siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

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 Montréal (Montréal, Canada). Participants signed an informed consent form.

1. Medium Preparation and Isolation of Primary Villous Cytotrophoblasts

1. Prepare culture medium for human primary villous cytotrophoblasts by supplementing Dulbecco’s Modified Eagle’s Medium (DMEM) with 25 mM HEPES, 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Filter the prepared medium through sterile 0.22 µm filter and store in a -20 °C freezer in aliquots of 50 ml.
2. Isolate primary villous cytotrophoblasts from fresh placentas following standard protocols ³.
   1. 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.
      1. Holding each cube with pliers, carefully remove the villous tissue from remaining vessels and fibrous tissue using scissors.
      2. For 15 to 30 mg of villous tissue, digest four times in Hank'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 µg/ml) for 30 min at 37 °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.
   2. 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.
   3. 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.
   4. Count the cells using a hemocytometer. Briefly, mix cells and add 10 µl in a new microcentrifuge tube. Add 10 µl of trypan blue and mix gently again. Draw up the cell suspension and fill the hemocytometer chambers. Count the cells under 10X objective.
   5. 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 °C and 5% CO₂ for a maximum of 4 days for other experiments.
3. Evaluation of the purity of isolated primary villous cytotrophoblasts
   1. 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.
   2. Add 1 ml of cold methanol to cells and incubate at -20 °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.
   3. Add 1 ml of PBS at room temperature to rehydrate cells, centrifuge at 300 x g and remove PBS by aspiration.
   4. 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.
   5. Wash twice with 500 µl of room temperature PBS, centrifuge cells at 300 x g for 5 min and remove PBS by aspiration. Resuspend cells in 100 µl room temperature PBS.
   6. 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.
   7. 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.

2. siRNA Transfection of Human Primary Villous Cytotrophoblasts

1. Transfection of human primary villous cytotrophoblasts using a microporation device
   1. Prepare 6-well plates containing 2 ml of supplemented DMEM medium for the incubation of microporated cells.
   2. After isolation of primary cytotrophoblasts (see section 1.2), take an aliquot of cells in suspension and determine cell number using a hemocytometer.
   3. 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's PBS (DPBS) (Mg²⁺, Ca²⁺-free) and pellet the cells by centrifugation at 300 x g for 5 min at room temperature.
   4. Remove the supernatant and resuspend the cells gently in 100 µl of resuspension buffer.
      Note: Avoid keeping cell suspension for longer than 15-30 min at room temperature, to maximize cell viability and transfection efficiency.
   5. 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 µl 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.
   6. Immediately transfer the cells to 6-well plates (prepared in step 2.1.1) containing 37 °C pre-warmed supplemented medium to avoid cell damage.
   7. Repeat steps 2.1.5 and 2.1.6 for the remaining samples.
   8. Gently rock the plate for 30 sec to ensure even distribution of the cells. Incubate the plate at 37 °C in a humidified CO₂ incubator for 24 to 48 hr. Refresh culture medium 24 hr after transfection.
   9. Evaluate transfection efficiency by Western blot analysis (see steps 3 to 6).
2. Lipofection of siRNA in human primary cytotrophoblasts
   1. 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.
   2. Dilute 300 ng of Syncytin-2-specific siRNA (or control-scrambled siRNA) and 5 µl of lipid-based transfection reagent in separate 1.5 ml tubes in a total volume of 10 µl of antibiotic- and serum-free medium. Incubate for 5 min.
   3. Mix the diluted reagents and incubate for an additional 20 min at room temperature to form the transfection complex. Add 90 µl of antibiotic- and serum-free medium to the mix to a final volume of 100 µl.
   4. Remove culture medium from the 6-well plates (step 2.2.1). Add 2 ml of fresh medium and the 100 µl transfection mix to each well. Incubate the cells at 37 °C in a CO₂ incubator for 24 to 48 hr. Refresh culture medium 24 hr after transfection.
   5. Evaluate transfection efficiency by Western blot analysis (see steps 3 to 6).

3. Preparation of Lysates from Whole Cell Culture

1. 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 µl of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA). Incubate for 1-3 min at 37 °C in a CO₂ incubator. Stop trypsin treatment by adding 500 µl 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.
2. 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.
3. Carefully remove supernatant without disturbing the pellet. Place the tube on ice. Resuspend the pellet in 50-200 µl 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.
4. Spin at 16,000 x g for 20 min at 4 °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.

4. Preparation of Nuclear Extracts from Cultured Cells

1. 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 µl of 0.25% trypsin/EDTA. Incubate for 1-3 min at 37 °C in a CO₂ incubator and stop trypsin treatment by adding 500 µl of serum containing growth media.
2. 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.
3. Extract nuclear protein according to manufacturer's instructions. Determine protein concentration of each sample using protein quantification assay (Bradford or bicinchoninic acid assay (BCA)).

5. Sample Preparation and Electrophoresis

1. Determine the protein concentration for each cell extract using a protein quantification assay (for example, Bradford ⁸ or BCA ⁹).
2. As per manufacturer'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 µg of total protein.
3. Boil cell lysates at 100 °C for 5 min. Aliquot 50-100 µl of lysate to avoid repeated freeze/thaw cycles and store the remaining tubes at -20 °C for future use.
4. While the samples are heating, prepare 1 L of 1x running buffer (Table 2).
5. Spin the samples at 16,000 x g for a few seconds and place them on ice.
6. 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.

