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 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.
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.
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
2. siRNA Transfection of Human Primary Villous Cytotrophoblasts
3. Preparation of Lysates from Whole Cell Culture
4. Preparation of Nuclear Extracts from Cultured Cells
5. Sample Preparation and Electrophoresis
6. Transferring the Protein from the Gel to the Membrane
7. Antibody Staining
8. Radiolabeling of Single Stranded Oligonucleotide
9. Preparation of a Non-denaturing Polyacrylamide Gel for EMSA
10. DNA Binding Reaction
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 of primary villous cytotrophoblasts, analyzed by standard flow cytometry. Following the density gradient step, >97% cells (in this case, 99%) are positively stained for cytokeratin-7 in standard experiments, thereby demonstrating a very high degree of efficiency in the purification of this cell type.
After their isolation, human primary cytotrophoblasts can be directly used for transfection. Two different protocols were evaluated, i.e., microporation and lipid-based transfection. Both protocols were tested with siRNA specific to Syncytin-2 mRNA and compared to a scrambled siRNA (negative control). Transfection efficiency was next evaluated by Western blot analysis. Results confirmed that Syncytin-2 expression was significantly silenced in cytotrophoblast-derived extracts upon microporation (Figure 2). On the other hand, no significant silencing was noted when siRNAs were transfected by the lipid-based transfection reagent.
We also conducted EMSA experiments on freshly isolated cytotrophoblasts. A radiolabeled probe that originated from a Syncytin-2 promoter region was synthesized. The synthesized probe (termed WT; 5'-CTCTAGGAACACCTGACTGATAAGGGAAAAATGTC-3') has been previously shown to bind CREB-related transcription factors 7. In the presence of nuclear extracts from 24 and 48 hr cultured villous cytotrophoblasts, the Syncytin-2-promoter-derived probe showed the presence of two DNA-protein complexes. Addition of 100x cold WT oligonucleotide competed for the formation of the complex, while no similar competition with excess of a cold unrelated oligonucleotide (mouse Neural Crest Enhancer 2; 5′-GATCCTGTGATTTTCGTCTTGGGTGGTCTCCCTCG-3') was observed, confirming the specificity of both signals (Figure 3).
Figure 1. Evaluation of the purity of primary villous cytotrophoblasts through flow cytometry. Freshly isolated preparations of villous cytotrophoblasts were first permeabilized and then labeled with isotype control antibody (red line) or monoclonal antibody specific for cytokeratin-7 (black line). Cells were analyzed by flow cytometry. Please click here to view a larger version of this figure.
Figure 2. Comparison of siRNA transfection efficiencies between lipid-based transfection and microporation. Freshly isolated villous cytotrophoblasts were transfected with 300 ng of Syncytin-2-specific siRNA vs. a scrambled control siRNA using the lipid-based reagent or microporation. (A) Western blot analyses were performed on cellular extracts from each transfection condition using anti-Syncytin-2 and anti-GAPDH antibodies. (B) Syncytin-2 protein levels from Western blot analyses were quantified in terms of band intensity following normalization with corresponding GAPDH signals (Error bars are defined as SD, ***p<0.001). Please click here to view a larger version of this figure.
Figure 3. DNA-protein complexes identified by EMSA analysis. Nuclear extracts from primary villous cytotrophoblasts cultured for 24 or 48 hr were incubated with a WT probe corresponding to a promoter region of Syncytin-2 with or without excess (100x) of specific or nonspecific cold oligonucleotides. DNA-protein complexes were separated upon migration on a native gel. Specific signals and free probes are indicated on the right side of the gel. WT probe incubated in the absence of nuclear extract was also used as a negative control. CT NE = cytotrophoblast nuclear extracts. Please click here to view a larger version of this figure.
% of Percoll final | Volume of Percoll (90%) (ml) | Volume of HBSS (ml) |
70 | 2.33 | 0.67 |
65 | 2.17 | 0.83 |
60 | 2.00 | 1.00 |
55 | 1.83 | 1.17 |
50 | 1.67 | 1.33 |
45 | 1.50 | 1.50 |
40 | 1.33 | 1.67 |
35 | 1.17 | 1.83 |
30 | 1.00 | 2.00 |
25 | 0.83 | 2.17 |
20 | 0.67 | 2.33 |
15 | 0.50 | 2.50 |
10 | 0.33 | 2.67 |
5 | 0.17 | 2.83 |
Table 1. 5%-70% polyvinylpyrrolidone-coated silica gradient setting.
