Summary

As análises siRNA transfecção e EMSA sobre recentemente isolados das vilosidades citotrofoblastos Humanos

Published: September 20, 2016
doi:

Summary

Este protocolo descreve um método para eficazmente transfectar siARN em citotrofoblastos vilosidades humanos isolados de fresco utilizando formação de microporos e identificação de complexos de ADN-proteína nestas células. As células transfectadas pode ser monitorado por análises Western blot e EMSA durante o tempo de cultura de 4 dias.

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.

Protocol

O comitê de ética UQAM aprovou estes protocolos, que estão em conformidade com as orientações do comitê de ética de St-Luc Hospital do Centre Hospitalier Universitaire de Montreal (Montreal, Canadá). Os participantes assinaram um termo de consentimento informado. 1. Médio Preparação e Isolamento de primárias das vilosidades citotrofoblastos Prepare meio de cultura para citotrofoblastos vilosidades primárias humanas, completando meio de Dulbecco modificado de Eagle (DMEM), com HEPES…

Representative Results

placentas frescos de gestação a termo foram usados ​​para isolar citotrofoblastos vilosidades primárias humanos para conduzir o conjunto de experimentos apresentados na seção Protocolo. Seguindo o seu isolamento, analisou-se primeiro a pureza de citotrofoblastos através da utilização do marcador citoqueratina-7 (Figura 1). Preparações celulares foram assim coradas utilizando um anticorpo monoclonal anti-citoqueratina-7. A Figura 1 represent…

Discussion

Estudos sobre a função da placenta humana e desenvolvimento têm sido muito melhorada através de protocolos destinados a optimizar o isolamento de várias populações de células da placenta. Um dos melhores população de células da placenta estudado continua a ser o citotrofoblastos vilosidades, o estudo de que muito tem beneficiado de protocolos otimizados que permitem o isolamento eficiente e confiável. Isto permitiu ainda um certo número de experiências, tais como estudos de transfecção e promotor. Usando…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Este trabalho foi apoiado por uma bolsa do Conselho Nacional de Ciências e Pesquisa de Engenharia do Canadá (NSERC) (# 298527) (BB). CT foi apoiado por uma bolsa FARE institucional. AV foi apoiado por um Ph.D. NSERC Graham Bell Bolsa. BB realizou uma Research Chair Canadá em Retrovirology Humano (Tier 2). Graças a Beatrix Beisner para ajudar na revisão do texto.

Materials

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
MgSO4.7H2 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

References

  1. Huppertz, B. Placental origins of preeclampsia: challenging the current hypothesis. Hypertension. 51, 970-975 (2008).
  2. Huppertz, B. IFPA Award in Placentology Lecture: Biology of the placental syncytiotrophoblast–myths and facts. Placenta. 31, S75-S81 (2010).
  3. Le Bellego, F., Vaillancourt, C., Lafond, J. Isolation and culture of term human cytotrophoblast cells and in vitro methods for studying human cytotrophoblast cells’ calcium uptake. Methods Mol. Biol. 550, 73-87 (2009).
  4. Morrish, D. W., et al. In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium. Placenta. 18, 577-585 (1997).
  5. Forbes, K., Desforges, M., Garside, R., Aplin, J. D., Westwood, M. Methods for siRNA-mediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines. Placenta. 30, 124-129 (2009).
  6. Vargas, A., et al. Syncytin-2 plays an important role in the fusion of human trophoblast cells. J. Mol. Biol. 392, 301-318 (2009).
  7. Toufaily, C., Lokossou, A. G., Vargas, A., Rassart, E., Barbeau, B. A CRE/AP-1-Like Motif Is Essential for Induced Syncytin-2 Expression and Fusion in Human Trophoblast-Like Model. PLoS One. 10, e0121468 (2015).
  8. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976).
  9. Smith, P. K., et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85 (1985).
  10. Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta. 21, 733-741 (2000).
  11. Desforges, M., et al. The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane. J. Physiol. 587, 61-72 (2009).
  12. Felgner, P. L., et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 84, 7413-7417 (1987).
  13. Guilbert, L. J., et al. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments. Placenta. 23, 175-183 (2002).
  14. Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. Isolation and culture of term human trophoblast cells. Methods Mol. Med. 121, 203-217 (2006).
  15. Ma, B., Zhang, S., Jiang, H., Zhao, B., Lv, H. Lipoplex morphologies and their influences on transfection efficiency in gene delivery. J. Control. Release. 123, 184-194 (2007).
  16. Freeley, M., Long, A. Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection. Biochem. J. 455, 133-147 (2013).

Play Video

Cite This Article
Lokossou, A. G., Toufaily, C., Vargas, A., Barbeau, B. siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts. J. Vis. Exp. (115), e53995, doi:10.3791/53995 (2016).

View Video