Name: isolation of human endometrial stromal cells for in vitro decidualization
Uploaded: 2018-09-01T12:30:00.000+00:00
Duration: 14 min 55 s
Description: Watch this Scientific Journal Video about Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization at JoVE.com

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<ol>
	<li>Maruyama, T., Yoshimura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+mechanisms+for+differentiation+and+regeneration+of+the+uterine+endometrium.">Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium.</a> Endocrine Journal. 55, 795-810 (2008).</li><li>Yu, J., 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=Endometrial+Stromal+Decidualization+Responds+Reversibly+to+Hormone+Stimulation+and+Withdrawal.">Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.</a> Endocrinology. 157, 2432-2446 (2016).</li><li>Ahn, 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=cAMP-Response+Element-Binding+3-Like+Protein+1+(CREB3L1)+is+Required+for+Decidualization+and+its+Expression+is+Decreased+in+Women+with+Endometriosis.">cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.</a> Current Molecular Medicine. 16, 276-287 (2016).</li><li>Gellersen, B., Brosens, J. 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+decidualization+of+the+human+endometrium+in+reproductive+health+and+failure.">Cyclic decidualization of the human endometrium in reproductive health and failure.</a> Endocrine Reviews. 35, 851-905 (2014).</li><li>Telgmann, R., Gellersen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marker+genes+of+decidualization:+activation+of+the+decidual+prolactin+gene.">Marker genes of decidualization: activation of the decidual prolactin gene.</a> Human Reproduction Update. 4, 472-479 (1998).</li><li>Camden, A. J., 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=Growth+regulation+by+estrogen+in+breast+cancer+1+(GREB1)+is+a+novel+progesterone-responsive+gene+required+for+human+endometrial+stromal+decidualization.">Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.</a> Molecular Human Reproduction. 23, 646-653 (2017).</li><li>Kommagani, R., 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=The+Promyelocytic+Leukemia+Zinc+Finger+Transcription+Factor+Is+Critical+for+Human+Endometrial+Stromal+Cell+Decidualization.">The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization.</a> PLoS Genetics. 12, e1005937 (2016).</li><li>Large, M. J., DeMayo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+embryo+implantation+and+endometrial+decidualization+by+progesterone+receptor+signaling.">The regulation of embryo implantation and endometrial decidualization by progesterone receptor signaling.</a> Molecular and Cellular Endocrinology. 358, 155-165 (2012).</li><li>Zhang, X. H., 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=The+mesenchymal-epithelial+transition+during+in+vitro+decidualization.">The mesenchymal-epithelial transition during in vitro decidualization.</a> Reproductive Sciences. 20, 354-360 (2013).</li><li>Kommagani, R., 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=Acceleration+of+the+glycolytic+flux+by+steroid+receptor+coactivator-2+is+essential+for+endometrial+decidualization.">Acceleration of the glycolytic flux by steroid receptor coactivator-2 is essential for endometrial decidualization.</a> PLoS Genetics. 9, e1003900 (2013).</li><li>Ramathal, C. Y., Bagchi, I. C., Taylor, R. N., Bagchi, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+decidualization:+of+mice+and+men.">Endometrial decidualization: of mice and men.</a> Seminars in Reproductive Medicine. 28, 17-26 (2010).</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=Novel+Function+of+a+Transcription+Factor+WT1+in+Regulating+Decidualization+in+Human+Endometrial+Stromal+Cells+and+Its+Molecular+Mechanism.">Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism.</a> Endocrinology. 158, 3696-3707 (2017).</li><li>Vinketova, K., Mourdjeva, M., Oreshkova, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Decidual+Stromal+Cells+as+a+Component+of+the+Implantation+Niche+and+a+Modulator+of+Maternal+Immunity.">Human Decidual Stromal Cells as a Component of the Implantation Niche and a Modulator of Maternal Immunity.</a> Journal of Pregnancy. , 8689436 (2016).</li><li>Wu, D., 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=Chronic+endometritis+modifies+decidualization+in+human+endometrial+stromal+cells.">Chronic endometritis modifies decidualization in human endometrial stromal cells.</a> Reproductive Biology and Endocrinology. 15, 16 (2017).</li><li>Garcia-Elias, 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=Defining+quantification+methods+and+optimizing+protocols+for+microarray+hybridization+of+circulating+microRNAs.">Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs.</a> Scientific Reports. 7, 7725 (2017).</li><li>Brosens, J. J., 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=Human+endometrial+fibroblasts+immortalized+by+simian+virus+40+large+T+antigen+differentiate+in+response+to+a+decidualization+stimulus.">Human endometrial fibroblasts immortalized by simian virus 40 large T antigen differentiate in response to a decidualization stimulus.</a> Endocrinology. 137, 2225-2231 (1996).</li><li>Koukouritaki, S. B., Margioris, A. N., Gravanis, A., Hartig, R., Stournaras, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+induces+rapid+actin+assembly+in+human+endometrial+cells+without+affecting+its+synthesis.">Dexamethasone induces rapid actin assembly in human endometrial cells without affecting its synthesis.</a> Journal of Cellular Biochemistry. 65, 492-500 (1997).</li></ol>

