The overall goal of this procedure is to derive TS cells from mouse blasts. This is accomplished by first isolating the uterine horns and recovering blast assist from pregnant mice. The second step of the procedure is to culture blast, assist on meth feeders and conditions favoring the growth of TS cells.
The third step of the procedure is to expand TS colonies and eventually culture TS cells in the absence of meth feeders. The final step of the procedure is to culture TS cells with maintenance of stem cell properties to perform cryo-preservation and to induce trophoblast differentiation. Ultimately, results can be obtained that show the expression of a trophoblast stem cell marker IE CDX two in undifferentiated TS cells, or a differentiated marker IEP four 50 SCC after their maturation into trophoblast giant cells through immunofluorescent microscopy.
Hi, I am Shani Chu from the lab of in the Department of Biomedical Genetics at the University of Rochester Medical Center. Today we'll show you a procedure of trophoblast stem cell ation. We use this procedure to study the growth and the differentiation of trophoblast and its role in extra embryonic tissue for the making of a healthy placenta.
So let's get started. To begin this procedure, set up a breeding cross between your mice of interest. Next, prepare mouse embryonic fibroblasts ORMs as feeder cells.
Two days before the collection of blast assist plate mes in 100 millimeter dishes on the following day. Treat these meth cells with 10 milliliters of TS medium containing 20 micrograms per milliliter of mitomycin C for two hours at 37 degrees Celsius, 5%carbon dioxide after two hours, wash cells twice with PBS resuspend, the mitomycin sea treated meth cells in 10 milliliters of TS medium and see the cells in a 12 well plate. On the day of blast assist collection, replace the meth culture medium with TS medium plus one X FGF four heparin.
We are now ready to collect blast assists from the mouse uterus. First, lay the euthanized animal on its back and clean the skin and fur with 70%ethanol. Next, make a small lateral incision and pull the skin toward the head and to expose the abdomen, cut through the peritoneum laterally.
To expose the abdominal cavity, locate the uterus. Remove the uterus by grasping it with forceps at the cervix located behind the bladder and cutting across the junction of the uterus and cervix. Pull the uterus and trim the membrane and fat tissues away.
Then cut the uterus below the junction with the oviduct. Place the utero in 0.2 milliliters of TS medium in a 60 millimeter plate. Now fill a one milliliter syringe with one milliliter of TS medium.
Attach a 26 gauge needle and insert the needle into the base of one uterus. Flush slowly into a new 60 millimeter plate. The uterus should be inflated and extended fully during flushing.
In the same way, flush the other uterine horn using a mouth controlled pipette. Collect and transfer the blast. Assist carefully into a clean 12 well plate containing TS medium wash blast assist three times by serial transfer through TS medium in 12 well plates.
Place one blast assist per well. In a 12 well plate containing the mitomycin C treated meth feeders culture at 37 degrees Celsius 5%carbon dioxide. The blast assists will hatch from the zona lucita and attach to the wells in 24 to 36 hours.
A small outgrowth will be readily observed on day three. Feed the cultures with 500 microliters of fresh TS medium plus one X FGF four heparin on day four or five. The blast assist outgrowth should be ready for disaggregation depending on its size.
The ideal size for TSL disaggregation is illustrated here. Wash the cultures once with 0.5 milliliters of PBS. Then add 100 microliters of 0.25%trips in EDTA and incubate for five minutes at 37 degrees Celsius, 5%carbon dioxide.
Now we are ready to break the blasts, which is probably the most critical part of this procedure. Next break the blast assist outgrowth into small clumps by pipetting up and down vigorously with a P 100 pipet men stop the trypsin ization by adding 0.5 milliliters of 70 cm 1.5 F four H medium into the well return cells immediately to the incubator. Eight hours after disaggregation change the medium.
Continue to feed the cells every two days between days six and 10. And this is highly variable. TSL colonies can be observed.
They have flat epithelial sheetlike morphology with a clear colony boundary. Continue to feed the cultures every two days When the TS cells reach 50%co fluency, usually between days 15 and 20. Wash them once with PBS and add 100 microliters of 0.25%tripsin EDTA, pipe it up and down during trypsin ization to ensure a near single cell suspension.
After five minutes, stop the trypsin by adding seven DCM 1.5 F four H medium. Then transfer the cells to new six well plates containing the previously prepared mitomycin C treated meth feeders and incubate at 37 degrees Celsius 5%Carbon dioxide change the medium eight hours after passage and continue to feed the cultures every two days. After one or two more passages on the meth feeders, the TSL may be cultured without them to separate TSL from the meth feeders.
After passing the cultured cells incubate them at 37 degrees Celsius, 5%carbon dioxide for 30 minutes. Because mes attached to the culture plate much faster than TS cells after trypsin, most mes will attach to the plate while the TS population remains in suspension. Transfer the supernatant to a new plate.
Repeat this process of separating the TS cells from the meth feeders for several passages to obtain a pure TS population. Maintain TS cells in 70 cm, 1.5 F four H medium and pass them every four days or when the culture reaches 70%co fluency. Finally, the identity of TS cells can be confirmed by immuno staining of a cell marker.
CDX two to freeze TS cells, collect them by ization, followed by centrifugation at 450 GS for six minutes. Resus suspend the cells in TS medium with an equal volume of two x freezing medium and transfer to cryo vials. A 60 millimeter plate that is 70%confluent is usually kept in two vials.
Freeze the cells slowly using an isopropyl alcohol chamber at minus 70 degrees Celsius. Transfer to liquid nitrogen after 24 to 48 hours. Thawing TSL should be done quickly by thawing the vial in 37 degrees Celsius.
Transfer the haw cells to a 15 milliliter tube containing 10 milliliters of TS media and collect the cells by centrifugation resuspend the cells in 70 cm, 1.5 F four H medium and seed each vial of cells in a 60 millimeter plate. Differentiation of TS cells into trophoblast giant cells or tgc can be induced by the withdrawal of MEC four and heparin. The T GCs are easy to identify in culture with enlarged nuclei and cell bodies.
The TGC phenotype can also be detected by immunos staining of A TGC marker P four 50 SCC shown in green counterstain with dappy shown in red as observed here. Differentiated cells express P four 50 SCC while undifferentiated cells do not flow. Cytometric analysis of the differentiated cells stained with propidium iodide further shows a higher DNA content with M1 representing two to four copies and M two representing more than four copies of DNA.
These results suggest that TSLs undergo endo reduplicating to become polyploid TCS represented by the M two population. We just show you how to collect blasts, ptosis, and derive mouse TS cells from blasts, ptosis. It's important to remember to keep an eye on the outgrowth when establishing the TSL lines, make sure their pluripotency is maintained in culture condition.
So that's it. Thank you for watching and good luck with your experiments.
