Nous remercions la collaboration de Dr Thao Dang et les membres de son laboratoire pour l&#39;utilisation de leur équipement d&#39;imagerie et d&#39;un microscope. Nous remercions également la banque de tissus biologiques et de tissus installation de recherche (BTRF) noyau, Jeff Harper, et les résidents de l&#39;Université de Virginie de nous fournir le tissu utérin. Nous remercions Karol Szlachta pour l&#39;aide avec vue d&#39;ensemble schématique.

Spécimens hystérectomie utilisés dans ce manuscrit ont été recueillies en concordance avec un protocole d&#39;éthique de l&#39;Université de la CISR approuvé numérotée IRB-RSS # 14424. 1. Acquisition des échantillons de clinique Source <ol><li> Obtenir gouvernement et des principes éthiques en établissement et de la documentation d&#39;approbation avant de commencer. </li><li> Réaliser toutes les étapes des conditions stériles. </li><li> Préserver le tissu dérivé du patient dans les médias (RPMI ou DMEM / High glucose) dans un tube de 50 ml à 4 ° C si l&#39;échantillon ne peut pas être traitée dans la culture immédiatement. Les échantillons peuvent être stockés dans cet état pendant un maximum de 24 heures. </li><li> Laver échantillon de tissu trois fois avec 1X stérile tamponnée au phosphate (PBS) et jeter la solution entre les lavages. </li></ol> 2. Préparation de lignées cellulaires primaires à l&#39;aide de la méthode Grattage <ol><li> Ajouter 4-10 ml de milieu de croissance (RPMI ou DMEM / High Glucose supplémenté avec 10% de sérum bovin fœtal et 1% de pénicilline-streptomycin) de tissu et ajouter Fungizone (0,25 ug / ml de concentration finale) pendant 30 min. </li><li> Jeter le milieu de croissance. </li><li> Laver le tissu deux fois avec du PBS 1X. </li><li> Placer le tissu sur une plaque de culture de 6 cm de cellules de distinguer entre les couches myomètre et l&#39;endomètre (se référer aux figures 2A à 2C pour une description plus détaillée). Séparer l&#39;endomètre. </li><li> L&#39;utilisation d&#39;un scalpel ou une lame de rasoir, sectionner le tissu en petits morceaux en grattant sur la boîte de culture de 6 cm de cellule. Ces rayures faciliter la fixation des cellules de l&#39;endomètre primaires émergents. Par rapport à la méthode de la trypsine, plus le tissu est nécessaire (voir la figure 2D pour une approximation). </li><li> Doucement, ajouter 2 ml de milieu de croissance à plat rayé (fragments de tissus seront visibles et idéalement immobilisé à partir du mouvement de grattage). </li><li> Transférer la plaque (s) à un incubateur de culture cellulaire désignée (37 ° C et 5% CO 2). Désigner un lieu pour les lignes de cellules primaires, loin de OTHer des boîtes de culture cellulaire. Les cultures primaires sont plus sensibles à l&#39;infection et de contamination. </li><li> Examiner les cellules au microscope optique quotidien. De petites populations de cellules devraient émerger de deux ou trois jours autour des tissus tranches (voir la figure 3B). </li><li> Pour maintenir les cultures, laver délicatement avec du PBS 1X et ajouter un milieu de croissance frais (2 ml) tous les trois jours. Lorsque l&#39;augmentation du taux de prolifération, des milieux de croissance supplémentaire (3-4 ml) peuvent être ajoutés à la plaque de culture de 6 cm (s). Remarque: Pendant le lavage et médias changements, des morceaux de tissu seront probablement aspiré. Ceci n&#39;affectera pas la croissance de cellules adhérentes. Des morceaux de tissu doivent être aspirés par l&#39;étape ultérieure (2,10) en tant que nouvelles colonies sont peu susceptibles d&#39;apparaître après une semaine. </li><li> Lorsque la plaque de 6 cm est d&#39;environ 75 - 80% de confluence (voir la figure 3B), passage en utilisant 0,05% de trypsine. Cellules primaires ne peuvent pas être passées indéfiniment - geler bas une plaque de cellules dès que deux ou trois plaques sont maintained. Si plusieurs passages sont nécessaires, conformément à un protocole d&#39;immortalisation (revue dans Ref 3). </li></ol> 3. Préparation de lignées cellulaires primaires à l&#39;aide de la méthode de la trypsine <ol><li> Placer un petit morceau de tissu endométrial (voir la figure 2D pour un rapprochement) dans 2 ml de trypsine à 0,25% additionné de kanamycine (0,03 mg / ml de concentration finale). </li><li> Incuber les tissus à 37 ° C sur une plateforme rotative pendant 30 minutes. </li><li> Vortex brièvement le tissu. </li><li> Centrifuger l&#39;échantillon à 200-400 g pendant 2 min et jeter le surnageant. </li><li> Ajouter trypsine frais et à la kanamycine. </li><li> Faire incuber le tissu à 37 ° C tout en tournant pour un hr. </li><li> Vortex le tissu pour 5-10 sec. </li><li> Ajouter 2 ml de milieu de croissance (supplémenté avec 10% de sérum bovin fœtal et 1% de pénicilline-streptomycine) pour désactiver la trypsine. </li><li> Centrifuger les cellules à 200-400 g pendant 2-3 min. </li><li> Jeter le surnageant et ajouter 2 ml de grandire médias à le culot cellulaire. </li><li> Plaque sur une boîte de culture de 6 cm de cellule. Grattage de la plaque comme dans l&#39;étape 2.5 de préparation de lignées cellulaires primaires en utilisant la méthode Grattage n&#39;est pas nécessaire, mais améliore la fixation et l&#39;excroissance de cultures primaires. </li><li> Surveillance de cellules sous un microscope optique quotidien. Les cellules sont généralement visibles au microscope optique après 24-48 heures (voir la figure 3C). </li><li> Pour maintenir les cultures, de surveiller les cellules et changer de support tous les 2-3 jours comme dans l&#39;étape 2.9. </li><li> Pour le passage des cellules, utilisez 0,05% ou 0,25% de trypsine lorsque les cellules atteignent 75-80% de confluence (voir la figure 3C). </li></ol> 4. Sauvegarde tissu supplémentaire pour l&#39;analyse (Snap congélation et la fixation au formol) <ol><li> Laver l&#39;échantillon deux fois avec du PBS 1X. </li><li> Aspirer autant de liquide que possible. </li><li> Snap congélation, placer l&#39;échantillon de tissu de l&#39;endomètre dans un tube de 1,7 centrifugeuse (voir les figures 2F et 2G). Placez le 1.7tube de centrifugeuse dans l&#39;azote liquide pendant 10 secondes ou jusqu&#39;à ce qu&#39;il puisse être considéré que le tissu a gelé le bas. Remarque: Prenez soin de ne pas toucher directement l&#39;azote liquide lors de la préparation des échantillons. Les échantillons peuvent être conservés à -80 ° C jusqu&#39;à leur traitement ultérieur ou d&#39;analyse. </li><li> Pour la fixation au formol, couper un petit morceau de tissu endométrial (voir