stem cell population found as a subset of the positive populations, these plots show us that we've achieved a good cell yield with 100 to 300 cells per yap sac.
And per a GM, we have just shown you how to dissect and isolate early hematopoietic stem cells from a mass embryo. When doing this procedure, it's important to remember that the time required to complete the protocol adversely affects cell viability. So in essence, the faster you can complete the dissections, the better your cell yield will be.
So that's it. Thanks for watching and good luck with your experiments.
