Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

All mouse experiments were conducted according to the German law of animal protection and in agreement with the approval of the local institutional animal care committees (Landesamt fuer Natur, Umwelt und Verbraucherschutz, North Rhine-Westphalia [approval ID number: AZ 84-02.04.2013.A428]).

1. Media Preparation

1. Prepare 293T medium: Dulbecco's Modified Eagle Medium (DMEM), 10% (v/v) FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin/streptomycin (1x).
2. Prepare MEF medium: DMEM, 10% (v/v) FBS, L-glutamine (2 mM), 1x non-essential amino acids, penicillin/streptomycin (1x).
3. Prepare TS medium (w/o FGF4/heparin): Roswell Park Memorial Institute medium (RPMI) 1640, 20% (v/v) FBS (stem cell culture grade), penicillin/streptomycin (1x), L-glutamine (2 mM), sodium pyruvate (1 mM), 2-mercaptoethanol (0.1 mM).
4. Prepare TS medium with FGF4/heparin (TS + F4H): RPMI 1640, 20% (v/v) FBS (stem cell culture grade), penicillin/streptomycin (1x), L-glutamine (2 mM), sodium pyruvate (1 mM), 2-mercaptoethanol (0.1 mM), 25 ng/ml FGF4, 1 µg/ml heparin.
   Note: FGF4 and heparin are added directly into the media immediately prior to use.
5. Prepare TS-CM with FGF4/heparin (TS-CM + F4H): 70% MEF-conditioned TS medium + 30% freshly prepared TS medium + 25 ng/ml FGF4, 1 µg/ml heparin.
   Note: FGF4 and heparin are added directly into the media immediately prior to use.
6. Prepare Transfection medium: Advanced DMEM, 2% (v/v) FBS (stem cell culture grade), L-glutamine (2 mM), penicillin/streptomycin (1x).
7. Prepare Virus-production medium: Advanced DMEM, 5% (v/v) FBS (stem cell culture grade), L-glutamine (2 mM), penicillin/streptomycin (1x).

2. Murine Embryonic Fibroblast Derivation

1. Set timed matings using wt 129S2SV females and homozygous male R26::rtTA mice (strain name; B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae/J 7}). On E13.5 sacrifice mice by cervical dislocation and isolate primary murine embryonic fibroblasts (MEFs) according to standard protocols⁸. Thereafter, plate cells from one embryo on a 150 mm dish in MEF media.
2. Once primary MEF cultures reach confluency on 150 mm dishes, wash cells twice with 10 ml PBS and add 4 ml 0.05% trypsin/EDTA solution. Incubate dishes for 3 – 4 min in the incubator at 37 °C.
3. After incubation check if cells detached from the dish using an inverted microscope (using a 10X lens). Add 5 ml of MEF media to stop trypsinization and collect cell suspension into a 50 ml conical tube.
4. Pool cell suspension from several dishes and collect into a 50 ml conical tube. Centrifuge cell suspension for 3 min at 200 x g and 10 °C.
5. Discard supernatant and resuspend cell pellet in FBS containing 10% (v/v) dimethyl sulfoxide. Freeze MEFs in aliquots for further use.
   Note: Alternatively, use wild type primary MEFs; however, an additional lentiviral vector expressing rtTA needs to be transduced together with the other lentiviral vectors (for example using the FUdeltaGW-rtTA plasmid described by Maherali et al. ⁹).
6. Preparation of MEF-conditioned TS medium
   Note: Prior to conversion experiments, MEF-conditioned (CM) TS media has to be prepared, which is required for subculture of individual iTSC lines (described in section 6).
   1. Plate 1 x 10⁷ mitotically inactivated MEFs per 150 mm dish in MEF medium on desired number of dishes. Inactivate MEFs by γ-irradiation with a dose of 9 Gy.
      Note: The yield per 150 mm dish is approximately 60 ml of CM.
   2. The day after plating, aspirate and discard MEF medium and add 20 ml freshly prepared TS medium (without FGF4/heparin) onto each dish of mitotically inactivated MEFs and place dishes back into the incubator at 37 °C.
   3. After incubating TS medium for three days on mitotically inactivated MEFs, collect medium in 50 ml conical tubes. Filter medium through a 0.2 µm filter and proceed with 2.6.4. Replace CM with 20 ml of fresh TS medium for preparation of a further batch of CM.
      Note: Mitotically inactivated MEFs can be used for CM preparation up to three times.
   4. Aliquot 35 ml of filtered CM into 50 ml conical tubes and store at -20 °C until needed. After thawing, add 15 ml of freshly prepared TS medium in order to obtain 70% TS-CM and store at 4 °C for up to one month.

