Name: Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells (Video) | JoVE Journal
Uploaded: 2016-07-25T15:00:00.000+00:00
Duration: 8 min 57 s
Description: Learn to convert murine embryonic fibroblasts into induced trophoblast stem cells using transient transcription factor expression. This protocol enables stable iTSC generation for studying placental development and lineage specification.

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5 minutes

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
<ol>
	<li>Prepare 293T medium: Dulbecco&#39;s Modified Eagle Medium (DMEM), 10% (v/v) FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin/streptomycin (1x).</li>
	<li>Prepare MEF medium: DMEM, 10% (v/v) FBS, L-glutamine (2 mM), 1x non-essential amino acids, penicillin/streptomycin (1x).</li>
	<li>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).</li>
	<li>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 &#181;g/ml heparin. 
	Note: FGF4 and heparin are added directly into the media immediately prior to use.</li>
	<li>Prepare TS-CM with FGF4/heparin (TS-CM + F4H): 70% MEF-conditioned TS medium + 30% freshly prepared TS medium + 25 ng/ml FGF4, 1 &#181;g/ml heparin. 
	Note: FGF4 and heparin are added directly into the media immediately prior to use.</li>
	<li>Prepare Transfection medium: Advanced DMEM, 2% (v/v) FBS (stem cell culture grade), L-glutamine (2 mM), penicillin/streptomycin (1x).</li>
	<li>Prepare Virus-production medium: Advanced DMEM, 5% (v/v) FBS (stem cell culture grade), L-glutamine (2 mM), penicillin/streptomycin (1x).</li>
</ol>
2. Murine Embryonic Fibroblast Derivation
<ol>
	<li>Set timed matings using wt 129S2SV females and homozygous male R26::rtTA mice (strain name; B6.Cg-Gt(ROSA)26Sortm1(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.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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).</li>
	<li>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).
	<ol>
		<li>Plate 1 x 107 mitotically inactivated MEFs per 150 mm dish in MEF medium on desired number of dishes. Inactivate MEFs by &#947;-irradiation with a dose of 9 Gy. 
		Note: The yield per 150 mm dish is approximately 60 ml of CM.</li>
		<li>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 &#176;C.</li>
		<li>After incubating TS medium for three days on mitotically inactivated MEFs, collect medium in 50 ml conical tubes. Filter medium through a 0.2 &#181;m 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.</li>
		<li>Aliquot 35 ml of filtered CM into 50 ml conical tubes and store at -20 &#176;C until needed. After thawing, add 15 ml of freshly prepared TS medium in order to obtain 70% TS-CM and store at 4 &#176;C for up to one month.</li>
	</ol>
	</li>
</ol>
3. Lentiviral Vector Production in 293T Cells
Note: All steps are carried out in laboratory space licensed to Biosafety Level 2.
<ol>
	<li>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.</li>
	<li>Coat five 100 mm dishes with poly-L-lysine (100 &#181;g/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 &#176;C for 30 min. After 30 min aspirate coating solution and rinse dishes once with PBS.</li>
	<li>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 &#176;C.</li>
	<li>The next morning, replace growth medium with 5 ml of transfection medium. Add 6 &#181;l of a 1,000x stock of chloroquine solution (25 mM) and place dishes back into the incubator at 37 &#176;C.</li>
	<li>Meanwhile, prepare transfection mixture:
	<ol>
		<li>For each lentiviral vector prepare two 5 ml reaction tubes, labeled A and B.</li>
		<li>Add 600 &#181;l of 2x HEPES buffered saline (HBS) to tube A.</li>
		<li>In tube B mix 61.5 &#181;l CaCl2 solution (2 M), with 18.5 &#181;g lentivector DNA and 9.25 &#181;g psPAX2 and 9.25 &#181;g of pMD2.G. Adjust volume to 600 &#181;l with sterile culture grade H2O.</li>
		<li>Add solution from tube B drop-wise to tube A while vortexing. Incubate transfection mixture for 15 min at RT.</li>
	</ol>
	</li>
	<li>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 &#176;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.</li>
	<li>The following morning, carefully remove and discard medium and replace with 7 ml of virus-production medium.</li>
	<li>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.</li>
	<li>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 &#181;m surfactant-free cellulose acetate -membrane filter. Aliquot filtered, virus-containing medium and store at -80 &#176;C for further use. 
	Note: Virus production can also be performed in 6-well dishes. Scale down volumes and cell numbers accordingly.</li>
</ol>
4. Fibroblast Transduction with 4 Factors and mCherry Control Vector
Note: All steps are carried out in a Biosafety Level 2 facility.
<ol>
	<li>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.</li>
	<li>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 &#181;l 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 &#181;l and additionally add 100 &#181;l of FUdeltaGW-rtTA virus containing supernatant to the 4F mix.</li>
	<li>Add 100 &#181;l of mCherry virus-containing media to the well labeled with mCherry. Add polybrene to a final concentration of 8 &#181;g/ml into each well. Place the dish back into the incubator at 37 &#176;C and incubate MEFs O/N with virus-containing supernatant. 
	Note: In case of wild type MEFs, reduce volume of preplated media by 100 &#181;l and additionally add 100 &#181;l of FUdeltaGW-rtTA virus-containing supernatant.</li>
	<li>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.</li>
</ol>
5. Fibroblast to iTSC Conversion
Note: All steps are carried out in laboratory space licensed to Biosafety Level 2.
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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 &#181;g/ml dox (TS medium + FGF4/heparin + dox).</li>
		<li>Alternatively, perform transdifferentiation on smaller dishes. Plate 3.6 x 103 cells per cm2 on desired dish size, scale volume accordingly.</li>
	</ol>
	</li>
	<li>The next day, using an epifluorescence microscope (using a 10X&#160;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.</li>
	<li>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).</li>
	<li>Using an inverted microscope monitor for appearance of &#39;transdifferentiated areas&#39; (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).</li>
</ol>
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.
<ol>
	<li>Add 40 &#181;l of 0.05% trypsin/EDTA solution into 12 wells of a 96 U-bottom well dish and place the dish on ice.</li>
	<li>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.</li>
	<li>Using a 100-&#181;l pipette set to 40 &#181;l 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 &#176;C for 5 min.</li>
	<li>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 &#181;l of TS-CM medium (30% fresh TS medium + 70% CM + FGF4/heparin).</li>
	<li>The following day replace medium with fresh TS-CM medium to remove dead cells. Change medium every other day.</li>
	<li>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.</li>
</ol>