6. Transferring the Protein from the Gel to the Membrane

1. Activate polyvinylidene fluoride (PVDF) membrane with methanol for 1 min and rinse with 1x transfer buffer/10% methanol solution. According to manufacturer'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.

7. Antibody Staining

1. Block the membrane for 1 hr at room temperature or overnight at 4 °C using 5% blocking solution.
2. Incubate the membrane with anti-Syncytin-2 antibodies (4 µg/ml; 1:5,000) ⁶ or with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 0.4 µg/ml; 1:500) in 5% blocking solution overnight at 4 °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.
3. 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.

8. Radiolabeling of Single Stranded Oligonucleotide

1. Phosphorylation reaction
   1. 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 µCi (γ-³²P) ATP and make up the reaction volume to 20 µl with nuclease-free water.
   2. Incubate at 37 °C for 30 min. Stop the reaction by adding 5 µl of 0.5 M EDTA. Make up the volume to 50 µl with nuclease-free water.
2. Removal of unincorporated nucleotides from oligonucleotides
   1. Prepare a G-25 column equilibrated with TE buffer (Table 2) following manufacturer's instructions. Place the column in a fresh DNase-free 1.5 ml microcentrifuge tube.
   2. Slowly add 50 µl 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 µl of nuclease-free water and spin for 2 min at 350 x g to collect it in the microcentrifuge tube.
3. Hybridization with the complementary strand oligonucleotide
   1. Transfer the radiolabeled probe (86 µl) 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 °C and leave overnight at room temperature for cooling.
   2. 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 µl with nuclease-free water. Heat and keep at room temperature as described in step 8.3.1.

9. Preparation of a Non-denaturing Polyacrylamide Gel for EMSA

1. 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.
2. Immediately pour the acrylamide solution in between the plates and allow polymerization to proceed (approximately 30 min).
3. 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.

10. DNA Binding Reaction

1. In a 1.5 ml microcentrifuge tube, add 10 µg of nuclear extract (step 4.3) in 1x Gel Shift Binding buffer (Table 2) and make up the final volume to 10 µl with nuclease-free water. As a control, prepare a tube with no nuclear extract. For the competition reaction, add 1 µl of cold non-specific or specific double-stranded oligonucleotide (step 8.3.2).
2. Add 1 µl of radioactive probe from step 8.3.1 to each reaction. Incubate the reaction at room temperature for 20 min. Add 1 µl of 10x gel loading buffer per reaction and load each sample on the gel prepared in section 9.
3. 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 °C for 24 hr.
4. Remove the screen from the exposure cassette and insert in the phosphor imaging device for scanning.

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Results

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...

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
HBSS without  Ca2+, Mg2+	Sigma	#H2387
HBSS (10x)	Sigma	#14060-057
DMEM High Glucose without Hepes	Gibco	#12100-061
Hepes (1 M)	Gibco	#15630-080
Penicillin-Streptomycin-Neomycin (100x)	Gibco	#15640-055
Amphotericin B	Sigma	#A2411
CaCl2	Sigma	#C4901
MgSO4.7H2O	Sigma	#M
Fetal bovine serum	Gibco	#16170-078
Percoll	Sigma	#P-1644	For density gradient
Syncytin-2 siRNA	Ambion Life technologies	#AM16708
Scrambled siRNA	Qiagen	# SI03650318
DNase I	Sigma-Aldrich	#D5025
Trypsine, type I	Sigma	#T8003
DharmaFECT Lipotransfection  reagents	GEhealthcare	# T-2001-01
Trypsin/EDTA	Life technologies	#25300-062
Protease Inhibitor Cocktail	Roche Diagnostic	#11873580001
Pierce BCA Protein Assay Kit	Thermo Scientific	#23225
BSA	Sigma	#A7906
TWEEN 20	Sigma	#P9416
Anti-rabbit IgG, HRP-linked antibody	Cell Signaling	#7074
BM Chemiluminescence Western Blotting Substrate (POD)	Roche Diagnostic	#11500708001
DPBS	Life technologies	#14287-080
T4 Polynucleotide Kinase	NEB	#M0201S
ATP, [γ-32P]	Perkin Elmer	#BLU002A100UC
Acrylmide	Sigma	#A9099
TEMED	Life technologies	#17919
Ammonium Persulfate	Sigma	#A3678
Anti-human cytokeratin-7 antibody clone LP5K, FITC conjugated	Millipore	CBL194F	Dilution 1:200
FcR blocking reagent	Miltenyi Biotec	130- 059-901	Dilution 1:10
Flow Cytometer BD Acuri system	Becton Dickinson
Microporator MP-100 apparatus	Digital Bio
Resuspension Buffer R (Neon Transfection System 100 µl Kit)	Life technologies	MPK10096
PVDF membrane	Millipore	IPVH00010	Activate with methanol
Anti-human GAPDH antibody	Santa Cruz Biotechnology	sc-137179	1:500
HorseRadish Peroxidase (HRP)-conjugated goat anti-rabbit antibody or anti-mouse antibody	Cell Signalling	#7074	1:10,000
HorseRadish Peroxidase (HRP)-conjugated goat anti-mouse antibody	Cell Signalling	#7076	1:10,000
NE-PER Nuclear and Cytoplasmic Extraction Reagent	Thermo Scientific	#78833
G-25 column	GE Healthcare	#27-5325-01
Chemiluminsescence and fluorescence imaging device	Montréal Biotech	Fusion FX5
4% native gel	Home made
PBS	Home made		1x
Personal Molecular Imager (PMI) System	BioRad