la figure 2H), et plonger dans 10% du formol tamponné de zinc. Après 24 heures, éliminer le formol et ajouter de l&#39;éthanol à 70% des échantillons de tissu jusqu&#39;à ce que le traitement ultérieur. </li></ol> 5. Immunofluorescence (IF) <ol><li> Cellule fixation <ol><li> Préparer les cellules sur une lame de culture ou de la plaque. </li><li> Doucement, laver les cellules avec du PBS 1X. </li><li> Ajouter pré-réfrigéré (4 ° C) et de l&#39;acétone Methanol (mélangés dans un rapport 1:1) pendant 5 min. </li><li> Air sécher la lame avant de poursuivre. Glisser peut être stocké à 4 ° C pendant 1 à 2 semaines si nécessaire. </li></ol></li><li> Traitement SI <ol><li> Réhydrater les cellules avec du PBS 1X pendant 10 min. Pour la following étapes 1-6, procéder à tous les lavages et les incubations sur une plate-forme tournante. </li><li> Bloc à l&#39;aide de PBS 1X supplémenté avec 1% de sérum-albumine bovine (BSA) et 1% d&#39;espèces sérum spécifique (2 ème source d&#39;anticorps) pendant 30 min à température ambiante (RT). </li><li> Incuber dans l&#39;anticorps primaire (voir les recommandations du fabricant pour la dilution de l&#39;anticorps). Reportez-vous aux documents d&#39;anticorps spécifiques. Diluer l&#39;anticorps primaire dans un tampon de blocage frais comme à l&#39;étape 5.2.2. Cette incubation peut être réalisée pendant au moins 2 heures à température ambiante ou une nuit à 4 ° C. </li><li> Laver 3 fois avec du PBS 1X pendant 5 min chacun. </li><li> Incuber dans l&#39;anticorps secondaire (voir les recommandations du fabricant pour la dilution de l&#39;anticorps). Diluer l&#39;anticorps secondaire dans du tampon de blocage frais comme à l&#39;étape 5.2.2. Pour empêcher la photo-blanchiment, il est important de procéder à des étapes ultérieures, et ce en l&#39;absence de lumière. </li><li> Laver 3 fois avec du PBS 1X pendant 5 min chacun. </li><li> Thoroughly sécher les diapositives. </li><li> Ajouter milieux de montage et solution de contre-coloration DAPI. </li><li> Appliquer la lamelle et sceller les bords à l&#39;aide d&#39;étanchéité (s). Lames peuvent être stockées à 4 ° C dans l&#39;obscurité pendant jusqu&#39;à 2 semaines. </li></ol></li></ol> 6. Extraction de l&#39;ARN <ol><li> Pellet 1 x 10 7 cellules par centrifugation. Tous les matériaux et les réactifs de ce protocole doivent être RNase. </li><li> Lyser les cellules dans 1 ml de réactif TRIZOL, et incuber les échantillons homogénéisés pendant 10 min à température ambiante pour compléter la dissociation des complexes de nucléoprotéines. </li><li> Ajouter 200 ul de chloroforme pour 1 ml de TRIZOL. Secouer vigoureusement à la main pendant 15 secondes et incuber pendant 2-3 min à température ambiante. </li><li> Centrifuger à la vitesse maximale (15 000 xg) pendant 15 min à 4 ° C. Trois couches en résulteront. </li><li> Transférer la phase aqueuse supérieure dans un tube à centrifuger frais. Ajouter 1 pi de glycogène pour augmenter le rendement de l&#39;ARN; Toutefois, cette étape n&#39;est nécessaire que lorsque une petitequantité d&#39;ARN est prévu. </li><li> Ajouter 0,5 ml d&#39;alcool isopropylique à la couche aqueuse, et inverser les échantillons 3 à 5 fois. Incuber les échantillons à température ambiante pendant 10 min. Pour augmenter le