3. Lentiviral Vector Production in 293T Cells

Note: All steps are carried out in laboratory space licensed to Biosafety Level 2.

1. Prior to conversion experiments, prepare lentiviral vectors for the doxycycline (dox) dependent over-expression of Tfap2c, Gata3, Eomes, Ets2 and mCherry. For each lentivirus, co-transfect 293T cells using the respective lentiviral transfer vector together with a packaging plasmid and a VSV-G expressing envelope plasmid (for plasmid information refer to the Materials Table).
   Note: For helpful information regarding virus production refer to Gavrilescu et al.¹⁰. Although it describes retrovirus production, general comments regarding quality of 293T cells and plasmid DNA are true for lentivirus production as well.
   CAUTION: Appropriate safety measures need to be taken, when working with lentiviral vectors and work has to be performed in a Biosafety Level 2 facility.
2. Coat five 100 mm dishes with poly-L-lysine (100 µg/ml) solution by covering the dishes with 5 ml of solution and gently rocking to evenly distribute coating solution. Place the dishes back into the incubator at 37 °C for 30 min. After 30 min aspirate coating solution and rinse dishes once with PBS.
3. Plate 7 x 10⁶ 293T cells per dish in 10 ml 293T-cell media, swirl dishes to evenly distribute cells and place dishes back into the incubator at 37 °C.
4. The next morning, replace growth medium with 5 ml of transfection medium. Add 6 µl of a 1,000x stock of chloroquine solution (25 mM) and place dishes back into the incubator at 37 °C.
5. Meanwhile, prepare transfection mixture:
   1. For each lentiviral vector prepare two 5 ml reaction tubes, labeled A and B.
   2. Add 600 µl of 2x HEPES buffered saline (HBS) to tube A.
   3. In tube B mix 61.5 µl CaCl₂ solution (2 M), with 18.5 µg lentivector DNA and 9.25 µg psPAX2 and 9.25 µg of pMD2.G. Adjust volume to 600 µl with sterile culture grade H₂O.
   4. Add solution from tube B drop-wise to tube A while vortexing. Incubate transfection mixture for 15 min at RT.
6. Add transfection mixture very carefully drop wise to the prepared 293T cells on the 100 mm dish. After 5 to 6 hr remove medium and replace with 10 ml of virus-production medium. Place dishes back into the incubator at 37 °C.
   Note: Be careful when removing and replacing medium since 293T cells easily detach from the dish. Medium change 5-6 hr after transfection is crucial, since prolonged exposure to chloroquine is toxic to the cells.
7. The following morning, carefully remove and discard medium and replace with 7 ml of virus-production medium.
8. The following morning harvest the first batch of virus-containing medium. Aspirate medium and collect in a 15 ml conical tube. Again, add 7 ml of virus-production medium onto the cells. Store virus-containing medium in the fridge until the second batch is harvested the following day.
9. The next morning aspirate the second batch of virus-containing medium adding it to the 15 ml conical tube containing the first batch. 293T cells can now be discarded. Filter virus-containing medium through a 0.45 µm surfactant-free cellulose acetate -membrane filter. Aliquot filtered, virus-containing medium and store at -80 °C for further use.
   Note: Virus production can also be performed in 6-well dishes. Scale down volumes and cell numbers accordingly.

4. Fibroblast Transduction with 4 Factors and mCherry Control Vector

Note: All steps are carried out in a Biosafety Level 2 facility.

