This work was supported by a grant from the National Sciences and Engineering Research Council of Canada (NSERC) (#298527) (BB). CT was supported by an institutional FARE scholarship. AV was supported by a NSERC Graham Bell Ph.D. scholarship. BB held a Canada Research Chair in Human Retrovirology (Tier 2). Thanks to Beatrix Beisner for help in revising the text.

The UQAM ethics committee has approved these protocols, which are in accordance with the guidelines of the ethics committee of St-Luc Hospital of the Centre Hospitalier Universitaire de Montr&#233;al (Montr&#233;al, Canada). Participants signed an informed consent form.
1. Medium Preparation and Isolation of Primary Villous Cytotrophoblasts
<ol>
	<li>Prepare culture medium for human primary villous cytotrophoblasts by supplementing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with 25 mM HEPES, 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Filter the prepared medium through sterile 0.22 &#181;m filter and store in a -20 &#176;C freezer in aliquots of 50 ml.</li>
	<li>Isolate primary villous cytotrophoblasts from fresh placentas following standard protocols 3.
	<ol>
		<li>Using pliers, remove the fetal membrane from the tissue by gently scratching it off, while being careful not to remove villous tissue. Cut the remaining placental villi in cubes of about 3 x 3 cm using scalpel and scissors. Thoroughly wash with water containing 0.9% NaCl to remove maternal blood.
		<ol>
			<li>Holding each cube with pliers, carefully remove the villous tissue from remaining vessels and fibrous tissue using scissors.</li>
			<li>For 15 to 30 mg of villous tissue, digest four times in Hank&#39;s balanced salt solution (HBSS) containing trypsin (from 9.6 x 105 to 1.8 x 106 U; final concentration 1.2 mg/ml) and deoxyribonuclease I (DNase I) (final concentration 200 &#181;g/ml) for 30 min at 37 &#176;C in a water bath with continuous shaking. 
			Note: Use 150 ml total volume for the first digestion, 100 ml for the second digestion and 75 ml for the two remaining digestions.</li>
		</ol>
		</li>
		<li>Collect the supernatant containing dispersed cells and centrifuge at 1,250 x g for 15 min without brake. Remove the supernatant and the resuspend cells in 1 ml of prewarmed DMEM containing 1% penicillin/streptomycin.</li>
		<li>Layer the dispersed cells on top of a discontinuous 5% - 70% polyvinylpyrrolidone-coated silica gradient (see Table 1 for gradient preparation) and centrifuge for 23 min at 507 x g without brake. Remove the density layer between 1.017 to 1.033 by aspiration and collect the layers between 40% and 50% in the gradient with a new pipette. Wash cells extensively with 10 ml of prewarmed DMEM containing 1% penicillin/streptomycin. 
		Note: The collected fraction corresponds to densities between 1.048 and 1.062, which harbors trophoblasts.</li>
		<li>Count the cells using a hemocytometer. Briefly, mix cells and add 10 &#181;l in a new microcentrifuge tube. Add 10 &#181;l of trypan blue and mix gently again. Draw up the cell suspension and fill the hemocytometer chambers. Count the cells under 10X objective.</li>
		<li>Place 1 x 106 and 4.5 x 106 cells into separate tubes for use in sections 1.3 and 2, respectively. Seed the remaining cells at a density of 1.5 x 106 cells/well in supplemented DMEM medium and culture at 37 &#176;C and 5% CO2 for a maximum of 4 days for other experiments.</li>
	</ol>
	</li>
	<li>Evaluation of the purity of isolated primary villous cytotrophoblasts
	<ol>
		<li>Spin 1 x 106 of the freshly isolated cytotrophoblasts in microcentrifuge tubes at 300 x g. Discard the supernatant and add 1 ml of prewarmed phosphate-buffered saline (PBS). Centrifuge cells at 300 x g and remove PBS by aspiration.</li>
		<li>Add 1 ml of cold methanol to cells and incubate at -20 &#176;C for 20 min. Centrifuge cells at 300 x g for 5 min and remove methanol by aspiration. 
