Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The overall goal of this procedure is to study the maintenance, migration and fate of heart and muscle progenitors in the pharyngeal mesoderm. This is accomplished by first dissecting the pharyngeal arches from a freshly harvested mouse embryo. Next, the arches are placed in a film of media to facilitate their attachment.

The cells within or migrating from the pharyngeal explan are then monitored daily. Ultimately, Cree based fluorescence lineage tracing can be used to track the migration differentiation and renewal of the heart and muscle progenitors within the pharyngeal arches. The main advantage of this technique is that it is the only ex vivo system for studying a renewing population of cardiac or muscle progenitors and their microenvironment in vitro.

Demonstration of this method is critical as the parental arches in the 9.5 day old mouse embryos are only two to 300 micrometers in size, making them very difficult to identify and dissect without prior visualization. Before beginning the procedure. Coda 12, well plate with PBS supplemented with 10%FBS.

After one hour, replace the coating solution with 200 microliters of serum free medium per well. Swirling the plate to make sure a film of medium covers the entire surface of the well bottoms. Next spray and clean the belly of a euthanized pregnant mouse with 70%ethanol.

Then using sterile tools, make a 0.5 centimeter incision at the naval region. Grab the skin above and below the incision and gently pull in opposite directions. Then make a V-shaped incision in the membrane from the navel to the ovaries on each side of the abdomen to reveal the uterus.

Pinch the oviduct connecting the uterus to the ovary with forceps and the oviduct on the ovary side with scissors to free the uterus from the ovary. Gently pulling up the uterus by the uct. Cut the uterus free from the bladder region.

Then pull the uterus further up by the uct and cut the UCT on the other side, releasing the uterus from the abdominal cavity. Transfer the uterus to a 50 milliliter tube containing 20 milliliters of cold, sterile PBS with calcium and magnesium. Gently shaking the tube for 10 seconds to wash out the blood.

Then use forceps to transfer the uterus into a 10 milliliter dish and add five milliliters of cold PBS to the tissue. Next, under a stereo microscope, use two pairs of forceps to gently open the uterus and dissect away the amniotic sacks containing the embryos one at a time. Then for each embryo in turn, pinch and gently remove the amniotic sack with one pair of forceps using the other pair to cut and release the sack from the embryo.

Place the embryo on its side and use forceps to cut the first and second arch posteriorly to the heart between the arch and the pharyngeal pouch. After flipping the embryo and cutting the arch on the other side is just demonstrated. Cut the cardiac outflow tract connecting the arches to the embryo and remove them.

Pinch and release each of the two pharyngeal arches from the remaining cardiac outflow tract and four gut endoderm. In turn, then transfer the tissues into the center of individual wells of the coated 12 well plate taking care that the arches are not covered by medium. To facilitate the attachment, incubate the arches under cell culture conditions.

After two hours, add 200 microliters of 37 degree Celsius, serum free medium down the sides of the wells, taking care that the arches remain attached and incubate the tissues overnight. The next day visually confirmed that the arches are still attached. Then gently add 200 more microliters of 37 degree Celsius serum free medium to the wells.

If the tissues become unattached and begin to float, gently remove the medium with a pipette without removing the arches until only a film of media is left. Then use the pipette tip to move the arches back into the middle of the well and incubate the tissues again. Every second day, replace any evaporated medium with 100 to 200 microliters of fresh, medium during development.

Facial muscle and heart progenitors can be traced as they proliferate and migrate from the first and second pharyngeal arch to become the head and heart musculature respectively. Culturing the pharyngeal arches offers a unique way to study heart and muscle development in detail ex vivo. Using the CRE lock system, mouse mesoderm progeny can be traced by fluorescent reporters upon Cree expression within 24 to 48 hours of ex vivo culture at 36 to 72 hours.

Cardiomyocyte formation can be visually confirmed by spontaneously contracting clusters of migrating cells within the second arch, as well as by the analysis of specific cardiomyocyte markers. After three to seven days of culture, myo tube and facial muscle formation can be visualized as elongated and spontaneously twitching cells within the first arch, and by the analysis of specific muscle markers After its development. This technique paved the way for researchers in the field of heart and muscle stem cell biology to directly monitor and study expanding progenitors and their niches in vitro.

After watching this video, you should have a good understanding of how to dissect foral arches from developing mouse embryos and to cultural in vitro, which allows the study of heart and muscle pro generative development, ex vivo.