עבודה זו נתמכת על ידי מימון של נבחרת מכונים לבריאות (NIH) / המכון הלאומי של גרנט ובריאות הילד והתפתחות האדם (NICHD) (R00 HD080742), בית הספר לרפואה של אוניברסיטת וושינגטון הזנק מממן את ר. ק ברצוננו להודות המרפאה לפוריות אוניברסיטת וושינגטון למתן את דגימות ביופסיה רירית הרחם.

המחזור החודשי הרבייה הנשי מאופיין על ידי עלייה ברמות הפרוגסטרון לאורך כל שלב luteal, ובכך גרימת את decidualization של ESC לתוך תאים הפרשה עגול, אפיתל דמויי3,8. אתחול של decidualization הוא מין התלויים. בבני אדם, decidualization מתרחשת באופן ספונטני על עליית ריכוז הפרוגסטרון, ואילו עכבר?...

בין השלבים של הוסת, גיל המעבר, נשים בגיל הפריון לעבור מחזורים חודשיים של הורמון מוסדר התפשטות רירית הרחם, בידול, ושפך הבאים לקראת הריון בתהליך המכונה הווסת1 ,2. ושינויים פיזיים אלו של רירית הרחם האדם נחוצים להשתלת עובר תקין לתוך הרחם1. שינויים של רירית הרחם, כולל עיבודים מורפולוגי וביוכימי, הן מתווכת לאורך המחזור החודשי באמצעות הורמונים סטרואידים השחלות אסטרוגן ופרוגסטרון (P4)3,4,5. במסגרת שלב המקדימות (או הזקיקים), רמות אסטרוגן preovulatory עלייה, ייזום עיבוי רירית הרחם. בעקבות ביוץ, השלב הפרשה (או luteal) מקדמת לעלייה משמעותית בריכוז P4, גרימת שינוי מורפולוגי תאי סטרומה רירית הרחם (ESC) של פיברובלסט דמוי מראה מעוגל, תאי אפיתל דמויי decidual בתהליך המכונה decidualization4,6. Decidualization לא תקין נוצרה כמו שורש לבעיה עבור ההשתלה כשל ו עוקבות בתחילת העובר הפלה4,7,8. לכן, להבין את המנגנונים המולקולריים שבבסיס decidualization הוא יתרון באבחון וטיפול של אובדן הריון מוקדם.
כיום, מספר מתודולוגיות מנוצלים כדי לחקור את ההשפעות הבסיסית של decidualization על תאי סטרומה רירית הרחם. In vivo, הרחם העכבר יכולה להיגרם באופן מלאכותי על decidualization באמצעות גירוי מכני (קרי,מגרד) או שמן ההזרקה הרחם הורמונאלית להתקפת9. נבדל בני אדם, גירוי מלאכותי זה מקדם את הבידול של לומן הרחם על ידי מתן את המראה של הבלסטוציסט נוכחות, צעד זה נדרש עבור אתחול של decidualization מכרסמים10,11. בהתאם לכך, עקב סיבוכים translational הקשורים בבעלי חיים של דילמות סביב ויוו המבוסס על מחקרים בבני אדם, decidualization מבוסס מודלים ביותר בהצלחה נלמדים במבחנה.