<ol><li>Cambuli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+memory+of+the+first+cell+fate+decision+prevents+complete+ES+cell+reprogramming+into+trophoblast%5BTitle%5D)+AND+%22Nat+commun%22%5BJournal%5D)">Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast.</a> Nat commun. 5, 5538(2014).</li>
<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+Pluripotent+Stem+Cells+from+Mouse+Embryonic+and+Adult+Fibroblast+Cultures+by+Defined+Factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors.</a> Cell. 126 (4), 663-676 (2006).</li>
<li>Kubaczka, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+Induction+of+Trophoblast+Stem+Cells+from+Murine+Fibroblasts%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.</a> Cell Stem Cell. 17 (5), 557-568 (2015).</li>
<li>Benchetrit, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extensive+Nuclear+Reprogramming+Underlies+Lineage+Conversion+into+Functional+Trophoblast+Stem-like+Cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells.</a> Cell Stem Cell. 17 (5), 543-556 (2015).</li>
<li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTanaka%2C+S+%22Science%22">Science</a>. 282 (5396), 2072-2075 (1998).</li>
<li>Erlebacher, A., Price, K. A., Glimcher, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+mouse+trophoblast+stem+cell+proliferation+by+TGF-beta%2Factivin%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Maintenance of mouse trophoblast stem cell proliferation by TGF-beta/activin.</a> Dev Biol. 275 (1), 158-169 (2004).</li>
<li>Hochedlinger, K., Yamada, Y., Beard, C., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ectopic+expression+of+Oct-4+blocks+progenitor-cell+differentiation+and+causes+dysplasia+in+epithelial+tissues%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues.</a> Cell. 121 (3), 465-477 (2005).</li>
<li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. (64), (2012).</li>
<li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-efficiency+system+for+the+generation+and+study+of+human+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">A high-efficiency system for the generation and study of human induced pluripotent stem cells.</a> Cell Stem Cell. 3 (3), 340-345 (2008).</li>
<li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. (10), e550(2007).</li>
<li>Kubaczka, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Derivation+and+Maintenance+of+Murine+Trophoblast+Stem+Cells+under+Defined+Conditions%5BTitle%5D)+AND+%22Stem+Cell+Rep%22%5BJournal%5D)">Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions.</a> Stem Cell Rep. 2 (2), 232-242 (2014).</li>
</ol>

The authors have nothing to disclose.