rendement, les échantillons peuvent être placés dans -20 ° C ou -80 ° C pendant 30 min. </li><li> Centrifuger à vitesse maximale pendant 10 minutes à 4 ° C. </li><li> À ce stade, une petite pastille d&#39;ARN doit être visible. Jeter le surnageant. </li><li> Laver l&#39;ARN avec 1 ml d&#39;éthanol à 75%. </li><li> Centrifuger à vitesse maximale pendant 5 min et jeter le surnageant. Les échantillons peuvent être lavées une fois de plus à éliminer les contaminants inorganiques, mais cette étape n&#39;est pas nécessaire. </li><li> En bref, sécher le culot d&#39;ARN jusqu&#39;à culot d&#39;ARN est sec (prend habituellement 7-10 min). Remarque: Une étape supplémentaire peut être utilisé pour diminuer la matière inorganique et de diminuer le temps de séchage. Après avoir jeté le surnageant du lavage à l&#39;éthanol, les échantillons de rotation à la vitesse maximale pendant une minute supplémentaire et aspirer le liquide résiduel. Prenez soin de ne pas aspirer à granulés. </li><li> Dissoudre l&#39;ARN dans l&#39;eau exempte de RNase, en fonction de la taille de la pastille d&#39;ARN. Les volumes varient généralement 10-50 pi. </li><li> Incuber les échantillons à 55 ° C pendant 10 min, et de mesurer la concentration de l&#39;ARN. </li><li> Conserver les échantillons à -20 ° C jusqu&#39;à leur traitement ultérieur. </li></ol> 7. Transcription inverse <ol><li> Amener l&#39;ARN de l&#39;échantillon (1-3 pg) jusqu&#39;à un volume de 9 pi avec H 2 O. </li><li> l&#39;ARN de la chaleur à 70 ° C pendant 10 min. </li><li> Pour générer un mélange maître AMV, combiner les réactifs suivants: 5 ul de dNTP (concentration de travail de 50 uM), 5 ul de oligos N6 ADN (concentration de travail de 80 uM), 5 ul de tampon 5X AMV, et 1 ul de AMV . Cette solution maître de mélange est conçu par un échantillon. </li><li> Ajouter 16 pi de mélange maître de l&#39;ARN chauffée. Le volume réactionnel final est de 25 ul de réaction. </li><li> Utiliser les conditions suivantes PCR pour la transcription inverse: (étape 1) de 42 ° C pendant 90 min, (Stage 2) 95 ° C pendant 5 min, et (étape 3) 4 ° C jusqu&#39;à traitement ultérieur. </li></ol> 8. PCR en temps réel <ol><li> Mener des réactions de PCR en utilisant les amorces, les températures de recuit, les nombres de cycles, et de réactifs figurant au tableau 1. Remarque: Il est idéal pour suivre les instructions du fabricant pour l&#39;utilisation de réactifs de PCR (voir à la société et numéros de catalogue de matériaux). </li></ol> 9. In vitro Protocole décidualisation (Dérivé de Ref 4 et 5) <ol><li> Plate cellules de l&#39;endomètre. </li><li> Atteindre une confluence de 75-85%. </li><li> Après que les cellules deviennent confluentes, laver une fois avec du PBS 1X. </li><li> Rapidement, ajouter des médias sans hormones (phénol libre RPMI supplémenté avec 5% de charbon bande de FBS et 1% de pénicilline-streptomycine). </li><li> Après 24 heures, ajouter des médias d&#39;hormone libre supplémenté avec de l&#39;acétate de médroxyprogestérone (MPA) à une concentration finale de 1 uM et 8 bromoadenosine-3 &#39;, 5&#39;-Monophosphate cyclique (AMPc) à une concentration finale de 0,5 mM. </li><li> Après au moins 48 heures, arrêter la réaction et sauver la plaque (s) pour un traitement ultérieur. </li></ol>