		Caution: Use canister mask, safety goggles and rubber gloves while handling methanol.</li>
		<li>Add 1 ml of PBS at room temperature to rehydrate cells, centrifuge at 300 x g and remove PBS by aspiration.</li>
		<li>Incubate cells in PBS containing FBS (1:50 [v/v] dilution) and a FcR blocking reagent (1:10) for 45 min at room temperature to eliminate nonspecific binding.</li>
		<li>Wash twice with 500 &#181;l of room temperature PBS, centrifuge cells at 300 x g for 5 min and remove PBS by aspiration. Resuspend cells in 100 &#181;l room temperature PBS.</li>
		<li>Incubate cells with a mouse monoclonal fluorescein isothiocyanate (FITC)-conjugated anti-human cytokeratin-7 antibody clone LP5K (1:200) or a control isotype-matched non-specific antibody in PBS containing 0.2% BSA for 45 min at room temperature in the dark. Wash cells in PBS.</li>
		<li>Analyze the cells by flow cytometry 3. Use cell preparations, which demonstrate a minimum of 96% CK-7-positive cells by flow cytometry for further experiments.</li>
	</ol>
	</li>
</ol>
2. siRNA Transfection of Human Primary Villous Cytotrophoblasts
<ol>
	<li>Transfection of human primary villous cytotrophoblasts using a microporation device
	<ol>
		<li>Prepare 6-well plates containing 2 ml of supplemented DMEM medium for the incubation of microporated cells.</li>
		<li>After isolation of primary cytotrophoblasts (see section 1.2), take an aliquot of cells in suspension and determine cell number using a hemocytometer.</li>
		<li>Transfer 1.5 x 106 cells to a microcentrifuge tube and pellet cells by centrifugation at 300 x g for 5 min at room temperature. Wash the cells with 1 ml Dulbecco&#39;s PBS (DPBS) (Mg2+, Ca2+-free) and pellet the cells by centrifugation at 300 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and resuspend the cells gently in 100 &#181;l of resuspension buffer. 
		Note: Avoid keeping cell suspension for longer than 15-30 min at room temperature, to maximize cell viability and transfection efficiency.</li>
		<li>Add 300 ng of Syncytin-2 siRNA (or control-scrambled siRNA) to the 1.5 ml microcentrifuge tube and aspirate the cell-siRNA mixture using a 100 &#181;l transfection pipette. Insert the pipette into the station (see List of Materials) and subject the cells to a 1,300 V single pulse of 30 msec.</li>
		<li>Immediately transfer the cells to 6-well plates (prepared in step 2.1.1) containing 37 &#176;C pre-warmed supplemented medium to avoid cell damage.</li>
		<li>Repeat steps 2.1.5 and 2.1.6 for the remaining samples.</li>
		<li>Gently rock the plate for 30 sec to ensure even distribution of the cells. Incubate the plate at 37 &#176;C in a humidified CO2 incubator for 24 to 48 hr. Refresh culture medium 24 hr after transfection.</li>
		<li>Evaluate transfection efficiency by Western blot analysis (see steps 3 to 6).</li>
	</ol>
	</li>
	<li>Lipofection of siRNA in human primary cytotrophoblasts
	<ol>
		<li>After isolation of villous cytotrophoblasts, seed the cells at a density of 1.5 x 106 cells per well in 6-well plates in 2 ml of culture medium and incubate for 18 hr.</li>
		<li>Dilute 300 ng of Syncytin-2-specific siRNA (or control-scrambled siRNA) and 5 &#181;l of lipid-based transfection reagent in separate 1.5 ml tubes in a total volume of 10 &#181;l of antibiotic- and serum-free medium. Incubate for 5 min.</li>
		<li>Mix the diluted reagents and incubate for an additional 20 min at room temperature to form the transfection complex. Add 90 &#181;l of antibiotic- and serum-free medium to the mix to a final volume of 100 &#181;l.</li>
		<li>Remove culture medium from the 6-well plates (step 2.2.1). Add 2 ml of fresh medium and the 100 &#181;l transfection mix to each well. Incubate the cells at 37 &#176;C in a CO2 incubator for 24 to 48 hr. Refresh culture medium 24 hr after transfection.</li>
		<li>Evaluate transfection efficiency by Western blot analysis (see steps 3 to 6).</li>
	</ol>
	</li>
</ol>
3. Preparation of Lysates from Whole Cell Culture