Buffer | Composition | Comments/Description |
PBS 1x | 0.9 mM CaCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2, 136.9 mM NaCl and 0.8 mM Na2HPO4 | |
RIPA Buffer | 150 mM NaCl, 1.0% NP-40 or 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulfate), 50 mM Tris-HCl pH 8.0 | |
Laemmli Sample Buffer | 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl, pH 6.8 | |
10x running buffer | 25 mM Tris base, 190 mM glycine, 0.1% SDS | |
10x transfer buffer | 25 mM Tris base, 192 mM Glycine | |
TBST 1x buffer | 10 mM Tris-HCl, 15 mM NaCl, 0.05% Tween 20 at pH 7 | Mix well and filter. Failure to filter can lead to “spotting” of the membrane. |
Blocking buffer | 5% milk or BSA (bovine serum albumin) added to TBST 1x buffer | |
TE Buffer | 10 mM Tris-HCl (pH 8.0), 1 mM EDTA | |
Annealing buffer | 1 M NaCl, 50 mM Tris-HCl pH 7.5, 100 mM MgCl2, 0,2 mM EDTA, 1 mM DTT | |
5x Gel Shift Binding Buffer | 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5) and 0.25 mg/ml poly(dI-dC). | |
Loading buffer | 250 mM Tris-HCl (pH 7.5), 0.2% bromophenol blue and 40% glycerol | |
TBE 5x | Dissolve 54 g of Tris Base and 27.5 g of boric acid in 1 L deionized water, which includes 20 ml 0.5 M EDTA, pH 8 | |
4% native gel | TBE 5x buffer | 6.0 ml |
29:1 acrylamide/bisacrylamide (30% w/v) | 10 ml | |
40% acrylamide (w/v) | 0.75 ml | |
100% glycerol | 1.5 ml | |
Distilled water | 41.5 ml | |
Ammonium persulfate 25% | 250 ml | |
TEMED | 75 ml | |
4% Stacking Gel (6 ml) for SDS-PAGE | ddH2O | 4.1 ml |
30% Acrylamide | 1.0 ml | |
1.0 M Tris | 0.75 ml | |
10% SDS | 60 μl | |
10% APS | 60 μl | |
TEMED | 6 μl | |
12% Separating Gel (10 ml) for SDS-PAGE | ddH2O | 3.3 ml |
30% Acrylamide | 4.0 ml | |
1.5 M Tris | 2.5 ml | |
10% SDS | 100 μl | |
10% APS | 100 μl | |
TEMED | 4 μl |
Table 2. Composition of buffers.
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 primary villous cytotrophoblasts can be obtained. CK-7 is an intermediate filament protein, which is expressed in cytotrophoblasts. Monoclonal antibodies against this protein are used to determine the purity of this trophoblast population after their isolation from the placenta 10. Preparations are defined to be pure when 96% of cells are positive for CK-7. Cells can subsequently be used directly for transfection. Transfection protocols involving lipid-based approaches are broadly used to transfer nucleic acids into primary cells 11,12. However, toxicity of lipid formulation in some cells becomes a disadvantage. Microporation is an electroporation technology using a pipette tip as an electroporation space, which generates minimal heat, metal ion dissolution, pH variation and oxide formation; all of which can be deleterious to cells. Based on the data presented herein, microporation represents a more efficient approach for siRNA delivery than the lipid-based transfection protocol, as judged by Western blot analyses. Using an EMSA approach, a probe designed against a selected region of the Syncytin-2 promoter that is known to bind important factors for its activity 7 was tested. The probe was incubated with nuclear extract from isolated cytotrophoblasts. As presented in Figure 3, specific signals were obtained.
The different protocols described herein have been optimized in recent years. However, they all rely on high quality cytotrophoblast preparations, which depend on a certain number of critical steps. First and foremost, following birth, placentas need to be kept in a buffer solution and cells must be isolated from them no more than 5 hours after being immersed in the solution. Trypsin and DNase treatments are also of great importance and should be carefully undertaken to maximize the quality of cytotrophoblast preparation. Extensive scrubbing of the placenta villi and reduction of the time needed for their isolation are both additional improvements, which can strongly upgrade the efficiency of cell number isolation. The yield of this protocol further depends on optimal centrifugation during the density gradient step, which should be closely monitored. Failure to properly balance the tubes will also lead to distortion of the gradient and severely reduce cell numbers.