<ol>
	<li>Heller, D., Heller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in The endometrium: a clinicopathologic approach. , 56-75 (1994).</li><li>Emera, D., Romero, R., Wagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+menstruation:+a+new+model+for+genetic+assimilation:+explaining+molecular+origins+of+maternal+responses+to+fetal+invasiveness.">The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.</a> Bioessays. 34, 26-35 (2011).</li><li>Gudjonsson, T., Villadsen, R., Ronnov-Jessen, L., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+protocols+used+in+cell+culture+models+of+human+breast+morphogenesis.">Immortalization protocols used in cell culture models of human breast morphogenesis.</a> Cell Mol Life Sci. 61, 2523-2534 (2004).</li><li>Brosens, J. J., Hayashi, N., White, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+regulates+decidual+prolactin+expression+in+differentiating+human+endometrial+stromal+cells.">Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.</a> Endocrinology. 140, 4809-4820 (1999).</li><li>Jones, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+SUMO+pathway+sensitizes+differentiating+human+endometrial+stromal+cells+to+progesterone.">Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone.</a> Proc Natl Acad Sci U S A. 103, 16272-16277 (2006).</li><li>Salamonsen, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+the+human+endometrium+and+uterine+fluid:+a+pathway+to+biomarker+discovery.">Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery.</a> Fertil Steril. 99, 1086-1092 (2013).</li><li>Haouzi, D., Dechaud, H., Assou, S., De Vos, J., Hamamah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+human+endometrial+receptivity+from+transcriptomic+and+proteomic+data.">Insights into human endometrial receptivity from transcriptomic and proteomic data.</a> Reprod Biomed Online. 24, 23-34 (2012).</li><li>Chen, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+midproliferative+and+midsecretory+human+endometrium.">Proteomic characterization of midproliferative and midsecretory human endometrium.</a> J Proteome Res. 8, 2032-2044 (2009).</li><li>Talbi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phenotyping+of+human+endometrium+distinguishes+menstrual+cycle+phases+and+underlying+biological+processes+in+normo-ovulatory+women.">Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.</a> Endocrinology. 147, 1097-1121 (2006).</li><li>Punyadeera, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oestrogen-modulated+gene+expression+in+the+human+endometrium.">Oestrogen-modulated gene expression in the human endometrium.</a> Cell Mol Life Sci. 62, 239-250 (2005).</li><li>Ponnampalam, A. P., Weston, G. C., Susil, B., Rogers, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+human+endometrium+during+the+menstrual+cycle.">Molecular profiling of human endometrium during the menstrual cycle.</a> Aust N Z J Obstet Gynaecol. 46, 154-158 (2006).</li><li>Zhang, L., Rees, M. C., Bicknell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+long-term+culture+of+normal+human+endometrial+epithelium+and+stroma.+Expression+of+mRNAs+for+angiogenic+polypeptides+basally+and+on+oestrogen+and+progesterone+challenges.">The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges.</a> J Cell Sci. 108 (1), 323-331 (1995).</li><li>Richards, R. G., Brar, A. K., Frank, G. R., Hartman, S. M., Jikihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+cells+from+term+human+decidua+closely+resemble+endometrial+stromal+cells:+induction+of+prolactin+and+insulin-like+growth+factor+binding+protein-1+expression).">Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression).</a> Biol Reprod. 52, 609-615 (1995).</li><li>Gellersen, B., Brosens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+and+progesterone+receptor+cross-talk+in+human+endometrium:+a+decidualizing+affair.">Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair.</a> J Endocrinol. 178, 357-372 (2003).</li><li>Marsh, M. M., Hampton, A. L., Riley, S. C., Findlay, J. K., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+endothelin+released+by+human+endometrial+epithelial+cells+in+culture.">Production and characterization of endothelin released by human endometrial epithelial cells in culture.</a> J Clin Endocrinol Metab. 79, 1625-1631 (1994).</li><li>Siegfried, J. M., Nelson, K. G., Martin, J. L., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+identification+of+cultured+cells+from+human+endometrium.">Histochemical identification of cultured cells from human endometrium.</a> In Vitro. 20, 25-32 (1984).</li><li>Rawdanowicz, T. J., Hampton, A. L., Nagase, H., Woolley, D. E., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+production+by+cultured+human+endometrial+stromal+cells:+identification+of+interstitial+collagenase,+gelatinase-A,+gelatinase-B,+and+stromelysin-1+and+their+differential+regulation+by+interleukin-1+alpha+and+tumor+necrosis+factor-alpha.">Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.</a> J Clin Endocrinol Metab. 79, 530-536 (1994).</li><li>Dimitriadis, E., Robb, L., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+11+advances+progesterone-induced+decidualization+of+human+endometrial+stromal+cells.">Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 8, 636-643 (2002).</li><li>Sakai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+histone+acetylation+in+ovarian+steroid-induced+decidualization+of+human+endometrial+stromal+cells.">Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells.</a> J Biol Chem. 278, 16675-16682 (2003).</li><li>Logan, P. C., Ponnampalam, A. P., Steiner, M., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyclic+AMP+and+estrogen/progesterone+on+the+transcription+of+DNA+methyltransferases+during+the+decidualization+of+human+endometrial+stromal+cells.">Effect of cyclic AMP and estrogen/progesterone on the transcription of DNA methyltransferases during the decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 19, 302-312 (2013).</li><li>Tamura, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IGFBP-1+expression+by+cAMP+is+associated+with+histone+acetylation+status+of+the+promoter+region+in+human+endometrial+stromal+cells.">Induction of IGFBP-1 expression by cAMP is associated with histone acetylation status of the promoter region in human endometrial stromal cells.</a> Endocrinology. 153, 5612-5621 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Society for Reproductive Medicine: Quick facts about infertility. , (2013).</li><li>Salker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+selection+of+human+embryos:+impaired+decidualization+of+endometrium+disables+embryo-maternal+interactions+and+causes+recurrent+pregnancy+loss.">Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.</a> PLoS One. 5, (2010).</li></ol>

