The overall goal of this procedure is to isolate primary hemogenic endothelial cells from murine embryonic tissues via florescence activated cell sorting. The main advantage of this technique is that it enables the reliable and specific isolation of viable hemogenic endothelial cells from embryonic hematopoietic tissues. These cells can then be used for subsequent analysis and culture.

The hemogenic endothelial cells as well as the hematopoietic stem and progenitor cells that can be isolated using this method will then be used to help answer key questions about the regulation of their development and function. Generally individuals new to this method may struggle with identifying the side population of cells that includes the hemogenic endothelial cells, and hematopoietic stem and progenitor cells. The embryonic tissue dissection and scanning procedures will be demonstrated by Kat Marcelo, a former graduate student from my lab.

As well as Emily Grits a current clinical research fellow. And Jen Fang a current post-doc. Begin by wiping down all of the work surfaces with 70%ethanol and placing an absorbent under pad on the lab bench surface.

Next placed a euthanized stem supine on the under pad and liberally spray the lower abdomen with 70%ethanol. Make a long horizontal incision perpendicular to the mid-line of the lower abdominal wall. Followed by two additional one inch horizontal incisions extending up and down from the midpoint of the horizontal incision.

Then dissect away the abdominal wall to fully expose the right and left uterine horns containing the multiple gestating embryos. Using forceps grab one of the two uterine horns and use scissors and forceps to separate the uterus from the surrounding myometrium. And transfer both horns into a sterile 60 millimeter polystyrene tissue culture plate on ice.

Place the plate under a standard light dissecting microscope, and use the forceps to separate each fetal placental unit. For each unit dissect away the muscular layer of each uterine sac, revealing the underlying gestational sac and decidua. To isolate the yolk sac, gently remove as much of the sac from the enclosed embryo as possible.

Removing the rest of the yoke sac tissue from the embryo proper at the embryonic origin of the vitelline vessels as necessary. To isolate the aorta-gonad-mesonephros or AGM, transect the embryo below the heart and forelimbs and discard the thorax and head region. Next transect the embryo just below the hind limbs, and remove and discard the tail tissue.

Then remove the hind limbs and excess ventral tissue from the remaining AGM containing section. As each yolk sac or AGM is harvested, pool the embryonic tissues in HPSS+in 1.5 milliliter tubes on ice. To generate a single cell suspension, centrifuge the embryonic tissue, and re-suspend the tissue pellet in one milliliter of Collagenase type two diluted in HPSS+Incubate the tube for 30 minutes in a 37 degree celsius water bath.

Mixing by inversion every five minutes. At the end of the incubation, pass the sample 10 times through a P1000 pipette to gently mechanically dissociate the tissue. Then spin down the sample again, re-suspending the pellet in one milliliter of ice cold HPSS+Now filter the sample though a 70 micron cell strainer, and count the cells.

After counting collect the cells again by centrifugation. And re-suspend the pellet in 37 degree celsius DMN+at a one times 10 to the sixth cells per milliliter concentration. To label the cells for fac sorting, aliquot at least 100 microliters of cells into 1.5 milliliter tubes and add verapamil diluted in 95%ethanol to the hoechst only plus verapamil control tube to a 50 micromolar final concentration.

Place all of the tubes at 37 degrees celsius for five minutes. Then add hoechst to the hoechst only and hoechst plus verapamil only control tubes, and the sample tube to a final concentration of five micrograms per milliliter and return the tubes to 37 degrees celsius for one hour protected from light, gently mixing the tubes by inversion every 15 minutes. At the end of the incubation centrifuge all of the samples and dilute the pellets to a one times 10 to the fifth cells per milliliter concentration in cold HPSS+Now add fluorescently conjugated antibodies to the appropriate antibody only control tubes, and to the sample tube to a final concentration of two micrograms per milliliter for a 30 minute incubation on ice protected from light.

At the end of the incubation, spin down the samples and re-suspend the pellets in 500 microliters of ice cold HPSS. Strain the samples through the mesh filter caps of five milliliter round bottom polystyrene facs tubes, and place the tubes on ice protected from light. Then immediately analyze the cells by flow cytometry.

Following standard live cell and doublet discrimination by forward and side scatter side population events are visualized in the linear hoechst red versus hoechst blue dot plot as a shoulder left shifted from non side population events. This side population is significantly reduced with verapamil treatment. The non side population cells are identified as the dense cluster of cells adjacent to the side population.

The non side population cell fraction contains non hemogenic endothelial cells which are further distinguished as CD31+CD45-events. To identify the hematopoietic stem and progenitor cells and hemogenic endothelial cells, daughter gates are drawn from the side population fraction revealing the CD45+and CD45-cells. The hemogenic endothelial cells are subsequently identified from CD45-cells in a differential cKit versus Flk1 daughter plot as double positive events.

The hematopoietic stem and progenitor cells are identified from the CD45+fraction in a separate daughter plot as Flk1-cKit+cells. Following this procedure methyl cellulose based cultures can be setup to provide a functional assessment of the hematopoietic capacity of the isolated cells or the cells can be immediately processed for gene and protein expression analyses. Once mastered this technique can be completed in about four to five hours if it is performed properly.

While attempting this procedure it is important to remember to maintain the samples protected from light and on ice unless otherwise indicated to maximize the cell viability. This technique has enable researchers in the field of Hemoto Vascular Biology to explore the hemogenic specification of endothelial cells and their production of hematopoietic stem and progenitor cells during embryonic development. After watching this video you should have a good understanding of how to isolate hemogenic endothelial cells from murine embryonic tissues using flow cytometry.

Thanks for watching and good luck with your experiments.