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/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 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).
The strength of this protocol is the use of doxycycline-inducible vectors, which allow for temporal control of transgene expression. This enables the generation of stable iTSC lines that activate the endogenous transcription factor network, maintaining TSC fate independent of transgene expression. This is in contrast to previously published protocols, describing the induction of TSC fate from embryonic stem cells, a method that to date only yields incompletely reprogrammed trophoblast stem cell-like cells 1.
However, a known limitation of the protocol described here is the variable number of emerging transdifferentiated colonies, making it difficult to predict the transdifferentiation efficiency. These limitations are due to&#160;several highly variable factors, which are critical for the transdifferentiation outcome: the transduction efficiency of the single lentiviral vectors, the amount of proliferation of the starting cell population, and the amount of cell death during transdifferentiation. Under some circumstances transdifferentiated iTSC colonies fail to emerge. In this case media and reagents should be tested for their ability to support genuine TSC culture, before their use in iTSC generation. Additionally, transduction with the 4F can be titrated, since too high amounts of transgene expression lead to cell death. In general, sub-culturing of individual iTSC lines requires some practice. In the event that difficulties with sub-culture of iTSCs occur (i.e., sub-cultured cells fail to maintain self-renewal and instead spontaneously differentiate), single isolated colonies can be alternatively plated on dishes with 50% cell density of growth inactivated feeder cells to support growth and attachment of cells. The iTSC lines can be then gradually weaned from feeders during subsequent passaging. Of note, we did not succeed in directly converting MEFs into iTSCs using defined TX medium conditions, since TX medium only supports established iTSC/TSC lines.
So far, the protocol for iTSC induction from fibroblasts is only established for murine cells. Hence, it will now be of great interest to adapt this protocol to other species and enable derivation of iTSC lines from human or other model systems. Further, analysis of the precise mechanisms of fibroblast to iTSC conversion will offer new insights into the nature of the somatic versus extra-embryonic lineage barrier and aid in understanding principles of cell fate determination during normal and pathological development.

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). 4F-iTSCs stain positive for trophoblast transcription factors Cdx2 and Tfap2c (Figure 2E). These iTSC lines can now be used for subsequent in vitro or in vivo analyses, i.e., in vitro differentiation assays or placental chimerization experiments.
<img alt="Figure 1" src="/files/ftp_upload/54277/54277fig1.jpg" /> 
Figure 1. Timeline of MEF to iTSC Conversion. Graphical representation of transdifferentiation of MEFs into iTSCs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54277/54277fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54277/54277fig2.jpg" /> 
Figure 2. Representative Changes in Morphology During MEF to iTSC Conversion. 
(A) Photomicrograph of rtTA-MEFs. (B) Photomicrograph of transdifferentiated 4F-MEFs. Example of a transdifferentiated area, highlighted in red. (C) Example 2 of a transdifferentiated colony on day 21 of transdifferentiation after ten days dox induction in 4F-MEFs. Area suitable for sub-culturing highlighted in red. (D) Photomicrograph of 4F-iTSCs after sub-culturing. All scale bars indicate 100 &#181;m. Images were taken on an inverted microscope using a 10X lens and phase contrast. (E) Immunofluorescence staining against the transcription factors Cdx2 and Tfap2c in 4F-iTSCs. Nuclei are stained with Hoechst. Scale bars indicate 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54277/54277fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

protocol-for-direct-conversion-murine-embryonic-fibroblasts-into

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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 ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...