1. For a schematic representation of the experimental outline refer to Figure 1. Thaw one aliquot of primary rtTA-MEFs and plate 3.33 x 10⁵ cells per well of a 6-well dish in a total volume of 2 ml of MEF media. Prepare at least 3 wells and label them with 4 factors (4F), mCherry and control.
   Note: Alternatively, wild type MEFs can be used, when an rtTA expressing lentiviral vector is added to the lentivector cocktail during step 4.2.
2. The following day after cells fully recovered from cryopreservation, replace MEF media in the three 6-wells with 0.6 ml (4F), 0.9 ml (mCherry) and 1 ml (control) of Transduction media, respectively. Thaw one aliquot of pLV-tetO-Tfap2c, -Gata3, -Eomes, -Ets and -mCherry virus at RT and add 100 µl of each virus-containing supernatant (Tfap2c, Gata3, Eomes, Ets2) into the 4F well.
   Note: In case of wild type MEFs, reduce volume of preplated media by 100 µl and additionally add 100 µl of FUdeltaGW-rtTA virus containing supernatant to the 4F mix.
3. Add 100 µl of mCherry virus-containing media to the well labeled with mCherry. Add polybrene to a final concentration of 8 µg/ml into each well. Place the dish back into the incubator at 37 °C and incubate MEFs O/N with virus-containing supernatant.
   Note: In case of wild type MEFs, reduce volume of preplated media by 100 µl and additionally add 100 µl of FUdeltaGW-rtTA virus-containing supernatant.
4. The next morning, carefully remove virus-containing media and wash three times with 2 ml of PBS. Finally, add 2 ml of TS media (without FGF4/heparin).
   Note: Transduced MEFs can either be directly used for iTSC induction experiments or frozen for further use.

5. Fibroblast to iTSC Conversion

Note: All steps are carried out in laboratory space licensed to Biosafety Level 2.

1. Wash transduced MEFs twice with 2 ml PBS and after adding 0.5 ml trypsin/EDTA solution incubate them in the incubator for 3 – 4 min. Check under an inverted microscope for proper detachment of the cells.
   1. Inactivate trypsin by adding 1 ml TS medium. Transfer the cell suspension into a 15 ml conical tube and centrifuge for 3 min at 200 x g. Discard the supernatant and resuspend the cells in 2 ml TS medium. Use the untransduced control sample to count the cells.
   2. Plate 4F-MEFs, mCherry-MEFs and untransduced control MEFs each at a density of 2 x 10⁵ cells per 100 mm cell culture dish in 10 ml TS medium containing 2 µg/ml dox (TS medium + FGF4/heparin + dox).
   3. Alternatively, perform transdifferentiation on smaller dishes. Plate 3.6 x 10³ cells per cm² on desired dish size, scale volume accordingly.
2. The next day, using an epifluorescence microscope (using a 10X lens and a filter suitable for red fluorescence excitation) monitor expression of mCherry protein in order to estimate transduction efficiency.
   Note: Transduction efficiency should be close to 100%. If increased cell death is apparent after transgene induction this means that virus titer was too high. Transduction should be repeated with reduced amount of virus-containing medium.
3. From now on change media on all dishes every other day until day 10 after plating. Thereafter, feed cells with TS medium without dox (TS medium + FGF4/heparin).
4. Using an inverted microscope monitor for appearance of 'transdifferentiated areas' (refer to Figure 2B and C) on 4F dish for up to 3 weeks. Such colonies should not be visible on the two control dishes (mCherry-MEFs and untransduced MEFs).

6. Isolation of Individual iTSC Lines

Note: For step 6.3 a tissue culture hood is not required. It is recommended to wipe down the inverted microscope with 70% ethanol to minimize the chances of bacterial contamination. Individual iTSC colonies can be isolated between days 21 and 28 days of transdifferentiation.

1. Add 40 µl of 0.05% trypsin/EDTA solution into 12 wells of a 96 U-bottom well dish and place the dish on ice.
2. Before colony isolation, aspirate medium on 4F-MEF transdifferentiation dish. Wash cells once with 10 ml PBS. Again, add 10 ml PBS and place dish under an inverted microscope.
3. Using a 100-µl pipette set to 40 µl lift individual colonies, by circling them with the pipette tip and aspirating the floating colony. Transfer cells of one colony each into one well of the prepared 96-well dish. After picking 12 colonies place 96-well dish in the incubator at 37 °C for 5 min.
4. Dissociate cells by pipetting up and down for several times and transfer cell suspension into one well of a 24-well dish with prepared 500 µl of TS-CM medium (30% fresh TS medium + 70% CM + FGF4/heparin).
5. The following day replace medium with fresh TS-CM medium to remove dead cells. Change medium every other day.
6. Monitor for appearance of epithelial colonies with bright boundaries (refer to Figure 2). Once colonies appear, culture until 70% confluent or for up to one week and gradually expand on larger dishes. From now on, culture cells as bona fide TSCs, according to standard protocols in serum containing media or in serum-free, defined media ^3,5,11.