Les auteurs n&#39;ont rien à divulgation.

D&#39;autres groupes ont décrit et adapté méthodologie pour la préparation de cultures stromales endométriales, dont la plupart utilisent la collagénase 4,12,13,15-18. Dans ce manuscrit, nous avons fourni la méthodologie et les données pour deux endomètre méthodes de culture de cellules stromales primaires simplifiées, qui sont tous deux utilisés par notre laboratoire pour des raisons économiques et de la disponibilité pratique de la trypsine et / ou une lame de rasoir. <p class="jove_conten...

Le corpus de l&#39;utérus humain est composé de trois couches, la perimetrium (ou séreuse), le myomètre et l&#39;endomètre. Distinguer chacune de ces couches est une étape importante pour établir des lignées cellulaires de l&#39;endomètre. Le perimetrium est la couche la plus externe de l&#39;utérus et composée de fines cellules séreuses. Le myomètre est la couche intermédiaire épaisse de l&#39;utérus et le composé de cellules musculaires lisses. L&#39;endomètre est identifié en tant que couche intérieure de l&#39;utérus et comprend des populations de cellules épithéliales et stromales. L&#39;endomètre est encore subdivisé en couche basalis dont la tige population cellulaire est l&#39;hypothèse de repeupler la couche de functionalis environ tous les 28 jours 1. La couche de functionalis de l&#39;endomètre humain subit des modifications biochimiques et morphologiques importantes en réponse aux hormones circulant. Ces hormones sont dérivées de la glande pituitaire et les ovaires. Laproduction coordonnée et la libération d&#39;hormones entraîne un cycle de reproduction. Le cycle de reproduction est conçu pour préparer l&#39;endomètre pour les événements potentiels d&#39;implantation embryonnaire. Chez les humains, le cycle de reproduction est connu comme &quot;le cycle menstruel&quot; et divisé en trois phases - proliférative, sécrétoires, et menstruels. La phase proliférative implique la prolifération de l&#39;endomètre functionalis couche tandis que la phase sécrétoire est marquée par functionalis maturation. Plus précisément, les modifications extracellulaires, les sécrétions, et la différenciation cellulaire signalent une implantation potentielle. Si l&#39;implantation ne se produit pas avant la fin de la phase de sécrétion, la couche functionalis de l&#39;endomètre est éliminée au cours de la phase menstruel. L&#39;importance de la menstruation et les événements qui déclenchent l&#39;effusion de la couche de functionalis sont encore en discussion. Chez l&#39;homme, il a été posé que la menstruation est le résultat d&#39;un événement à mi-sécrétoire différenciation de phase spécifique connuecomme &quot;décidualisation spontanée» 2. Dans ce manuscrit, nous proposons une méthodologie détaillée pour les deux méthodes d&#39;isolement de cellules stromales de l&#39;endomètre, et utilisons une combinaison de l&#39;immunofluorescence et des images numériques pour démontrer l&#39;efficacité de ces approches. En outre, nous appliquons un modèle couramment utilisé dans vitro de décidualisation spontanée pour confirmer l&#39;isolement de cellules stromales de l&#39;endomètre.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">0.25 Trypsin or 0.05% Trypsin&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH3023602 or SH30004202&#160;&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="27">
			<td dir="LTR" height="27" style="height:27px;width:235px;">1.7 micro Centrifuge Tube&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">22-272A&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">1&#181;l,20&#181;l, 200ml and 1000&#181;l Pipette&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">24-401,24-402, 24-412, 24-430&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">15ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339650</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">50ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339652</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">6cm Cell Culture Dish&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-556-002&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">8 well Chambers&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Acetate&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher scientific&#160;</td>
			<td dir="LTR" style="width:216px;">C4-100&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">AMV RT Enzyme/Buffer&#160;</td>
			<td dir="LTR" style="width:201px;">Bio Labs&#160;</td>
			<td dir="LTR" style="width:216px;">M077L&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Bovine Serum Albumin (BSA)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-1605-100&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Buffered Zinc Formalin&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">59201ZF&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Charcoal strip FBS&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher&#160;</td>
