Name: protocol for the direct conversion of murine embryonic fibroblasts into trophoblast stem cells
Uploaded: 2016-07-25T15:00:00
Description: Watch this Scientific Journal Video about Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells at JoVE.com

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...

<ol>
	<li>Cambuli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+memory+of+the+first+cell+fate+decision+prevents+complete+ES+cell+reprogramming+into+trophoblast.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Pluripotent+Stem+Cells+from+Mouse+Embryonic+and+Adult+Fibroblast+Cultures+by+Defined+Factors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Induction+of+Trophoblast+Stem+Cells+from+Murine+Fibroblasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Nuclear+Reprogramming+Underlies+Lineage+Conversion+into+Functional+Trophoblast+Stem-like+Cells.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+mouse+trophoblast+stem+cell+proliferation+by+TGF-beta/activin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+Oct-4+blocks+progenitor-cell+differentiation+and+causes+dysplasia+in+epithelial+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+system+for+the+generation+and+study+of+human+induced+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+Maintenance+of+Murine+Trophoblast+Stem+Cells+under+Defined+Conditions.">Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions.</a> Stem Cell Rep. 2 (2), 232-242 (2014).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Doxycycline hyclate</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:101px;">D9891-10G</td>
			<td style="width:180px;">Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Chloroquine diphosphate salt&#160;</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>C6628-25G</td>
			<td style="width:180px;">Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Polybrene (Hexadimethrine bromide)</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td style="width:180px;">Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">human recombinant Fibroblast Growth Factor 4</td>
			<td style="width:93px;">ReliaTech</td>
			<td>300-131L</td>
			<td style="width:180px;">Dissolve in sterile 0.1% BSA in PBS, prepare 25 &#181;g/ml stock solution. Store aliquots at -80&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Heparin sodium salt</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td style="width:180px;">Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">ProFection Mammalian Transfection System</td>
			<td style="width:93px;">Promega</td>
			<td>E1200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">PBS, no calcium, no magnesium</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>1419-094</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>31966-021</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">RPMI 1640, no glutamine</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>31870-025</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Advanced DMEM (1x)</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>12491-015</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>25300-054</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Poly-L-lysine</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>P4707</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Fetal bovine serum</td>
			<td style="width:93px;">Hyclone</td>
			<td>SH30071.03IR</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Surfactant-free cellulose acetate-membrane filter</td>
			<td style="width:93px;">Corning</td>
			<td>431220</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Non-essential amino acids</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>11140-035</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Penicillin Streptomycin</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>15140-122</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Sodium Pyruvate&#160;</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>11360-039</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">L-glutamine&#160;</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>25030-24</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">2-Mercaptoethanol</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>31350-010</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Dimethyl sulfoxide (DMSO), cell culture grade</td>
			<td style="width:93px;">Panreac AppliChem GmbH</td>
			<td>A3672,0100</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Tfap2c</td>
			<td style="width:93px;">Addgene</td>
			<td>70269</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Gata3</td>
			<td style="width:93px;">Addgene</td>
			<td>70270</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Eomes</td>
			<td style="width:93px;">Addgene</td>
			<td>70271</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Ets2</td>
			<td style="width:93px;">Addgene</td>
			<td>70272</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-mCherry</td>
			<td style="width:93px;">Addgene</td>
			<td>70273</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">psPAX2</td>
			<td style="width:93px;">Addgene</td>
			<td>12260</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pMD2.G</td>
			<td style="width:93px;">Addgene</td>
			<td>12259</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">FUdeltaGW-rtTA&#160;</td>
			<td style="width:93px;">Addgene</td>
			<td>19780</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Mice B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J 7</td>
			<td style="width:93px;">JAX Mice</td>
			<td>6965</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">129S2SV</td>
			<td style="width:93px;">Charles River</td>
			<td>129</td>
			<td style="width:180px;"></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...

Watch this Scientific Journal Video about Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells at JoVE.com

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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