Human Trophoblast primaire Culture cellulaire de modèle pour étudier les effets protecteurs de la mélatonine contre l'hypoxie / perturbation induite par la réoxygénation-
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
Tout au long de la grossesse humaine, les cellules placentaires cytotrophoblaste, qui sont des cellules souches mononucléées, prolifèrent rapidement et se différencient en cellules soit de cytotrophoblaste villeux ou extravilleux. cytotrophoblastes extravillositaire envahissent et remodeler les artères spiralées de la paroi utérine. Cytotrophoblastes villeux, d'autre part, continuent à proliférer, se différencient et fusionnent pour former syncytiotrophoblaste multinucléés (le syncytium) 1. Le maintien de l'homéostasie villous trophoblaste est essentiel pour le bien-être du fœtus et la grossesse en bonne santé. En fait, trophoblaste villeux permettent l'échange d'oxygène et de nutriments materno-fœtale, et produisent des hormones essentielles pour la grossesse. Par ailleurs, le syncytiotrophoblaste est le seul type de cellule en contact direct avec la circulation sanguine maternelle et fournit une barrière physique et immunologiques essentielles. Par conséquent, le syncytiotrophoblaste doit subir l'apoptose et de remplacement pour l'entretien homéostatique et Avoid placentaire pathologies 2-5.
La technique développée par Kliman et al. , 6 en 1986 pour isoler cytotrophoblastes villosités primaires de placentas humains a provoqué une révolution dans la recherche placentaire en permettant l'étude des mécanismes moléculaires impliqués dans la différenciation du trophoblaste villeux. Cette technique classique, sur la base des digestions enzymatiques séquentielles avec de la trypsine et de l'ADNase, suivie d'un isolement en milieu de centrifugation de densité (particules de silice colloïdale revêtue par de la polyvinylpyrrolidone ou de Percoll) est maintenant reconnu comme l'étalon-or pour isoler des cellules du cytotrophoblaste villeux. La technique peut être optimisée par immunopurification magnétique, une procédure qui sépare cytotrophoblastes villeux des cellules non trophoblastiques basées sur l'expression différentielle des antigènes spécifiques sur les surfaces de ces cellules. Nous avons choisi le leucocyte humain antigène ABC (HLA-ABC) en raison de l'absence de son expression sur le Membran cellulaire trophoblastiquee 7,8.
Le placenta est un organe qui subit des variations considérables dans les niveaux d'oxygène au cours de la grossesse. Au cours du premier trimestre de la grossesse, le taux d'oxygénation est physiologiquement très faible (2% d' O 2) , mais augmente à des niveaux d'oxygénation légers (8% O 2) dans le deuxième et le troisième trimestre de la grossesse. Tuuli et al. 9 décrit que la reproduction in vitro de l'environnement à l' intérieur du trophoblaste villosités placentaires est un défi et des variations dans les niveaux d'oxygénation peuvent même conduire à des changements phénotypiques. Il est donc suggéré d'adopter 8% d' oxygène en normoxie pour imiter la tension d'oxygène trouvé dans villosités placentaires au cours du troisième trimestre de la gestation 8,9. Chen et al. 10 longuement étudié plusieurs variables liées à la tension d'oxygène dans la culture de cellules de trophoblaste et ont démontré l'importance de la détermination des niveaux d'oxygène dans un environnement péricellulaire. Les niveaux d'oxygène dans les villosités tendent à accroîtreen raison de la vasculogenèse. Le flux sanguin dans les villosités placentaires augmente constamment et le niveau de peroxyde d'hydrogène (une espèce d'oxygène réactif abondant) est un signal important qui contrôle la vasculogenèse 11,12. Dans complications de la grossesse, un manque de vasculogenèse génère l'hypoxie, et plus important encore, les variations intermittentes de l'oxygénation (appelé hypoxie / réoxygénation). Ces conditions conduisent à une augmentation anormale du stress oxydatif, ce qui compromet la viabilité fœtale placentaire et 13,14. Les modifications que les cellules trophoblastiques subissent in vivo au cours des épisodes d'hypoxie / réoxygénation peuvent être imitées in vitro comme suit: cytotrophoblastes villosités sont maintenues dans des conditions normoxiques (8% de O 2) jusqu'à ce qu'ils se différencient en syncytiotrophoblaste. Elles sont ensuite soumises à des conditions hypoxiques (0,5% O 2) pendant 4 heures, suivie d'une 18 heure supplémentaire normoxie (réoxygénation). En utilisant cette approche hypoxie / réoxygénation, trophoblastes exHiBit déréglementé état redox et des niveaux accrus d'apoptose intrinsèque 8, comme cela a été observé dans certaines complications de la grossesse. Par conséquent, cela est un utile dans le modèle in vitro pour évaluer de nouvelles approches préventives et thérapeutiques pour lutter contre les complications de la grossesse associés à placentaire hypoxie / réoxygénation.
Les cellules placentaires produisent la mélatonine, qui a plusieurs fonctions importantes telles que la capacité à prévenir le stress oxydatif et à un dysfonctionnement placentaire 15. Ici, nous présentons les modèles d'approche et de cellules expérimentales utilisées pour démontrer les effets protecteurs de la mélatonine dans les cellules trophoblastiques placentaires au niveau moléculaire, cellulaire et fonctionnelle 8.
Protocol
Representative Results
Discussion
Chez les mammifères, le développement du foetus est directement dépendante de la fonction placentaire adéquate. Les origines développementales des troubles de santé sont fondées sur l'hypothèse que la cause des maladies qui se manifestent plus tard dans la vie peut être retracée au développement précoce et que le placenta a un rôle mécanique dans la programmation fœtale 30-32. Le placenta est le médiateur clé de la croissance et le développement du fœtus: il régule le transfert des nut…
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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