			<td dir="LTR" style="width:216px;">NC9019735&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Chloroform&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cover slip&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Brand&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-544D&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cyclic AMP (cAMP)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">B7880</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">DMEM/High Glucose&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH30243FS&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">dNTP&#160;</td>
			<td dir="LTR" style="width:201px;">Bioline&#160;</td>
			<td dir="LTR" style="width:216px;">BIO-39025&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Anti Goat -TRITC&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-3855&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Serum&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s lab&#160;</td>
			<td dir="LTR" style="width:216px;">&#160;017-000-002&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">E Cadherin Antibody&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">1702-1</td>
			<td></td>
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		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Ethanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">BP2818-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific</td>
			<td dir="LTR" style="width:216px;">03-600-511&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fungizone Amphotericin B&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">15290-018&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">GAPDH Probe&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">HS99999905&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Glycogen&#160;</td>
			<td dir="LTR" style="width:201px;">5Prime&#160;</td>
			<td dir="LTR" style="width:216px;">2301440</td>
			<td></td>
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		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Goat Anti Mouse -FITC&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s Lab&#160;</td>
			<td dir="LTR" style="width:216px;">115-096-003&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Isopropanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP2618-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Kanamycin&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP906-5&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Medroxyprogesterone acetate (MPA)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">M1629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">MeOH (Methanol)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">A4-08-1&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Mounting Media (w/DAPI)&#160;</td>
			<td dir="LTR" style="width:201px;">Vector Labratories&#160;</td>
			<td dir="LTR" style="width:216px;">H-1500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">N6 DNA Oligos&#160;</td>
			<td dir="LTR" style="width:201px;">Invitrogen&#160;</td>
			<td dir="LTR" style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Number 15 Scraper&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">BD&#160;</td>
			<td dir="LTR" style="width:216px;">371615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Pan Cytokeratin&#160; Mouse mAB&#160;</td>
			<td dir="LTR" style="width:201px;">Cell Signaling&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">4545</td>
			<td></td>
		</tr>
		<tr height="29">
			<td dir="LTR" height="29" style="height:29px;width:235px;">PBS (phosphate buffered saline)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-399-4&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Penicillin-Streptomycin Glutamine Solution 100X&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">SV30082.01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">PML Anti Goat Anti body&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-9862&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Primer(s)&#160;</td>
			<td dir="LTR" style="width:201px;">Eurofins&#160;</td>
			<td dir="LTR" style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">RPMI&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">SH30027FS&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">RPMI (Phenol free)&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">11835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Sybr Green&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Taqman&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Trizol&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Vimentin Antibody&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">4211-1</td>
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		</tr>
	</tbody>
</table>

