Stem cells are pluripotent and specialized cells characterized by their long-term self-renewal and their ability to give rise to differentiated cells from the three germ layers under certain conditions. Due to these properties, stem cells have a great potential in regenerative medicine, disease modeling, and animal engineering. For years, embryonic stem cell lines destined to research purposes have been mainly derived from mouse embryos, but in general the derivation efficiencies achieved have been low.
In this video, we show a simple protocol to derive mouse embryonic stem cell lines using standard conditions that provides reasonably high derivation efficiencies comparable to the efficiencies obtained when using modulators of signaling pathways, such as 2i. In this protocol, we'll human foreskin fibroblasts as feeder cells for ESC derivation and culture. Before starting the ESC derivation process, it's necessary to inactivate the feeder cells.
First, take a cryogenic vial of human foreskin fibroblasts stored in liquid nitrogen, and thaw it by immersing the vial in a water bath at 37 degrees C.Once thawed, dilute the content in DMEM supplemented with fetal bovine serum. Seed the dilution in a Falcon flask, and culture it until reaching the confluence stage. Next, to inactivate the fibroblasts, dilute mitomycin C in DMEM supplemented with fetal bovine serum and incubate the cells for three hours in this inactivation solution.
Then, remove the inactivation solution and rinse the cells three times with Hanks'Balanced Salt Solution to eliminate mitomycin C remnants. To detach the inactivated feeder cells, add one milliliter of trypsin-EDTA solution and incubate the cells for five minutes. After that, observe the cells under an inverted microscope to ensure they are detached from the culture flask.
Then, pipette the cells with a Pasteur pipette to dissociate the cell clumps and neutralize the enzymatic solution by adding four milliliters of DMEM supplemented with fetal bovine serum. Take a small aliquot, dilute it in saline solution, and determine the number of cells using a Neubauer chamber. In parallel, centrifuge the rest of the sample.
Discard the supernatant, and dilute the pellet in freezing solution at a final concentration of one million cells per milliliter. Aliquot the suspension in cryogenic vials. And finally, place the cryogenic vials in a freezing container and put them at minus 80 degrees C.The next day, store them in liquid nitrogen.
One to four days before starting the derivation process, take a cryotube of inactivated feeder cells stored in liquid nitrogen and thaw it in a water bath at 37 degrees C.Then, dilute the cells in pre-warmed DMEM supplemented with fetal bovine serum and pipette gently to ensure a homogeneous mixture. Add 500 microliters of pre-warmed gelatin solution to each well of a four-well plate, and keep it at 37 degrees C for 10 minutes. Right after, remove the gelatin from each well.
Add 500 microliters of the feeder cells dilution to each well, and gently rock the plate to ensure a uniform distribution of the feeder cells on the bottom of the wells. Avoid making circular movements, as this would concentrate the cells in the center of the well. Then, place the plate in the incubator at 37 degrees and 5%carbon dioxide for at least 24 hours.
The next day, observe the cells under an inverted microscope and check they're attached and thoroughly distributed. Finally, change the medium of the feeder cells with pre-warmed DMEM supplemented with fetal bovine serum to remove DMSO remnants. Before embryo collection, induce mouse female superovulation by intraperitoneal injection of five international units of pregnant mare serum gonadotropin and human chorionic gonadotropin 48 hours apart.
Mate the females with males, and separate them 24 hours later. To collect two-cell stage embryos, euthanize the females 48 hours post coitum, dissect the female mice to obtain the oviducts and a short portion of the uterine horns, and place them in a dish with HEPES-buffered CZB medium. Then, one by one, flush the oviducts with HEPES-buffered CZB medium under the stereo microscope to collect the embryos.
Transfer the two-cell embryos to a culture dish prepared the previous day with several drops of embryo culture medium covered with mineral oil. Keep the embryos in the incubator until the blastocyst stage. Monitor the embryo development every 24 hours.
