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

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Alle menneskelige endometrium biopsier samlet denne studien var oppnådd fra Washington University i St. Louis, Institutt for obstetrikk og gynekologi ved hjelp av en institusjonell Review Board (IRB) godkjent skriftlig samtykke.
1. forberedelse
<ol><li>Forberede 500 mL 1 x Hanks balansert Salt løsning (HBSS) ved å legge til 100 U/mL penicillin og 100 µg/mL streptomycin media inneholder flasken (videre referert til som HBSS + medium).</li>
	<li>Forberede 500 mL Dulbeccos endret Eagle Medi...

<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>

Kvinnelige reproduktive menstruasjonssyklusen er preget av en økning i progesteronnivåer gjennom luteal fase, og dermed indusere decidualization av ESC i runde, epithelial-lignende sekretoriske celler3,8. Initiering av decidualization er avhengig. Hos mennesker oppstår decidualization spontant på fremveksten av progesteron konsentrasjon, mens mus kreve blastocysten tilstedeværelse10,11. Denne inkonse...

Mellom stadier av menarke og menopause gjennomgå kvinner i reproduktiv alder månedlige sykluser av hormon-regulert endometrial spredning, differensiering og påfølgende blodsutgytelse i forberedelse til graviditet i en prosess som kalles menstruasjon1 ,2. Slike fysiske endringer av menneskelig endometrium er nødvendig for riktig embryoet implantasjon inn i livmor veggen1. Endringer av endometrium, inkludert både morfologiske og biokjemiske tilpasninger, er formidlet gjennom menstruasjonssyklusen via ovarian steroidhormoner østrogen og progesteron (P4)3,4,5. Innenfor den proliferativ (eller follikulær) fasen, preovulatory estrogen nivåer øker, starte endometrium jevning. Etter eggløsning fremmer sekretoriske (eller luteal) fasen en betydelig økning i P4 konsentrasjoner inducing morfologiske transformasjonen av endometrial stromal celler (ESC) fra fibroblast-lignende utseende til avrundet, epithelial-lignende decidual celler i en prosess kjent som decidualization4,6. Feil decidualization har blitt etablert som en rotårsak for implantasjon feil og påfølgende tidlig embryo spontanabort4,7,8. Derfor er forstå molekylære mekanismer underliggende decidualization fordelaktig for diagnostisering og behandling av tidlig graviditet.
For tiden benyttes flere metoder for å utforske underliggende virkningene av decidualization på endometrial stromal celler. I vivo, musen livmoren kan være kunstig indusert for decidualization via mekanisk stimulering (dvs., skrape) eller olje injeksjon i hormonally primet livmoren9. Forskjellig fra mennesker, fremmer dette syntetiske stimulering differensiering av livmor lumen ved å gi utseendet av blastocysten tilstedeværelse, et skritt som kreves for oppstart av decidualization i Red10,11. Følgelig, på grunn av translasjonsforskning komplikasjoner knyttet dyremodeller og etiske dilemmaer rundt i vivo studier i mennesker, er decidualization basert modeller mest vellykket studerte i vitro.
I denne studien er fag rekruttert gjennom plasseringen av annonser i både engelsk og spansk lokalaviser. Emner som egnet kandidater til denne studien er brakt i å møte den koordinatoren, som en full avsløring av potensielle risikoer er omtalt. Idet en fullstendig forståelse av risiko involvert, er fag samtykke oppnådd i både skriftlig og muntlig former. Emnet samtykke inkluderer tillatelse (1) gjennomgå phlebotomy (2) langsiktig lagring av deres vev for fremtidige forskningsformål og (3) samtykke til etableringen av primære kulturer fra samlet vevsprøver. Etter samtykke får fag et skjema fylle ut som tillatt selv-identifikasjon av rase/etnisitet og/eller til høyre for taushetsplikt. Etterfølgende besøk er planlagt å oppnå endometrium biopsi basert på emnet menstrual syklus. Frivillige rekruttert til denne studien reflektere både etniske og rasemessige demografi av St. Louis hovedstadens som dokumentert av folketellingen i 2012 og involverer ikke deltakelse av alle sårbare befolkningen inkludert gravide, fostre, embryo, barn under 18 år, eller andre sårbare grupper. Kravene til deltagelse i samlingen biopsi prøven inkluderer (1) å være i alderen 18-45 år (2) å ha vanlig menstrual sykluser (25-32 dager) (3) har ingen gjeldende graviditet eller bruk av hormonelle/intrauterine enhet piller i 30 dager før registrering (4) ingen gjeldende vaginal infeksjon eller seksuelt overførbare sykdommer (5) har ingen aktuelle antibiotika behandlinger, og (6) har ingen gjeldende unormal Pap smear.
I denne studien er menneskelige endometrial stromal celler (HESC) kultivert og kunstig overtalt til å gjennomgå i vitro decidualization gjennom tilskudd av hormoner (østradiol (E2) medroxyprogesterone acetate (MPA) og syklisk adenosin monofosfat (cAMP)) til mediet. I denne metoden endres graden av decidualization basert på antall dager av hormonell behandling. I forbindelse med cellen cytoskjelett omorganisering induserer hormonelle kosttilskudd biokjemiske tilpasninger som decidual cellene oppleve sekretoriske-lignende egenskaper2,4. Uttrykket av hallmark gener som prolaktin (PRL) og insulin-lignende vekstfaktor bindende protein 1 (IGFBP1), kan brukes til å bekrefte og kvantifisere graden av HESC decidualization5,12, 13 , 14. viktigst, levedyktigheten til denne protokollen å gjennomføre genet bestemt knockdown er også demonstrert.