<ol>
	<li>Remove the media and rinse each well (from steps 2.1.8 and 2.2.4) using pre-warmed DPBS (Mg2+, Ca2+ free). Aspirate the DPBS and add 500 &#181;l of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA). Incubate for 1-3 min at 37 &#176;C in a CO2 incubator. Stop trypsin treatment by adding 500 &#181;l of serum containing growth media. 
	Note: Use appropriate media volume according to the type of flask/dish: 1 ml per 107 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5 x 106 cells /60 mm dish /75 cm2 flask.</li>
	<li>Transfer cells to a 1.5 ml microcentrifuge tube, pellet cells by centrifugation at 300 x g for 2 min at room temperature. Wash the cells with DPBS (Mg2+, Ca2+-free) and pellet cells by centrifugation at 300 x g for 2 min at room temperature.</li>
	<li>Carefully remove supernatant without disturbing the pellet. Place the tube on ice. Resuspend the pellet in 50-200 &#181;l ice-cold Radio-Immunoprecipitation Assay (RIPA) buffer (see Table 2 for buffer composition) containing freshly added protease and phosphatase inhibitors at a 1x final concentration. Briefly, vortex the tube and leave on ice for 30 min.</li>
	<li>Spin at 16,000 x g for 20 min at 4 &#176;C. Carefully place the tube on ice. Using a pipette, transfer the supernatant to a fresh pre-cooled microcentrifuge tube kept on ice. Discard the pellet.</li>
</ol>
4. Preparation of Nuclear Extracts from Cultured Cells
<ol>
	<li>For each well (step 1.2.5), wash cultured cytotrophoblasts twice with 1 ml of pre-warmed DPBS (Mg2+, Ca2+ free). Aspirate DPBS and add 500 &#181;l of 0.25% trypsin/EDTA. Incubate for 1-3 min at 37 &#176;C in a CO2 incubator and stop trypsin treatment by adding 500 &#181;l of serum containing growth media.</li>
	<li>Transfer cells to a 1.5 ml microcentrifuge tube, pellet cells by centrifugation at 300 x g for 2 min at room temperature. Wash the cells with DPBS (Mg2+, Ca2+-free) and pellet cells by centrifugation at 300 x g for 2 min at room temperature. Carefully remove all supernatant without disrupting the pellet and place the tube on ice.</li>
	<li>Extract nuclear protein according to manufacturer&#39;s instructions. Determine protein concentration of each sample using protein quantification assay (Bradford or bicinchoninic acid assay (BCA)).</li>
</ol>
5. Sample Preparation and Electrophoresis
<ol>
	<li>Determine the protein concentration for each cell extract using a protein quantification assay (for example, Bradford 8 or BCA 9).</li>
	<li>As per manufacturer&#39;s instructions (if antibodies could be used in reduction and denaturation conditions), add an equal volume of 2x Laemmli Sample Buffer (Table 2) to 20-30 &#181;g of total protein.</li>
	<li>Boil cell lysates at 100 &#176;C for 5 min. Aliquot 50-100 &#181;l of lysate to avoid repeated freeze/thaw cycles and store the remaining tubes at -20 &#176;C for future use.</li>
	<li>While the samples are heating, prepare 1 L of 1x running buffer (Table 2).</li>
	<li>Spin the samples at 16,000 x g for a few seconds and place them on ice.</li>
	<li>Load equal amount of protein into each well of a 12% SDS-PAGE gel (see Table 2 for gel composition), along with a molecular weight marker. Run the gel for 1 to 2 hr at 100 V.</li>
</ol>
6. Transferring the Protein from the Gel to the Membrane
<ol>
	<li>Activate polyvinylidene fluoride (PVDF) membrane with methanol for 1 min and rinse with 1x transfer buffer/10% methanol solution. According to manufacturer&#39;s instructions, transfer for 30 min to 1 hr depending on the protein size. 
	Note: Transfer efficiency can be assessed using Ponceau Red staining before the blocking step.</li>
</ol>
7. Antibody Staining
<ol>
	<li>Block the membrane for 1 hr at room temperature or overnight at 4 &#176;C using 5% blocking solution.</li>