במחקר זה, נושאים מגויסים דרך המיקום של פרסומות בעיתונים מקומיים בשתי באנגלית ובספרדית. נושאים מזוהה מועמדים מתאימים למחקר זה מובאים להיפגש עם מתאם מחקר, שבו מתוארים על גילוי מלא של סיכונים פוטנציאליים. אישור ההזמנה הבנה מלאה של הסיכונים הפוטנציאליים, מושגת ההסכמה של הנבדקים בצורות בכתב ומילולית. נושא הסכמה כוללת רשות (1) עוברים דם ורידי אחסון (2) לטווח ארוך של הרקמות שלהם למטרות מחקר עתידי, (3) מסכים ליצירתו של תרבויות העיקרי של דגימות רקמה שנאספו. בעקבות הסכמה, נותנים לנסיינים טופס כדי להשלים איזו self-identification המותר של גזע/מוצא אתני ו/או הזכות של סודיות. ביקור עוקבות מתוזמנת כדי להשיג את הביופסיה רירית הרחם בהתבסס על המחזור החודשי של הנושא. מתנדבים מגויס מחקר זה לשקף שני אתנית וגזעית דמוגרפיים של אזור המטרופולין סנט לואיס כפי שתועדו בידי מפקד 2012 ו לא היה כרוך ההשתתפות של כל אוכלוסיה פגיעה כולל נשים בהריון, העוברים, עוברי, ילדים מתחת לגיל 18 שנים של גיל או קבוצות פגיעות אחרות. דרישות הכשירות להשתתפות האוסף דגימת ביופסיה כוללים (1) להיות בין הגילאים 18-45 שנים (2) שיש מווסת סדירה (25-32 ימים) (3) לאחר ההריון הנוכחי וללא שימוש באמצעי מניעה הורמונליים/תוך רחמי ההתקן 30 ימים לפני ההרשמה (4) אין זיהום נרתיקי הנוכחי או מחלות המועברות במגע מיני (5) נתקל אין טיפולים אנטיביוטיים הנוכחי, ו (6) נתקל אין פאפ חריגות הנוכחי.
במסגרת מחקר זה, תאי סטרומה רירית הרחם (HESC) אדם תרבותי, המושרה באופן מלאכותי לעבור במבחנה decidualization דרך תוספת של הורמונים (אסטרדיול (E2) medroxyprogesterone אצטט (MPA), אדנוזין מחזורית monophosphate (מחנה)) למדיום. בשיטה זו, היא מידת decidualization שונה בהתבסס על המספר הכולל של ימים של טיפול הורמונלי. יחד עם התמורות cytoskeletal, תוספי ההורמונלי גורם עיבודים ביוכימי שבו התאים decidual לחוות הפרשה כמו איכויות2,4. הביטוי של גנים הולמרק, פרולקטין (PRL) ו פקטורי גדילה דמויי אינסולין מחייב חלבון 1 (IGFBP1), יכול להיות מנוצל כדי לאשר ולכמת את מידת HESC decidualization5,12, 13 , 14. חשוב, הכדאיות של פרוטוקול זה לנהל גנים ספציפיים נוקאאוט גם הוכח.