The transfection protocol described herein has been tested for over several years. Although conditions needed for microporation are generally straightforward, optimization of transfection for the various primary cells are nonetheless required. This also involves the optimization of the amount of transfected nucleic acid (siRNA or plasmid DNA). Thus, for the protocol described here, optimization of various siRNAs concentrations is recommended to identify an efficiently repressing siRNA. Addition of an appropriate suspension volume to the cell pellet before microporation and complete suspension of the cells are additional factors, which affect transfection efficiency.
EMSA experiments have also necessitated optimization. Given the heterogeneity associated with placenta donors and the varying cytotrophoblast isolation efficiencies and purities, EMSA experiments need to be repeated up to 5 times to confirm the results. In addition, having high cell numbers is important to ensure sufficient protein levels in nuclear extract preparations and subsequent detection of protein/ DNA complexes. Furthermore, highly radioactive oligonucleotide probes should be prepared, and should be prepared fresh from recently purchased radioactive ATP.
A series of limitations must be underscored in the presented protocol. It first needs to be stressed that flow cytometry analyses cannot assess the potential presence of syncytiotrophoblast fragments in cell preparations. Other protocols have been proposed to solve this issue, such as sorting using flow cytometry or the use of antibody-bound magnetic beads 13,14. It should be mentioned though that these fragments do not normally adhere to cell culture wells and are often removed following the cell washing step. Another limitation to the presented protocol is that primary villous cytotrophoblasts have a very limited survival time in cell culture. Hence, regardless of the selected method, stable transfection of villous cytotrophoblasts will remain unattainable. EMSA analyses, as described in this protocol, also have certain limitations. Although specificity of the signals can easily be assessed by competition experiments with excess unlabeled oligonucleotides, bound factors can only be identified using antibodies against specific transcription factors resulting in supershifted signals. EMSA experiments furthermore remain limited as it studies in vitro DNA-protein interactions. Experiments with more representative settings, such as ChIP assay, and more recent in vivo protocols are needed to further confirm the EMSA result.
The protocols described herein for cytotrophoblast isolation and transfection have important applications for studies in which transient silencing or overexpression of specific genes is needed to understand their role in function, proliferation or differentiation of a specific trophoblast cell type. In addition, upon transfection, tagged versions of expressed proteins will be suitable for intracellular tracking and analyses by standard techniques, such as confocal microscopy and flow cytometry. The EMSA protocol can be used to study intricate details pertaining to promoter regulation and the implicated transcription factors. Furthermore, this experimental approach will allow researchers to identify factors involved in the development of certain diseases related to placentation deficiency and the altered regulation of genes expressed in villous cytotrophoblasts.
In conclusion, human primary cytotrophoblasts can be easily isolated from placentas and are amenable to standard protocols, such as siRNA transfection and EMSA. Although different transfection procedures, such as lipofection 15 and nucleofection 16 are available, microporation seems a very efficient method for siRNA transfection of isolated villous cytotrophoblasts, offering an approach with limited cell death and relatively low cost. In the context of villous cytotrophoblasts, this approach should permit the silencing of different genes and the assessment of their implication in various functions or characteristics of the cell type. In addition, transfection of expression vectors is also feasible using this protocol and will yield complementary data to the siRNA-based approach. With respect to EMSA, this technique provides a more rapid and simpler method of evaluating protein-DNA complexes when compared to alternative approaches, such as DNase I footprinting, methylation interference assay, chromatin immunoprecipitation and chromatin conformation analyses. Hence, this technique remains valuable in standard promoter analyses or testing of any other enhancer/suppressor-containing DNA regions. Our demonstration of its reliable use for villous cytotrophoblasts is important, as it adds information on the regulation of genes, with potential implications at the functional level. Despite their limited time in culture, primary villous cytotrophoblasts are suitable for standard studies and should be adaptable to more recent as well as informative methods.
The authors have nothing to disclose.
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.
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 | Dilution1:200 |
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 |