	<li>Incubate the membrane with anti-Syncytin-2 antibodies (4 &#181;g/ml; 1:5,000) 6 or with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 0.4 &#181;g/ml; 1:500) in 5% blocking solution overnight at 4 &#176;C (anti-Syncytin-2) or for 45 min at room temperature. Wash the membrane three times in Tris-buffered saline Tween (TBST; see Table 2 for buffer composition) for 5 min.</li>
	<li>Incubate the membrane with appropriate horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibody (1:10,000) in 5% blocking buffer in TBST at room temperature for 1 hr. Wash the membrane three times in TBST for 5 min and then rinse in TBS. Detect signals using the chemiluminescence-based blotting substrate.</li>
</ol>
8. Radiolabeling of Single Stranded Oligonucleotide
<ol>
	<li>Phosphorylation reaction
	<ol>
		<li>In a microcentrifuge tube, add 50 ng of a forward oligonucleotide along with 1x T4 polynucleotide kinase buffer, 1 U T4 polynucleotide kinase and 20 &#181;Ci (&#947;-32P) ATP and make up the reaction volume to 20 &#181;l with nuclease-free water.</li>
		<li>Incubate at 37 &#176;C for 30 min. Stop the reaction by adding 5 &#181;l of 0.5 M EDTA. Make up the volume to 50 &#181;l with nuclease-free water.</li>
	</ol>
	</li>
	<li>Removal of unincorporated nucleotides from oligonucleotides
	<ol>
		<li>Prepare a G-25 column equilibrated with TE buffer (Table 2) following manufacturer&#39;s instructions. Place the column in a fresh DNase-free 1.5 ml microcentrifuge tube.</li>
		<li>Slowly add 50 &#181;l of probe (from step 8.1.2) over the resin, being careful not to disturb the resin bed. Spin for 2 min at 350 x g to collect the purified sample in the 1.5 ml microcentrifuge tube. Wash the G-25 column with 36 &#181;l of nuclease-free water and spin for 2 min at 350 x g to collect it in the microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Hybridization with the complementary strand oligonucleotide
	<ol>
		<li>Transfer the radiolabeled probe (86 &#181;l) to a 1.5 ml microcentrifuge tube and add 200 ng of the reverse oligonucleotide and 1x of annealing buffer (Table 2). Heat 5 min at 95 &#176;C and leave overnight at room temperature for cooling.</li>
		<li>Prepare non-radioactive double-stranded oligonucleotides by adding 50 ng of unlabeled forward and reverse oligonucleotides and 1x of annealing buffer in a 1.5 ml microcentrifuge tube. Make up the volume to 100 &#181;l with nuclease-free water. Heat and keep at room temperature as described in step 8.3.1.</li>
	</ol>
	</li>
</ol>
9. Preparation of a Non-denaturing Polyacrylamide Gel for EMSA
<ol>
	<li>Prepare a 4% native gel (10 x 12 cm) with a 1.5 mm space-comb (see Table 2 for gel composition). Clean all glass plates using distilled water. 
	Note: It is critical that the plates be completely free of ionic detergent, like SDS.</li>
	<li>Immediately pour the acrylamide solution in between the plates and allow polymerization to proceed (approximately 30 min).</li>
	<li>Assemble the electrophoresis apparatus and fill the tank with 0.5x TBE. Pre-run the gel for 30 min to 1 hr at 150 V before sample loading.</li>
</ol>
10. DNA Binding Reaction
<ol>
	<li>In a 1.5 ml microcentrifuge tube, add 10 &#181;g of nuclear extract (step 4.3) in 1x Gel Shift Binding buffer (Table 2) and make up the final volume to 10 &#181;l with nuclease-free water. As a control, prepare a tube with no nuclear extract. For the competition reaction, add 1 &#181;l of cold non-specific or specific double-stranded oligonucleotide (step 8.3.2).</li>
	<li>Add 1 &#181;l of radioactive probe from step 8.3.1 to each reaction. Incubate the reaction at room temperature for 20 min. Add 1 &#181;l of 10x gel loading buffer per reaction and load each sample on the gel prepared in section 9.</li>
	<li>Run the gel for 1 1/2 to 2 hr at 150 V. After migration, wrap the gel and the glass in a plastic wrap and position in an exposure cassette below a phosphor screen. Leave cassette at 4 &#176;C for 24 hr.</li>
	<li>Remove the screen from the exposure cassette and insert in the phosphor imaging device for scanning.</li>
</ol>