<table><tbody><tr><td>Opti-MEM I (1X) Reduced Serum Medium</td><td>Gibco</td><td>31985-070</td><td>For decidualization media</td></tr><tr><td>DMEM / F12 (1:1) (1X)</td><td>Gibco</td><td>11330-032</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td>For cell count</td></tr><tr><td>PureLink RNA Mini Kit</td><td>Invitrogen</td><td>12183018A</td><td>For RNA Isolation/Purification</td></tr><tr><td>TaqMan 2X Universal PCR Master Mix</td><td>Applied Biosystems</td><td>4304437</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4374967</td><td></td></tr><tr><td>Eukaryotic 18S rRNA Endogenous Control</td><td>Life Technologies</td><td>4319413E</td><td>For qPCR internal control</td></tr><tr><td>TaqMan Gene Expression Assay Prolactin Probe</td><td>Applied Biosystems</td><td>4331182</td><td>For qPCR internal control</td></tr><tr><td>TaqMan Gene Expression Assay IGFBP1 Probe</td><td>Applied Biosystems</td><td>4331182</td><td>For qPCR internal control</td></tr><tr><td>Phalloidin-iFluor 488 Reagent - CytoPainter</td><td>Abcam</td><td>ab176753</td><td></td></tr><tr><td>Human Prolactin ELISA Kit</td><td>Invitrogen</td><td>EHIAPRL</td><td></td></tr><tr><td>16% Paraformaldehyde</td><td>Alfa Aesar</td><td>30525-89-4</td><td>Fixative</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778150</td><td>Transfection Reagent</td></tr><tr><td>ProLong Gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P36935</td><td>For mounting</td></tr><tr><td>0.25% Trypsin-EDTA (1X)</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Charcoal Stripped Fetal Bovine Serum</td><td>Sigma</td><td>F6765-500ML</td><td></td></tr><tr><td>Sodium Bicarbonate (7.5%)</td><td>Gibco</td><td>25080-094</td><td></td></tr><tr><td>Ficoll-Paque PLUS Reagent</td><td>Fisher Scientific</td><td>45001749</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma</td><td>C0130-1G</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>DN25-100MG</td><td></td></tr><tr><td>Medroxyprogesterone 17-acetate</td><td>Sigma</td><td>M1629-1G</td><td>For EPC Media</td></tr><tr><td>Estradiol</td><td>Sigma</td><td>E1024-1G</td><td>For EPC Media</td></tr><tr><td>cAMP</td><td>Sigma</td><td>A6885-100MG</td><td>For EPC Media</td></tr><tr><td>Fine straight stitch scissors</td><td>Fine Science Tools</td><td>15396-00</td><td></td></tr><tr><td>TaqMan Gene Expression Assay NCOA2 Probe</td><td>Applied Biosystems</td><td>4351372</td><td></td></tr><tr><td>Fetal Bovine Serum, heat inactivated</td><td>Gibco</td><td>10-082-147</td><td></td></tr><tr><td>Antibiotic-antimycotic</td><td>Thermo Scientific</td><td>15240062</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Corning</td><td>21-021-cv</td><td></td></tr><tr><td>50 mL conical polypropylene Falcon tube</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td></td></tr><tr><td>100 x 15 mm Glass Petri dish</td><td>VWR</td><td>75845-546</td><td></td></tr><tr><td>40 micron cell strainer</td><td>Midsci</td><td>229481</td><td></td></tr><tr><td>Isotemp 215 Water bath</td><td>Fisher Scientific</td><td>FS-215</td><td></td></tr><tr><td>LSE Centrifuge&amp;nbsp;</td><td>Corning</td><td>6755</td><td></td></tr><tr><td>Human NCOA2 siRNA&amp;nbsp;</td><td>Dharmacon</td><td>L-020159-00</td><td>Gene specific qPCR probe</td></tr><tr><td>Steri-cycle i160 CO2 Incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>51030301</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td>For RNA quantification</td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>Fisher Scientific</td><td>50146771</td><td></td></tr><tr><td>75 cm Canted Neck Cell Culture flask</td><td>Corning</td><td>430641U</td><td></td></tr><tr><td>6 well Non-Pyrogenic Cell Culture Plate</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Pipet Controller Ultra</td><td>Corning</td><td>4099</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>19.0.1.334</td><td></td></tr><tr><td>25 cm Canted Neck Cell Culture flask</td><td>Corning</td><td>7200876</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-1L</td><td></td></tr><tr><td>15 mL conical polypropylene Falcon tube</td><td>Corning</td><td>352099</td><td></td></tr><tr><td>10 mL Syringe</td><td>BD</td><td>302995</td><td></td></tr><tr><td>1300 Series A2 Biological Safety Hood</td><td>Thermo Scientific</td><td>1377</td><td></td></tr><tr><td>accuspin Micro17 centrifuge</td><td>Fisher Scientific</td><td>13100675</td><td></td></tr><tr><td>7500 Fast real time PCR system</td><td>Applied Biosystems</td><td>3052632</td><td></td></tr><tr><td>EVOS FL Immunofluorescence Microscope</td><td>Life Technologies</td><td>01414-155G-291</td><td></td></tr><tr><td>Leica Inverted Light Microscope</td><td>Leica</td><td>DMi1</td><td></td></tr><tr><td>Illrustrator</td><td>Adobe</td><td>22.0.1.253</td><td></td></tr><tr><td>Excel Spreadsheets</td><td>Microsoft</td><td>2016</td><td></td></tr><tr><td>Deckglaser 18 mm cover glasses</td><td>NeuVitro</td><td>GG-18</td><td></td></tr><tr><td>Reichert Bright-Line Hemacytometer</td><td>Sigma</td><td>Z359629-1EA</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Fisher Bioreagents</td><td>BP176-100</td><td>For RNA lysis buffer</td></tr><tr><td>1.2mL External Threaded Polypropylene Cryogenic Vial</td><td>Corning</td><td>430658</td><td>For freezing HESC cells&amp;nbsp;</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>D2650-100mL</td><td>For freezing media</td></tr></tbody></table>