Embryos are expected to reach the blastocyst stage 48 hours after the start of the in vitro culture. If possible, select only expanded blastocysts to start the derivation process. On the day of embryo seeding, prepare the medium for stem cell derivation.
Add leukemia inhibitory factor, or LIF, to DMEM supplemented with 2-beta-mercaptoethanol non-essential amino acids and KnockOut serum replacement. If desired, add an MAPK inhibitor and a GSK-3 inhibitor to obtain a 2i medium. Take the four-well plate with the monolayer of feeder cells from the incubator.
Remove the medium, and add 800 microliters of the freshly prepared derivation medium. Before seeding the blastocysts on the feeder cells, the zona pellucida must be removed. To do so, transfer one embryo from the culture plate to a drop of acidic Tyrode's solution and keep it there until the zona pellucida is digested.
Make sure that the zona pellucida has been completely removed by adjusting the focus and the contrast of the stereo microscope. Then, rinse the zona-free embryo in several drops of derivation medium and seed it on a monolayer of feeder cells. Repeat the process with the rest of the embryos.
Culture the plate at 37 degrees C and 5%carbon dioxide. Monitor the cultures every 24 hours under an inverted microscope to assess their growth and to identify potential early signs of differentiation, such as swollen refractive cells surrounding the outgrowth or even pushing the feeder cells aside. Change the medium of the culture every other day.
To do so, take the four-well plate from the incubator, remove the medium from each well, and add 800 microliters of pre-warmed derivation medium supplemented with LIF and 2i if used. Return the plates to the incubator, and maintain the culture until outgrowths are observed. Six to eight days after blastocyst seeding, an outgrowth should be observed, and it must be subcultured.
For this procedure, cull-like handles and stem cells manipulation pipettes are required. To make them, take a long-tip Pasteur pipette and heat its thinner part using a Bunsen burner. Once soft, withdraw the pipette from the flame and pull the two ends apart until they separate.
Use the thicker part as the stem cell manipulation pipette. Heat the tip of the thinner part, and shape it until obtaining a hook to prepare cull-like handle. To subculture the outgrowth, locate it under the stereo microscope.
With the help of the handle, push away the differentiated cells that surround the outgrowth. To avoid an eventual differentiation, it's important to be strict when removing the differentiated cells from the edge of the outgrowth. Using a stem cell manipulation pipette, aspirate the outgrowth and place it in a drop of pre-warmed trypsin-EDTA solution.
With the help of a scalpel, cut the outgrowth in several pieces to disaggregate it mechanically and enzymatically. Recover the outgrowth fragments, and place them in a drop of pre-warmed derivation medium in order to neutralize the trypsin action. Finally, seed the outgrowth fragments into the four-well plate with a monolayer of feeder cells and culture them in a derivation medium at 37 degrees C and 5%carbon dioxide.
Stem cell-like cultures must be maintained for at least six weeks before an ESC line can be considered as established. During this period, it's necessary to change the medium every other day and subculture them once a week. As passage numbers increase, the morphology of the colonies progressively changes from a dome shape to a more elongated and flattened morphology.
After six passages, mouse ESC lines constitutively express pluripotency markers, such as Oct 4 and Sox 2. Moreover, after inducing ESC in vitro differentiation by culturing them in DMEM supplemented with 10%fetal bovine serum and without feeder cells, differentiation markers representative of the three germ layers can be detected by immunofluorescence. Here, we show the detection of the anti-tubulin beta-3 protein as a marker for ectoderm, alpha-smooth muscle actin for mesoderm, and alpha-fetoprotein for endoderm.
Following this simple procedure, embryonic stem cells can be derived from mouse blastocysts with efficiencies ranging from 60%to 78%While attempting this procedure, it's important to control the cells daily or every other day because the timings provided here are just illustrative. If embryonic stem cell colonies overgrow, they become darker because of cell accumulation and tend to differentiate more easily. After watching this video, you should have a good understanding of mouse embryonic stem cell derivation and culture using standard conditions.
