The overall goal of this procedure is to generate bonafide induced trophoblast stem cells or iTSCs from murine fibroblasts. This method can help answer key questions in the stem cell field regarding the nature of the barrier separating the embryonic from the extra embryonic lineage. The main advantage of this technique is that the transgenic expression is only activated transiently, facilitating the selection of stably converted cells.
In a bio safety level two laboratory using the appropriate personal protective equipment, cover five 100 milliliter dishes with five milliliters of Poly-L-lysine solution, gently rocking the plates to evenly distribute the coating solution. Place the dishes into a 37 degree Celsius incubator for 30 minutes, then aspirate the Poly-L-lysine and rinse the dishes one time with PBS. Next, plate seven times 10 to sixth 293 T cells per dish in 10 milliliters of 293 T cell medium, swirl the dishes to evenly distribute the cells and return the dishes to the incubator.
The next morning, replace the growth medium with five milliliters of transfection medium. Then treat the cultures with six microliters of a 1000X stock of Chloroquine solution and place the plates back into the incubator at 37 degrees Celsius. To prepare the transfection mixture, for each Lentiviral vector, add 600 microliters of Lentivector DNA solution drop wise into 600 microliters of 2X HEPES buffered saline while vortexing and incubate the transfection mixture at room temperature.
After 15 minutes, very carefully add the mixture drop wise to the prepared 293 T cells, and return the dishes to the incubator. After five to six hours, replace the medium with 10 milliliters of virus production medium, and place the plates back in the incubator. The following morning, carefully replace the medium with seven milliliters of fresh virus production medium for another overnight incubation.
Next, transfer the medium from each plate into individual 15 milliliter conical tubes and add seven milliliters of fresh virus production medium onto the plates for another overnight incubation. The next morning, harvest the second batch of virus containing medium, pooling the supernatant with the first batch of virus and strain the virus containing medium through a 0.45 micron surfactant free cellulose acetate membrane filter for storage in 500 microliter Aliquots at minus 80. To convert the fibroblasts to induced trophoblast stem cells, wash the transduced mass embryonic fibroblasts or MEFs two times with two milliliters of PBS.
Then detach the cells with 0.5 milliliters of Trypsin EDTA solution at 37 degrees Celsius. After three to four minutes, confirm that the cells have lifted from the plate under an inverted microscope and then activate the Trypsin with one milliliter of TS medium. Transfer the cell suspensions into individual 15 milliliter conical tubes and centrifuge the cells.
Then re-suspend the pellets in two milliliters of TS medium, and plate two times 10 to the fifth four factor mCherry or control untransduced MEFs per 100 milliliter dish in 10 milliliters of TS medium containing fibroblast growth factor four and Heparin supplemented with Doxycycline for overnight culture. The next day, under an epifluorescence microscope, use the mCherry protein expression to estimate the transduction efficiency. If the transduction efficiency is about 100%return the cultures to the incubator, changing the medium in all of the dishes every other day until day 10.
Thereafter, feed the cells with fresh TS medium containing fibroblast growth factor four and Heparin without Doxycycline using the inverted microscope to monitor the appearance of transdifferentiated areas on the four factors dish for up three weeks. Identification of the transdifferentiated areas is critical, since at this point, MEFs do not morphologically resemble trophoblast stem cells. Look for densely piled up colonies adjacent to the trophoblast giant cells which are identified by their cell size and large nucleus.
To isolate individual iTSC lines, first wipe down the inverted microscope with 70%ethanol to minimize the chance of bacterial contamination. Next, add 40 microliters of 05%Trypsin EDTA into 12 wells of a 96 U bottom well plate, and place the plate on ice. Now aspirate the medium from the four factor's MEF transdifferentiation dish and wash the cells one time with 10 milliliters of PBS.
After the wash, add 10 milliliters of fresh PBS to the cells and place the plate on the inverted microscope stage. Using a 100 microliter pipette set to 40 microliters, circle each individual colony with the pipette tip and aspirate the resulting floating colony. Transfer each colony into individual wells of the 96 well plate until 12 colonies have been picked.
Then incubate the colonies at 37 degrees Celsius for five minutes. At the end of the incubation, dissociate the cells with pipetting and transfer the cell suspensions into individual wells of a 24 well plate containing 500 microliters of complete TS plus fibroblast growth factor and Heparin medium for overnight culture. The next day, replace the medium with fresh complete TS plus fibroblast growth factor and Heparin medium to remove the dead cells and return the plate to the incubator.
Monitoring the cultures daily for the appearance of Epithelial colonies with bright boundaries. When the colonies are observed, culture the cells for up to one week or until they are 70%confluent. In cultures where the four factor's transgene expression is activated, the cells rapidly change morphology.
Around days 14 to 21, distinct transdifferentiated areas emerge. These primary colonies lack the typical trophoblast stem cell morphology, however once they are subcultured, a characteristic epithelial morphology with tight edges and bright boundaries, highly reminiscent of bonafide trophoblast stem cells is observed. When the four factor's induced trophoblast stem cells stay positive for the trophoblast transcription factor, Cdx2, and Tfap2c, the idea of these lines can be used for subsequent In vitro or InVivo analyses.
Once mastered, this technique can be completed in six weeks if it is performed properly. Following this procedure, other methods like blast assisted injection can be performed to answer additional questions about the chimerization potential of the generated iTSCs. After its development, this technique paved the way for researchers in the field or early lineage segregation to explore the embryonic versus extra embryonic cell-fate conversion in mice.
After watching this video you should have a good understanding of how to directly convert new embryonic fibroblasts into induced trophoblast stem cells. Don't forget that working with Lentiviral vectors can be extremely hazardous and that precautions such as using bio safety level two appropriate working procedures should always be followed.