isolation of human endometrial stromal cells for in vitro decidualization

הבידול של רירית הרחם סטרומה תאים אנושיים (HESC) ממראה כמו פיברובלסט. לתוך רותמי הפרשה היא שינוי נדרש השרשת לתוך הרחם רירית רחם אימהי. Decidualization לא תקין נוצרה כמו שורש לבעיה כשל השרשה, הבאים הפלה העובר מוקדם. לכן, להבין את המנגנונים המולקולריים שבבסיס decidualization הוא יתרון בשיפור שיעור לידות מוצלחות. In vivo המבוסס על מחקרים של decidualization מלאכותיים הם הגבלת לעיתים קרובות עקב דילמות אתיות המשויך מחקר אנושי, כמו גם הסיבוכים translational בתוך חייתיים. כתוצאה מכך, במבחנה מבחני דרך התרבות התא הראשי מנוצלים לעיתים קרובות כדי לחקור את האפנון של decidualization באמצעות הורמונים. מחקר זה מספק נוהל מפורט בידודו של HESC ו- decidualization מלאכותיים עוקבות באמצעות תוספת של הורמונים למדיום culturing. עוד יותר, מחקר זה מספק שיטה מעוצב היטב נוקאאוט בכל הגן עניין על-ידי ניצול siRNA מבוססי השומנים transfections. פרוטוקול זה מאפשר אופטימיזציה של טוהר התרבות, כמו גם התשואה מוצר, ובכך למקסם את היכולת לנצל את המודל הזה בתור שיטה אמינה כדי להבין את המנגנונים המולקולריים שבבסיס decidualization, כימות עוקבות של סוכנים מופרשים על-ידי decidualized תאי סטרומה רירית הרחם.

Decidualization בתרבות HESC
בעקבות בידוד, תאי סטרומה רירית הרחם אדם היו בתרבית היווצרות חד שכבתי עם 80-90% confluency, שנגזרות עבור במבחנה decidualization בטיפול עם 10 ננומטר E2 1 מיקרומטר MPA, מחנה 50 מיקרומטר (EPC). משמרות מורפולוגי המשויך במבחנה decidu...

Watch this Scientific Journal Video about Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization at JoVE.com

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

מחקר זה מציג שגרה מאומת וממוטב עבור הבידוד של התרבות האנושית תאי סטרומה רירית הרחם לנהל במבחנה decidualization assay. יתרה מזאת, מחקר זה מספק שיטה מפורט ביעילות תמונות ציפורים גנים ספציפיים באמצעות siRNAs בתאי סטרומה רירית הרחם אדם.

לבידוד של תאי סטרומה רירית הרחם אדם במבחנה Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

isolation-human-endometrial-stromal-cells-for-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

10.1K Views.  Washington University School of Medicine. מחקר זה מציג שגרה מאומת וממוטב עבור הבידוד של התרבות האנושית תאי סטרומה רירית הרחם לנהל במבחנה decidualization assay. יתרה מזאת, מחקר זה מספק שיטה מפורט ביעילות תמונות ציפורים גנים ספציפיים באמצעות siRNAs בתאי סטרומה רירית הרחם אדם.

Video: Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

בידוד וההעשרה של אדם שומן המופק מתאי סטרומה לOsteogenesis משופר

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

בידוד הרחבת גזע mesenchymal / תאי סטרומה נגזרו רקמה אנושית שליה

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

בידוד מהיר של BMPR-IB + השומן שמקורם בתאי סטרומה לשימוש במודל ריפוי פגם calvarial

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

בידוד של עכבר רירית הרחם אפיתל סטרומה תאים עבור<em&gt; במבחנה</em&gt; Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

בידוד ואפיון של תאי סטרומה mesenchymal מן טבורי אדם העובר שליה

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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

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 culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

שתי שיטות לקביעת אדם תאי רירית הרחם סטרומה ראשיים מדוגמאות כריתת הרחם

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 ...

Medicine

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

יצירת מודל דצידואליזציה מלאכותית של עכבר עם כריתת שחלות למחקר דצידואליזציה של רירית הרחם

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

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.

בידוד של ראשי תאים Decidual האנושי בקרום עוברי של המונח Placentae

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

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

הקמת אורגנואידים 3D רירית הרחם מרחם העכבר

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Biology

<meta charset="utf8"></head><body>מודל פיגום מודפס בתלת מימד לחקירת מנגנוני השתלת עוברים

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

<meta charset="utf8"></head><body>זרקור המחבר: מודלים של רירית הרחם במבחנה לחקר אינטראקציות אפיתל-סטרומה ועוברים

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

מודל תרבית משותפת באמצעות שני סוגים של קווי תאים דבקים

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...