<table border="0" cellpadding="0" cellspacing="0" style="width:935px;" width="934">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Opti-MEM I (1X) Reduced Serum Medium</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">31985-070</td>
			<td style="width:271px;">For decidualization media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DMEM / F12 (1:1) (1X)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">15250-061</td>
			<td>For cell count</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PureLink RNA Mini Kit</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">12183018A</td>
			<td>For RNA Isolation/Purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan 2X Universal PCR Master Mix</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4304437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4374967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Eukaryotic 18S rRNA Endogenous Control</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:131px;">4319413E</td>
			<td>For qPCR internal control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan Gene Expression Assay Prolactin Probe</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4331182</td>
			<td>For qPCR internal control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan Gene Expression Assay IGFBP1 Probe</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4331182</td>
			<td>For qPCR internal control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phalloidin-iFluor 488 Reagent - CytoPainter</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:131px;">ab176753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Human Prolactin ELISA Kit</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">EHIAPRL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">16% Paraformaldehyde</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:131px;">30525-89-4</td>
			<td>Fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lipofectamine RNAiMAX</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">13778150</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ProLong Gold antifade reagent with DAPI</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:131px;">P36935</td>
			<td>For mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Penicillin Streptomycin</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">F6765-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sodium Bicarbonate (7.5%)</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">25080-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ficoll-Paque PLUS Reagent</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">45001749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">C0130-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">DN25-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Medroxyprogesterone 17-acetate</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">M1629-1G</td>
			<td>For EPC Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Estradiol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">E1024-1G</td>
			<td>For EPC Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">cAMP</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">A6885-100MG</td>
			<td>For EPC Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fine straight stitch scissors</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:131px;">15396-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TaqMan Gene Expression Assay NCOA2 Probe</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">4351372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fetal Bovine Serum, heat inactivated</td>
			<td style="width:167px;">Gibco</td>
			<td style="width:131px;">10-082-147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-antimycotic</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">21-021-cv</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical polypropylene Falcon tube</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dumont #5/45 Forceps</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:131px;">11251-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">100 x 15 mm Glass Petri dish</td>
			<td style="width:167px;">VWR</td>
			<td style="width:131px;">75845-546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">40 micron cell strainer</td>
			<td style="width:167px;">Midsci</td>
			<td style="width:131px;">229481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isotemp 215 Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">FS-215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">LSE Centrifuge&#160;</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">6755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Human NCOA2 siRNA&#160;</td>
			<td style="width:167px;">Dharmacon</td>
			<td style="width:131px;">L-020159-00</td>
			<td>Gene specific qPCR probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Steri-cycle i160 CO2 Incubator&#160;</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">51030301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">NanoDrop 2000</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">ND-2000</td>
			<td>For RNA quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phosphate Buffered Saline&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">50146771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">75 cm Canted Neck Cell Culture flask</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">430641U</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">6 well Non-Pyrogenic Cell Culture Plate</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Pipet Controller Ultra</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">4099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Photoshop</td>
			<td style="width:167px;">Adobe</td>
			<td style="width:131px;">19.0.1.334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">25 cm Canted Neck Cell Culture flask</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">7200876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Triton X-100</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">X100-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">15 mL conical polypropylene Falcon tube</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">10 mL Syringe</td>
			<td style="width:167px;">BD</td>
			<td style="width:131px;">302995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1300 Series A2 Biological Safety Hood</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:131px;">1377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">accuspin Micro17 centrifuge</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:131px;">13100675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">7500 Fast real time PCR system</td>
			<td style="width:167px;">Applied Biosystems</td>
			<td style="width:131px;">3052632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">EVOS FL Immunofluorescence Microscope</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:131px;">01414-155G-291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Leica Inverted Light Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:131px;">DMi1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Illrustrator</td>
			<td style="width:167px;">Adobe</td>
			<td style="width:131px;">22.0.1.253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Excel Spreadsheets</td>
			<td style="width:167px;">Microsoft</td>
			<td style="width:131px;">2016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Deckglaser 18 mm cover glasses</td>
			<td style="width:167px;">NeuVitro</td>
			<td style="width:131px;">GG-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Reichert Bright-Line Hemacytometer</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">Z359629-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2-mercaptoethanol</td>
			<td style="width:167px;">Fisher Bioreagents</td>
			<td style="width:131px;">BP176-100</td>
			<td>For RNA lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">1.2mL External Threaded Polypropylene Cryogenic Vial</td>
			<td style="width:167px;">Corning</td>
			<td style="width:131px;">430658</td>
			<td>For freezing HESC cells&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:131px;">D2650-100mL</td>
			<td>For freezing media</td>
		</tr>
	</tbody>
</table>

