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

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...

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

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 ...

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7.6K Views.  Institute of Pathology, University of Bonn Medical School. 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.

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

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

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.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Biology

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

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

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Przeglądaj więcej filmów

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions