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Abstract
Introduction
Protocol
Results
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Developmental Biology

Umana Trophoblast primaria coltura cellulare modello per studiare gli effetti protettivi della melatonina contro ipossia / riossigenazione Disruption-indotta

Published: July 30, 2016
doi:
10.3791/54228
, , , , , , , , ,
1INRS-Institut Armand-Frappier

Summary

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.

Abstract

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.

Introduction

Nel corso della gravidanza umana, le cellule placentari citotrofoblasto, che sono le cellule staminali mononucleate, rapidamente proliferano e si differenziano in cellule sia citotrofoblasto villi o extravilloso. citotrofoblasti extravilloso invadono e rimodellano le arterie a spirale della parete uterina. Cytotrophoblasts villi, invece, continuano a proliferare, differenziare e fusibile per formare syncytiotrophoblast multinucleate (il sincizio) 1. Il mantenimento della omeostasi dei villi trofoblasto è essenziale per il benessere fetale e gravidanza sana. In realtà, trophoblasts villi consentono lo scambio materno-fetale di ossigeno e nutrienti, e producono ormoni essenziali per la gravidanza. Inoltre, il syncytiotrophoblast è l'unico tipo cellulare in diretto contatto con la circolazione del sangue materno e fornisce una barriera fisica e immunologica essenziale. Pertanto, il sinciziotrofoblasto deve subire apoptosi e la sostituzione per la manutenzione omeostatico e di AVOid placentare patologie 2-5.

La tecnica sviluppata da Kliman et al. 6 nel 1986 per isolare citotrofoblasti villi primari da placenta umana ha causato una rivoluzione nel campo della ricerca placentare, consentendo lo studio dei meccanismi molecolari coinvolti nella differenziazione dei villi trofoblasto. Questa tecnica classica, sulla base di digestioni enzimatiche sequenziali con tripsina e DNasi, seguita da isolamento nei media densità centrifugazione (particelle di silice colloidale rivestito da polivinilpirrolidone, o Percoll) è ormai riconosciuto come il gold standard per isolare le cellule citotrofoblasto villi. La tecnica può essere ottimizzata immunopurificazione magnetica, una procedura che separa cytotrophoblasts villi da cellule non trofoblastiche basati sull'espressione differenziale di antigeni specifici sulla superficie di queste cellule. Abbiamo scelto l'antigene leucocitario umano ABC (HLA-ABC) per l'assenza della sua espressione sulla membran cellulare trofoblasticae 7,8.

La placenta è un organo che subisce drammatiche variazioni nei livelli di ossigeno durante la gravidanza. Nel primo trimestre, il rapporto di ossigenazione è fisiologicamente molto bassa (2% O 2), ma aumenta a livelli lievi di ossigenazione (8% O 2) nel secondo e terzo trimestre. Tuuli et al. 9 descritto che la riproduzione in vitro dell'ambiente trofoblasto all'interno del villi della placenta è una sfida e le variazioni dei livelli di ossigenazione può anche portare a cambiamenti fenotipici. È pertanto suggerito di adottare dell'8% ossigeno come normossia per imitare la tensione di ossigeno trovato in villi placentari durante il terzo trimestre di gestazione 8,9. Chen et al. 10 ampiamente studiato diverse variabili legate alla tensione di ossigeno nelle cellule in coltura trofoblasto e dimostrato l'importanza di determinare i livelli di ossigeno in un ambiente pericellulare. I livelli di ossigeno nei villi tendono ad aumentarea causa di vasculogenesi. Il flusso di sangue placentare villi aumenta costantemente e il livello di perossido di idrogeno (un'abbondante specie reattive) è un segnale importante che controlla vasculogenesi 11,12. In complicazioni della gravidanza, la mancanza di vasculogenesi genera ipossia, e ancora più importante, le variazioni intermittenti di ossigenazione (chiamato ipossia / riossigenazione). Queste condizioni portano ad un aumento anomalo stress ossidativo, che compromette placentare e fetale 13,14 vitalità. Le alterazioni che le cellule del trofoblasto subiscono in vivo durante gli episodi di ipossia / riossigenazione possono essere imitate in vitro come segue: citotrofoblasti villi sono mantenute in condizioni di normossia (8% O 2) fino a quando non si differenziano in sinciziotrofoblasto. Essi vengono quindi sottoposti a condizioni di ipossia (0,5% O 2) per 4 ore, seguito da un ulteriore 18 ore di normossia (riossigenazione). Usando questo approccio ipossia / riossigenazione, trofoblasto exvietare liberalizzato stato redox e aumento dei livelli di apoptosi intrinseca 8, come è stato osservato in alcune complicazioni durante la gravidanza. Quindi, questo è un utile modello in vitro per valutare nuovi approcci preventivi e terapeutici per combattere le complicazioni della gravidanza associate a ipossia placentare / riossigenazione.

Cellule placentari producono melatonina, che ha diverse funzioni importanti, come la capacità di ovviare stress ossidativo e disfunzione placentare 15. Qui, presentiamo i modelli di approccio e cellulari sperimentali utilizzate per dimostrare gli effetti protettivi della melatonina in cellule del trofoblasto placentare a livello molecolare, cellulare e funzionale 8.

Protocol

Placente sono stati ottenuti immediatamente dopo parto vaginale spontaneo da gravidanze complicate al CHUM-St-Luc Hospital, Montreal, QC, Canada, con consenso informato del paziente e l'approvazione dei comitati etici (CHUM-St-Luc Hospital e INRS-Institut Armand-Frappier, Laval, QC, Canada). 1. isolamento e la purificazione delle cellule dei villi citotrofoblasto Soluzioni e supporti Preparare mezzi di trasporto, completandolo Dulbecco Modified Eagle Medio Alto-glucosio (DMEM-HG) con 1% <…

Representative Results

Isolamento e immunopurificazione di cellule citotrofoblasto villi da una normale placenta termine ottenuti da parto vaginale ha prodotto 1 x 10 8 cellule vitali. La placenta pesava 350 g, era di 19 cm di diametro, 4 cm di altezza con forma discoidale e le membrane trasparenti. è stata rilevata alcuna cotyledon malformazione. Il cordone ombelicale aveva localizzazione paracentrale e una lunghezza di 56 cm. La purezza è stata valutata mediante citometria a flusso utilizzando v…

Discussion

Nei mammiferi, lo sviluppo fetale è direttamente dipendente adeguata funzione placentare. Le origini dello sviluppo di disturbi di salute sono basate sull'ipotesi che la causa delle malattie che si manifestano più tardi nella vita può essere fatta risalire al primo sviluppo e che la placenta ha un ruolo meccanicistico nella programmazione fetale 30-32. La placenta è il mediatore chiave della crescita e lo sviluppo fetale: regola il trasferimento dei nutrienti, protegge contro le esposizioni dannose, e…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (no. 262011-2009) to CV and March of Dimes Social and Behavioral Sciences Research grant (#12-FY12-179) to CV and JTS; by studentships to LSF from the Ministère de l’éducation, de l’Enseignement supérieurs et de la recherche (MEESR)-Fonds de recherche du Québec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the Réseau Québécois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Santé, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cientìfico e Tecnològico (CNPq) and the Programme de bourses d’excellence pour étudiants étrangers MEESR-FRQNT.

Materials

Curved Metzenbaum Scissors Shandon 9212 surgical equipment (cell isolation) (2 units)
Splinter Forceps Fine 41/2in Fisherbrand 13-812-42 surgical equipment (cell isolation) (2 units)
Scissors 4.5 Str Dissection Fisherbrand 08-940 surgical equipment (cell isolation) (2 units)
Gauze Sponge 10cm X 10cm Cardinal Health 361020733
Oblong Glass Baking Dish Pyrex 1105397 Glassware (2.8L)
Funnel Buchner  Coorstek Inc 10-356E Glassware (114MM DIAMeter)
Watch Glass  pyrex 9985100EMD Glassware
Formalin solution, neutral buffered, 10% Sigma-Aldrich HT501128-4L histological tissue fixative solution
Trypsinizing Flasks Wheaton 355395 Glassware (1 unit)
Disposable Culture Tubes Kimble 73750-13100 Glassware
Borosilicate Glass Pasteur Pipet (22.8 Cm)  Fisherbrand K63B1367820C Glassware
250 Ml Glass Beakers  Fisherbrand KFS14005250 Glassware
Glass Media Bottles With Cap Fisherbrand KFS14395250 Glassware (8 units)
50 Ml Corex Tube  Corning 8422-A (1 unit)
15 Ml Polystyrene Centrifuge Tube Corning 430791
50 Ml Polystyrene Centrifuge Tube Corning 430829
10ml Serological Pipet Corning 11415038
Cell Strainer 100μm Nylon Corning 431752
Absorbant Liner Scienceware 1199918
500 Ml Bottles Top Filter  Corning Pore: 0,22 µm / medium and HBSS preparation
2 Ml Criogenic Vials Corning 430488
Freezing Container, Nalgene Mr. Frosty Sigma-Aldrich C1562-1EA
Peristaltic Pump Pharmacia Fine Chemicals P3 model
Shaking Water Bath Fisher Model 127
Vacuum Pump ABM 4EKFS6CX-4
Sodium Chloride Fisherbrand EC231-598-3 Saline solution 0.9%
Hank’s Buffered Salt Solution (Hbss) Sigma-Aldrich H2387 Quantity: 9.25 (one vial) for 1L of digestion solution
Hydroxypiperazineethansulphonic Acid (Hepes) Life Technologies 15630-080 25mL (1M) for 1L of digestion solution
Trypsin Type I Sigma-Aldrich T8003 9,888U
Deoxyribonuclease Type Iv Roche 10-104-159-001 402,000U
Calcium Chloride Sigma-Aldrich C4901 100mM
Magnesium Sulfate Baker 2500-01 800mM
Dulbecco’s Modified Eagle Medium High Glucose (Dmem) Life Technologies 10564-045
Penicillin/Streptomycin Sulphate Hyclone SV30010
Fetal Bovine Serum Corning 35-010-CV
Percoll Sigma-Aldrich P1644  Density centrifugation media gradient. Volume: 36mL
Isopropanol Acros 42383-0010 50mL
Dimethyl Sulfoxide Sigma-Aldrich 472301
Automacs Magnetic Separator  Miltenyi Biotec Model 003
Automacs Columns  Miltenyi Biotec 130-021-101
Automacs Running Buffer  Miltenyi Biotec 130-091-221 http://www.miltenyibiotec.com/~/media/Images/Products/Import/0001100/IM0001131.ashx?force=1
Automacs Rinsing Solution  Miltenyi Biotec 130-091-222 http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-buffers/automacs-rinsing-solution.aspx
Anti-Human Hla Abc Purified Clone W6/32 Affymetrix eBioscience 14-9983-82 anti-mouse antibody
Anti Mouse Igg Microbeads Miltenyi Biotec 130048401
Multiple Well Plate -  6 Well With Lid Corning 3335 Cell Bind surface
Multiple Well Plate -  24 Well With Lid Corning 3337 Cell Bind surface
Multiple Well Plate -  96 Well With Lid Corning 3300 Cell Bind surface
Modular Incubator Chamber  Billups-Rothenberg MIC-101 A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions
Single Flow Meter Billups-Rothenberg SFM3001
50 Mm In-Line Filter  Whatman 6721-5010 PTFE, pore: 1.0 µm
Gas Regulator Pro Star PRS301233 A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions
Gas Hose Class Vi Clear 5/16  Parker 100-05070102 3 pieces with ~ 0.5 m
17 Mm Adjustable Gas Hose Clamp Tiewraps THCSS-16
Normoxia Gas Cylinder  Praxair NI CDOXR1U-K Size K (3rd trimester‘s composition: 5% CO2, 8% O2, Bal. N2)
Normoxia Gas Cylinder  Praxair NI CDOXR1U-K Size K (3rd trimester‘s composition: 5% CO2, 0.5% O2, Bal. N2)
Oxygen Microelectrode Mi-730 Microelectrodes INC 84477
Oxygen Adapter Microelectrodes INC 3572
ROS Detection Reagent: CM-H2DCFDA  Invitrogen C-400
β-hCG ELISA kit  DRG internatinal EIA-4115
Anti-Vimentin ourified antibody eBioscience 14-9897 Host: mouse
Anti-Cytokeratin 7 (FITC) antibody  Abcam ab119697 Host: mouse
Alexa Fluor 488 Goat Anti-mousse IgG H&L antibody Life Technologies A-11029
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References

  1. Vaillancourt, C., Lanoix, D., Le Bellego, F., Daoud, G., Lafond, J. Involvement of MAPK signalling in human villous trophoblast differentiation. Mini Rev Med Chem. 9 (8), 962-973 (2009).
  2. Gauster, M., Moser, G., Orendi, K., Huppertz, B. Factors involved in regulating trophoblast fusion: potential role in the development of preeclampsia. Placenta. 30, 49-54 (2009).
  3. Huppertz, B., Kadyrov, M., Kingdom, J. C. Apoptosis and its role in the trophoblast. Am J Obstet Gynecol. 195 (1), 29-39 (2006).
  4. Lanoix, D., Lacasse, A. A., Reiter, R. J., Vaillancourt, C. Melatonin: the smart killer: the human trophoblast as a model. Mol Cell Endocrinol. 348 (1), 1-11 (2012).
  5. Huppertz, B., Frank, H. G., Reister, F., Kingdom, J., Korr, H., Kaufmann, P. Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous cytotrophoblast and syncytial fragments in vitro. Lab Invest. 79 (12), 1687-1702 (1999).
  6. Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F., 3rd, Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology. 118 (4), 1567-1582 (1986).
  7. Lanoix, D., Beghdadi, H., Lafond, J., Vaillancourt, C. Human placental trophoblasts synthesize melatonin and express its receptors. J Pineal Res. 45 (1), 50-60 (2008).
  8. Lanoix, D., Lacasse, A. A., Reiter, R. J., Vaillancourt, C. Melatonin: The watchdog of villous trophoblast homeostasis against hypoxia/reoxygenation-induced oxidative stress and apoptosis. Mol Cell Endocrinol. 381 (1-2), 35-45 (2013).
  9. Tuuli, M. G., Longtine, M. S., Nelson, D. M. Review: Oxygen and trophoblast biology–a source of controversy. Placenta. 32, 109-118 (2011).
  10. Chen, B., Longtine, M. S., Nelson, D. M. Pericellular oxygen concentration of cultured primary human trophoblasts. Placenta. 34 (2), 106-109 (2013).
  11. Roberts, J. M., Hubel, C. A. Is oxidative stress the link in the two-stage model of pre-eclampsia. Lancet. 354 (9181), 788-789 (1999).
  12. Burton, G. J., Jauniaux, E. Oxidative stress. Best Pract Res Clin Obstet Gynaecol. 25 (3), 287-299 (2011).
  13. Ji, L., Brkic, J., Liu, M., Fu, G., Peng, C., Wang, Y. L. Placental trophoblast cell differentiation: Physiological regulation and pathological relevance to preeclampsia. Mol Aspects Med. 34 (5), 981-1023 (2013).
  14. Redman, C. W., Sargent, I. L. Placental stress and pre-eclampsia: a revised view. Placenta. 30, 38-42 (2009).
  15. Sagrillo-Fagundes, L., Soliman, A., Vaillancourt, C. Maternal and placental melatonin: actions and implication for successful pregnancies. Minerva Ginecol. 66 (3), 251-266 (2014).
  16. Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta. 21 (7), 733-741 (2000).
  17. Potgens, A. J., Gaus, G., Frank, H. G., Kaufmann, P. Characterization of trophoblast cell isolations by a modified flow cytometry assay. Placenta. 22 (2-3), 251-255 (2001).
  18. 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).
  19. Maldonado-Estrada, J., Menu, E., Roques, P., Barre-Sinoussi, F., Chaouat, G. Evaluation of Cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry. J Immunol Methods. 286 (1-2), 21-34 (2004).
  20. 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).
  21. Mounier, C., Barbeau, B., Vaillancourt, C., Lafond, J. Endocrinology and cell signaling in human villous trophoblast. Methods Mol Biol. 550, 89-102 (2009).
  22. Chen, B., et al. Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts. Am J Physiol Endocrinol Metab. 302 (9), 1142-1152 (2012).
  23. Reti, N. G., et al. Effect of high oxygen on placental function in short-term explant cultures. Cell Tissue Res. 328 (3), 607-616 (2007).
  24. Pidoux, G., et al. Biochemical characterization and modulation of LH/CG-receptor during human trophoblast differentiation. J Cell Physiol. 212 (1), 26-35 (2007).
  25. Pidoux, G., et al. ZO-1 is involved in trophoblastic cell differentiation in human placenta. Am J Physiol Cell Physiol. 298 (6), 1517-1526 (2010).
  26. Williams, J. L., Fyfe, G. K., Sibley, C. P., Baker, P. N., Greenwood, S. L. K+ channel inhibition modulates the biochemical and morphological differentiation of human placental cytotrophoblast cells in vitro. Am J Physiol Regul Integr Comp Physiol. 295 (4), 1204-1213 (2008).
  27. Schild, R. L., Schaiff, W. T., Carlson, M. G., Cronbach, E. J., Nelson, D. M., Sadovsky, Y. The activity of PPAR gamma in primary human trophoblasts is enhanced by oxidized lipids. J Clin Endocrinol Metab. 87 (3), 1105-1110 (2002).
  28. Menendez-Pelaez, A., Reiter, R. J. Distribution of melatonin in mammalian tissues: the relative importance of nuclear versus cytosolic localization. J Pineal Res. 15 (2), 59-69 (1993).
  29. Perrone, S., Stazzoni, G., Tataranno, M. L., Buonocore, G. New pharmacologic and therapeutic approaches for hypoxic-ischemic encephalopathy in the newborn. J Matern Fetal Neonatal Med. 25, 83-88 (2012).
  30. Nelissen, E. C., van Montfoort, A. P., Dumoulin, J. C., Evers, J. L. Epigenetics and the placenta. Hum Reprod Update. 17 (3), 397-417 (2011).
  31. Barker, D. J. Intrauterine programming of adult disease. Mol Med Today. 1 (9), 418-423 (1995).
  32. Barker, J. R., Thomas, C. F., Behan, M. Serotonergic projections from the caudal raphe nuclei to the hypoglossal nucleus in male and female rats. Respir Physiol Neurobiol. 165 (2-3), 175-184 (2009).
  33. Yui, J., et al. Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells. Placenta. 15 (3), 231-246 (1994).
  34. Kilani, R. T., Chang, L. J., Garcia-Lloret, M. I., Hemmings, D., Winkler-Lowen, B., Guilbert, L. J. Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection. J Virol. 71 (9), 6359-6372 (1997).
  35. Knofler, M., Stenzel, M., Husslein, P. Shedding of tumour necrosis factor receptors from purified villous term trophoblasts and cytotrophoblastic BeWo cells. Hum Reprod. 13 (8), 2308-2316 (1998).
  36. Douglas, G. C., King, B. F. Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J Immunol Methods. 119 (2), 259-268 (1989).
  37. Li, L., Schust, D. J. Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta. Reprod Biol Endocrinol. 13, 71 (2015).
  38. Stenqvist, A. C., et al. An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies. Am J Reprod Immunol. 60 (1), 33-42 (2008).
  39. Potgens, A. J., Kataoka, H., Ferstl, S., Frank, H. G., Kaufmann, P. A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity. Placenta. 24 (4), 412-423 (2003).
  40. Lanoix, D., Vaillancourt, C. Cell culture media formulation and supplementation affect villous trophoblast HCG release. Placenta. 31 (6), 558-559 (2010).
  41. Vaillancourt, C., Lafond, J. Human embryogenesis: overview. Methods Mol Biol. 550, 3-7 (2009).
  42. Armant, D. R., et al. Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor. Development. 133 (4), 751-759 (2006).
  43. McCaig, D., Lyall, F. Hypoxia upregulates GCM1 in human placenta explants. Hypertens Pregnancy. 28 (4), 457-472 (2009).
  44. Burton, G. J., et al. Optimising sample collection for placental research. Placenta. 35 (1), 9-22 (2014).
  45. Lanoix, D., et al. Quantitative PCR pitfalls: the case of the human placenta. Mol Biotechnol. 52 (3), 234-243 (2012).
  46. Bilban, M., et al. Trophoblast invasion: assessment of cellular models using gene expression signatures. Placenta. 31 (11), 989-996 (2010).
  47. Novakovic, B., et al. Wide-ranging DNA methylation differences of primary trophoblast cell populations and derived cell lines: implications and opportunities for understanding trophoblast function. Mol Hum Reprod. 17 (6), 344-353 (2011).
  48. Burleigh, D. W., et al. Microarray analysis of BeWo and JEG3 trophoblast cell lines: identification of differentially expressed transcripts. Placenta. 28 (5-6), 383-389 (2007).
  49. Hung, T. H., Skepper, J. N., Charnock-Jones, D. S., Burton, G. J. Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia. Circ Res. 90 (12), 1274-1281 (2002).
  50. Heazell, A. E., Moll, S. J., Jones, C. J., Baker, P. N., Crocker, I. P. Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species. Placenta. 28, 33-40 (2007).
  51. Heazell, A. E., Lacey, H. A., Jones, C. J., Huppertz, B., Baker, P. N., Crocker, I. P. Effects of oxygen on cell turnover and expression of regulators of apoptosis in human placental trophoblast. Placenta. 29 (2), 175-186 (2008).
  52. Chen, B., Longtine, M. S., Nelson, D. M. Hypoxia induces autophagy in primary human trophoblasts. Endocrinology. 153 (10), 4946-4954 (2012).
  53. Lanoix, D., Guerin, P., Vaillancourt, C. Placental melatonin production and melatonin receptor expression are altered in preeclampsia: new insights into the role of this hormone in pregnancy. J Pineal Res. 53 (4), 417-425 (2012).
  54. Galano, A., Tan, D. X., Reiter, R. J. Melatonin as a natural ally against oxidative stress: a physicochemical examination. J Pineal Res. 51 (1), 1-16 (2011).
  55. Alers, N. O., Jenkin, G., Miller, S. L., Wallace, E. M. Antenatal melatonin as an antioxidant in human pregnancies complicated by fetal growth restriction–a phase I pilot clinical trial: study protocol. BMJ Open. 3 (12), 004141 (2013).
  56. Hobson, S. R., Lim, R., Gardiner, E. E., Alers, N. O., Wallace, E. M. Phase I pilot clinical trial of antenatal maternally administered melatonin to decrease the level of oxidative stress in human pregnancies affected by pre-eclampsia (PAMPR): study protocol. BMJ Open. 3 (9), 003788 (2013).

Tags

Primary Trophoblast Cell CultureHypoxia/reoxygenationMelatoninPregnancy ComplicationsPlacentaOxidative StressVillous Cytotrophoblast IsolationDensity CentrifugationTrophoblast CultureFirst Trimester Placenta

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Sagrillo-Fagundes, L., Clabault, H., Laurent, L., Hudon-Thibeault, A., Salustiano, E. M. A., Fortier, M., Bienvenue-Pariseault, J., Wong Yen, P., Sanderson, J. T., Vaillancourt, C. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption. J. Vis. Exp. (113), e54228, doi:10.3791/54228 (2016).
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