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The authors have nothing to disclose.

Studies on human placental function and development have been greatly improved by protocols aimed at optimizing isolation of various placental cell populations. One of the best studied placental cell population remains the villous cytotrophoblasts, the study of which has greatly benefited from optimized protocols permitting efficient and reliable isolation. This has further allowed a number of experiments, such as transfection and promoter studies. Using a previously described protocol 3, pure populations of p...

Human placental dysfunction is associated with the development of several pregnancy-associated diseases like preeclampsia and intrauterine growth restriction 1. An important cell constituent of the placenta is the trophoblasts, which can be classified as either extravillous or villous cytotrophoblasts. Upon fusion, villous cytotrophoblasts further differentiate into the syncytiotrophoblast layer, a multinuclear cell structure with an important role in feto-maternal exchange and hormone production 2. Human primary villous cytotrophoblasts and their differentiated counterparts represent important biological samples and allow researchers to study a number of placenta-related processes, such as cell fusion, in culture. Furthermore, substantial efforts are ongoing to identify markers that will facilitate appropriate management and improve preventive therapies specific to pregnancy-related diseases. Laboratories routinely isolate primary human villous cytotrophoblasts from fresh placentas, using a standard isolation procedure based on trypsin digestion of placenta villi 3. As cultured cytotrophoblasts lose their capacity to proliferate and quickly differentiate in a syncytiotrophoblast-like layer upon culture 4, very efficient transfection methods and optimized analysis approaches are needed. Previous studies have determined optimal conditions of transfection of these primary cytotrophoblasts 5. Herein, a different method of siRNA transfection, which has been previously tested in this cell type 6, is presented. In comparison to a lipofection-based approach, this microporation method improves transfection, as assessed by the extent of silencing of specific genes.
Promoter and gene expression studies also provide a better understanding of placental function. Although more difficult to use owing to the short time frame for which primary villous cytotrophoblasts can be cultured, promoter analyses using standard protocols can nonetheless be addressed, as previously published 7. Electrophoretic Mobility Shift Assay (EMSA) is one of these commonly used in vitro methods, allowing for fast and easy monitoring of DNA-protein interactions. Nuclear extracts from these primary trophoblasts were used to test a region of the Syncytin-2 promoter for specific interactions. Results revealed that bound factors could be detected at different time points of culture and in a specific and reproducible manner.
Data presented in this protocol confirm that our transfection approach and the EMSA protocol can be used for isolated primary villous cytotrophoblasts and will be of great use to study the diverse functions of villous cytotrophoblasts in normal or pathological conditions.