The 4 factor (Tfap2c, Gata3, Eomes, Ets2) based transdifferentiation protocol presented here offers a reliable method to generate faithfully converted iTSCs from mouse embryonic fibroblasts. Further, the method is also applicable for post-natal tail fibroblasts, although with a drop in efficiency compared to embryonic fibroblasts 3. In general, quality of primary fibroblasts is a critical factor of transdifferentiation outcome and care should be taken to use early passage cells (passage two to three).
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Recently, a study comparing several approaches of mouse embryonic stem cell to trophoblast stem cells conversion has revealed that in all analyzed systems, lineage conversion remained incomplete. Instead of induced trophoblast stem cells (iTSCs) so called trophoblast stem cell-like cells have been generated retaining a memory of the cell fate of origin1. Here, we followed a different approach of iTSC generation. Similar to the direct induction of pluripotent stem cells from murine embryonic fibroblasts (MEFs) 2, iTSCs have been directly converted from differentiated somatic tissue. First, we identified 12 candidate factors inducing TSC fate when overexpressed in MEFs. Later on, the factors Tfap2c, Gata3, Eomes and Ets2 have been identified to be necessary and sufficient for the iTSC induction 3. Simultaneously, another group independently found Tfap2c, Gata3 and Eomes to be sufficient to convert MEFs into iTSCs. However, in that study, the time required for transgene expression is considerably longer compared to our study, indicating different conversion kinetics, when Ets2 is absent from the transdifferentiation cocktail 4.
Conventional fetal bovine serum (FBS) containing culture of induced and blastocyst derived trophoblast stem cells relies on the presence of factors secreted by growth-inactivated MEFs 5,6. During the iTSC induction, these factors are provided by MEFs, which lack the full combination of transgenes and are not undergoing transdifferentiation. However, once individual iTSC colonies are sub-cultured, they require media, which has been preconditioned by growth-inactivated MEFs. From there on, iTSCs can be cultured and treated like blastocyst derived TSCs according to standard protocols. Of note, in contrast to Tanaka et al. 5, we routinely culture TSCs and iTSCs without gelatinizing cell culture dishes.

<table><tbody><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-10G</td><td>Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>Chloroquine diphosphate salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C6628-25G</td><td>Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>Polybrene (Hexadimethrine bromide)</td><td>Sigma-Aldrich</td><td>107689-10G</td><td>Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>human recombinant Fibroblast Growth Factor 4</td><td>ReliaTech</td><td>300-131L</td><td>Dissolve in sterile 0.1% BSA in PBS, prepare 25 &amp;micro;g/ml stock solution. Store aliquots at -80 &amp;deg;C</td></tr><tr><td>Heparin sodium salt</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td>Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80&amp;deg;C</td></tr><tr><td>ProFection Mammalian Transfection System</td><td>Promega</td><td>E1200</td><td></td></tr><tr><td>PBS, no calcium, no magnesium</td><td>ThermoFisher Scientific</td><td>1419-094</td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>ThermoFisher Scientific</td><td>31966-021</td><td></td></tr><tr><td>RPMI 1640, no glutamine</td><td>ThermoFisher Scientific</td><td>31870-025</td><td></td></tr><tr><td>Advanced DMEM (1x)</td><td>ThermoFisher Scientific</td><td>12491-015</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>ThermoFisher Scientific</td><td>25300-054</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30071.03IR</td><td></td></tr><tr><td>Surfactant-free cellulose acetate-membrane filter</td><td>Corning</td><td>431220</td><td></td></tr><tr><td>Non-essential amino acids</td><td>ThermoFisher Scientific</td><td>11140-035</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>ThermoFisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Sodium Pyruvate&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11360-039</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>25030-24</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>ThermoFisher Scientific</td><td>31350-010</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO), cell culture grade</td><td>Panreac AppliChem GmbH</td><td>A3672,0100</td><td></td></tr><tr><td>pLV-tetO-Tfap2c</td><td>Addgene</td><td>70269</td><td></td></tr><tr><td>pLV-tetO-Gata3</td><td>Addgene</td><td>70270</td><td></td></tr><tr><td>pLV-tetO-Eomes</td><td>Addgene</td><td>70271</td><td></td></tr><tr><td>pLV-tetO-Ets2</td><td>Addgene</td><td>70272</td><td></td></tr><tr><td>pLV-tetO-mCherry</td><td>Addgene</td><td>70273</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>FUdeltaGW-rtTA&amp;nbsp;</td><td>Addgene</td><td>19780</td><td></td></tr><tr><td>Mice B6.Cg-Gt(ROSA)26Sor&lt;sup&gt;tm1(rtTA*M2)Jae/J 7&lt;/sup&gt;</td><td>JAX Mice</td><td>6965</td><td></td></tr><tr><td>129S2SV</td><td>Charles River</td><td>129</td><td></td></tr></tbody></table>

protocol for the direct conversion of murine embryonic fibroblasts into trophoblast stem cells

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

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

On the dish where transgene expression of the 4F is activated, cells rapidly change morphology (compare Figure 2A and B). Around day 14 - 21 distinct transdifferentiated areas emerge (two examples are given in Figure 2B and C). These primary colonies lack typical TSC morphology; however once they are sub-cultured, characteristic epithelial morphology with tight edges and bright boundaries highly reminiscent of bona fide TSCs emerges (Figure 2D</st...

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

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.

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Research

JoVE Journal

Developmental Biology

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts.  We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

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Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

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Learn to convert murine embryonic fibroblasts into induced trophoblast stem cells using transient transcription factor expression. This protocol enables stable iTSC generation for studying placental development and lineage specification.

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

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