two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

Beaucoup d&#39;efforts ont été consacrés à établir des systèmes de culture cellulaire in vitro. Ces systèmes sont conçus pour modéliser un grand nombre de procédés in vivo. systèmes de culture cellulaire résultant de l&#39;endomètre échantillons humains ne font pas exception. Les applications vont de processus physiologiques cycliques normales à des pathologies de l&#39;endomètre telles que les cancers gynécologiques, les maladies infectieuses et les carences en matière de reproduction. Ici, nous proposons deux méthodes pour établir des cellules stromales de l&#39;endomètre primaires à partir d&#39;échantillons d&#39;hystérectomie endomètre résection chirurgicale. Le premier procédé est appelé «le procédé de raclage&quot; et intègre raclage mécanique à l&#39;aide des lames chirurgicales ou rasoir tandis que le second procédé est appelé «le procédé de la trypsine.&quot; Cette dernière méthode utilise l&#39;activité enzymatique de la trypsine, de promouvoir la séparation des cellules et primaire excroissance cellulaire. Nous illustrons la méthodologie étape par étape à travers des images numériques et la microscopie. Nous également providexemples de e pour la validation des lignées de cellules stromales de l&#39;endomètre par des réactions quantitatives en temps réel de la chaîne de la polymérase (qPCR) et immunofluorescence (IF).

Comme l&#39;a souligné dans la section Protocole, assurez-vous de procéder à toutes les méthodes sous gouvernement, institutionnels, et des principes éthiques lors de la manipulation et la préparation de tissus humains. Sont inclus dans ce manuscrit est une illustration du processus général de &quot;la méthode de raclage&quot; (figure 1A) et &quot;le procédé de la trypsine&quot; (Figure 1B) utilisé pour établir des cultures prim...

Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Établir des systèmes primaires de l&#39;endomètre stromales de cellules de culture à partir d&#39;échantillons hystérectomie est une technique biologique précieux et une étape cruciale avant de poursuivre une vaste gamme de recherche vise. Ici, nous décrivons deux méthodes utilisées pour établir les cultures stromales de tissus de l&#39;endomètre résection chirurgicale des patients humains.

Deux méthodes de fixation des cellules humaines de l&#39;endomètre stromales primaires de spécimens hystérectomie

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

two-methods-for-establishing-primary-human-endometrial-stromal-cells

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Medicine

22.5K vues.  University of Virginia.  Établir des systèmes primaires de l'endomètre stromales de cellules de culture à partir d'échantillons hystérectomie est une technique biologique précieux et une étape cruciale avant de poursuivre une vaste gamme de recherche vise. Ici, nous décrivons deux méthodes utilisées pour établir les cultures stromales de tissus de l'endomètre résection chirurgicale des patients humains. 

Vidéo: Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Génération expérimentale de carcinome Associated fibroblastes (CAF) à partir des fibroblastes mammaires

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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Médecine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocole amélioré pour la microdissection laser d&#39;îlots pancréatiques humains à partir de spécimens chirurgicaux

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

L&#39;analyse transcriptomique de prélèvements rétine humaine en utilisant Journal

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Méthode pour l&#39;obtention de cellules cancéreuses ovariennes primaires à partir d&#39;échantillons solides

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Enrichissement pour chimiorésistantes cancer de l&#39;ovaire des cellules souches à partir de lignées cellulaires humaines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Une méthode d’isolement roman de Grade clinique pour les cellules stromales de rein humain périvasculaires

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolement de cellules primaires de Decidual humaines des Membranes fœtales des placentas à terme

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolement d’adipocytes viables et d’une fraction vasculaire stromale à partir de tissu adipeux viscéral humain adapté à l’analyse de l’ARN et au phénotypage des macrophages

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...