<table><tbody><tr><td>HBSS without&amp;nbsp; Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Sigma</td><td>#H2387</td><td></td></tr><tr><td>HBSS (10x)</td><td>Sigma</td><td>#14060-057</td><td></td></tr><tr><td>DMEM High Glucose without Hepes</td><td>Gibco</td><td>#12100-061</td><td></td></tr><tr><td>Hepes (1 M)</td><td>Gibco</td><td>#15630-080</td><td></td></tr><tr><td>Penicillin-Streptomycin-Neomycin (100x)</td><td>Gibco</td><td>#15640-055</td><td></td></tr><tr><td>Amphotericin B&amp;nbsp;</td><td>Sigma</td><td>#A2411</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>#C4901</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;.&lt;/sup&gt;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>#M</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>#16170-078&amp;nbsp;</td><td></td></tr><tr><td>Percoll&amp;nbsp;</td><td>Sigma</td><td>#P-1644</td><td>For density gradient</td></tr><tr><td>Syncytin-2 siRNA</td><td>Ambion Life technologies</td><td>#AM16708</td><td></td></tr><tr><td>Scrambled siRNA</td><td>Qiagen</td><td># SI03650318</td><td></td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>#D5025</td><td></td></tr><tr><td>Trypsine, type I&amp;nbsp;</td><td>Sigma</td><td>#T8003</td><td></td></tr><tr><td>DharmaFECT Lipotransfection&amp;nbsp; reagents&amp;nbsp;</td><td>GEhealthcare</td><td># T-2001-01</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life technologies</td><td>#25300-062</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Roche Diagnostic</td><td>#11873580001</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>#23225</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>#A7906</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma</td><td>#P9416&amp;nbsp;</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked antibody&amp;nbsp;</td><td>Cell Signaling</td><td>#7074</td><td></td></tr><tr><td>BM Chemiluminescence Western Blotting Substrate (POD)</td><td>Roche Diagnostic</td><td>#11500708001</td><td></td></tr><tr><td>DPBS&amp;nbsp;</td><td>Life technologies</td><td>#14287-080</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>#M0201S</td><td></td></tr><tr><td>ATP, [&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]</td><td>Perkin Elmer</td><td>#BLU002A100UC</td><td></td></tr><tr><td>Acrylmide</td><td>Sigma&amp;nbsp;</td><td>#A9099</td><td></td></tr><tr><td>TEMED</td><td>Life technologies</td><td>#17919</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma&amp;nbsp;</td><td>#A3678</td><td></td></tr><tr><td>Anti-human cytokeratin-7 antibody clone LP5K, FITC conjugated</td><td>Millipore</td><td>CBL194F</td><td>Dilution 1:200</td></tr><tr><td>FcR blocking reagent&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130- 059-901</td><td>Dilution 1:10</td></tr><tr><td>Flow Cytometer BD Acuri system</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Microporator MP-100 apparatus&amp;nbsp;</td><td>Digital Bio</td><td></td><td></td></tr><tr><td>Resuspension Buffer R (Neon Transfection System 100&amp;nbsp;&amp;micro;l Kit)</td><td>Life technologies</td><td>MPK10096</td><td></td></tr><tr><td>PVDF membrane&amp;nbsp;</td><td>Millipore</td><td>IPVH00010&amp;nbsp;</td><td>Activate with methanol</td></tr><tr><td>Anti-human GAPDH antibody</td><td>Santa Cruz Biotechnology</td><td>sc-137179</td><td>1:500</td></tr><tr><td>HorseRadish Peroxidase (HRP)-conjugated goat anti-rabbit antibody or anti-mouse antibody</td><td>Cell Signalling</td><td>#7074</td><td>1:10,000</td></tr><tr><td>HorseRadish Peroxidase (HRP)-conjugated goat anti-mouse antibody</td><td>Cell Signalling</td><td>#7076&amp;nbsp;</td><td>1:10,000</td></tr><tr><td>NE-PER Nuclear and Cytoplasmic Extraction Reagent</td><td>Thermo Scientific</td><td>#78833</td><td></td></tr><tr><td>G-25 column&amp;nbsp;</td><td>GE Healthcare</td><td>#27-5325-01</td><td></td></tr><tr><td>Chemiluminsescence and fluorescence imaging device</td><td>Montr&amp;eacute;al Biotech</td><td>Fusion FX5</td><td></td></tr><tr><td>4% native gel</td><td>Home made</td><td></td><td></td></tr><tr><td>PBS</td><td>Home made</td><td></td><td>1x</td></tr><tr><td>Personal Molecular Imager (PMI) System</td><td>BioRad</td><td></td><td></td></tr></tbody></table>

sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

Fresh placentas from term pregnancies were used to isolate human primary villous cytotrophoblasts to conduct the set of experiments presented in the Protocol section. Following their isolation, we first analyzed the purity of cytotrophoblasts through the use of the cytokeratin-7 marker (Figure 1). Cell preparations were thus stained using a monoclonal anti-cytokeratin-7 antibody. Figure 1 represents results from a typical experiment following purification...

Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

sirna-transfection-emsa-analyses-on-freshly-isolated-human-villous

Research

JoVE Journal

Genetics

10.5K Views.  University of Quebec in Montreal.  Dit protocol beschrijft een werkwijze voor het efficiënt siRNA transfectie van vers geïsoleerde menselijke villous cytotrofoblasten behulp microporatie en het identificeren van DNA-eiwitcomplexen in deze cellen. Getransfecteerde cellen kunnen worden gecontroleerd door Western blot en EMSA analyses gedurende de 4 dagen cultuurtijd. 

Video: siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Developmental Biology

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Improving Ex Vivo Placental Explant Culture Using a Flow-Based Approach

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

Author Spotlight: Advancing Research Through Single Cell Sequencing and Spatial Histology in Placental Tissues 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Biology

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

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