isolation of human endometrial stromal cells for in vitro decidualization

Differensiering av menneskelige endometrial stromal celler (HESC) fra fibroblast utseende i sekretoriske decidua er en forandring nødvendig for implantasjon av embryoet i livmor lining av mors livmor. Feil decidualization har blitt etablert som en rotårsak for implantasjon svikt og påfølgende tidlig embryo spontanabort. Derfor er forstå molekylære mekanismer underliggende decidualization en fordel å forbedre frekvens vellykket fødsler. I vivo studier av kunstig decidualization er ofte begrenser på grunn etiske dilemmaer knyttet til menneskelige forskning, samt translasjonsforskning komplikasjoner i dyremodeller. Resultatet benyttes i vitro analyser gjennom grunnskolen cellekultur ofte til å utforske modulering av decidualization via hormoner. Denne studien gir en detaljert protokoll for isolering av HESC og påfølgende kunstig decidualization via kosttilskudd av hormoner til culturing medium. Videre, denne studien gir en velutformet metode til knockdown noen genet av interesse ved å benytte lipid-baserte siRNA transfections. Denne protokollen tillater optimalisering av kultur renhet samt produkt avkastning, og dermed maksimere muligheten til å utnytte denne modellen som en pålitelig metode for å forstå molekylære mekanismer underliggende decidualization og den påfølgende kvantifiseringen av utskilles agenter av decidualized endometrial stromal celler.

Decidualization i HESC kultur
Etter isolasjon, endometrial stromal menneskeceller ble kultivert å en monolayer med 80-90% confluency og indusert for i vitro decidualization ved å behandle med 10 nM E2, 1 µM MPA og 50 µM leiren (EPC). Morfologiske Skift knyttet i vitro decidualization var visualisert i figur 1A. På decidualization gjennom HESCs cellen cytoskjel...

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

Denne studien presenterer en validert og optimalisert prosedyre for isolasjon og kultur av menneskelige endometrial stromal celler å gjennomføre i vitro decidualization analysen. Videre, denne studien gir en detaljert metode for å effektivt knockdown et bestemt gen bruker siRNAs i menneskeceller endometrial stromal.

Isolering av Endometrial Stromal menneskeceller for In Vitro Decidualization

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

This method can be used to answer key questions in the reproductive medicine field about regarding the underlying molecular mechanisms of decidualization, and understanding the roles of specific genes in endometrial stromal cell decidualization. This study provides a detailed protocol for the isolation of human endometrial stromal cells and subsequent artificial decidualization by the supplementation of hormones to the culturing media. The expression of hormonal change, such as prolactin and insulin-like growth factor binding protein one can be utilized to confirm and quantify the degree of decidualization.Importantly, the viability of this protocol to conduct gene specific knockdown is also demonstrated. Transfer the biopsy with sterilized forceps into a 50 milliliter tube containing ten milliliters fresh HBSF, followed by centrifugation at 500x G for 90 seconds. Using a one mL pipette, remove the media and add three mL pre-warmed hES isolation media.Decant the biopsy into a 10 centimeter dish containing five mL of hES isolation media, and begin chopping using Dumont 5 forceps and fine straight stitch scissors for about 20 minutes. Using one mL pipette, transfer the chopped biopsy into a new 50 mL tube, and add an additional seven mL of hES isolation media to the dish, to transfer the remaining pieces to the 50 mL tube. Then centrifuge the sample for two minutes at 800x G, and remove the supernatant using a one mL pipette.Next filter the DNase-1 and collagenase digestion mix through a 0.2 micron filter attached to a 10 milliliter syringe directly onto the pellet. Then vortex the tube for 10 seconds to resuspend the pellet, and incubate for 90 minutes in a water bath. After every 10 minutes, vortex the tube for 10 seconds to make sure the tissue is properly digested.After incubation, filter the suspension through a 40 micron cell strainer stacked over a new 50 milliliter tube to recover the stromal cells, then centrifuge the collected cells at 800x G for 2.5 minutes. Following centrifugation, gently remove the supernatant by using a one milliliter pipette, making sure not to disturb the cell pellet. Then resuspend the cell pellet in 10 milliliters of fresh hES isolation media, followed by centrifugation at 800x G for 2.5 minutes.After centrifugation, aspirate the supernatant carefully, and resuspend the cells in six milliliters fresh hES media. Next, to remove blood cells, slowly layer resuspended cells onto three milliliters of density gradient media, and centrifuge the sample at 400x G for 30 minutes. Following centrifugation, collect the supernatant without disturbing the density gradient media.Then add hES media to make the total volume up to 10 milliliters, and centrifuge the sample again at 800x G for 2.5 minutes. After centrifugation, remove the supernatant and resuspend the cells in six milliliters of hES media. Next plate the cells in a 75 centimeter flask by adding six milliliters of the cell resuspension and 10 milliliters of hES media to the flask, followed by gentle mixing through rocking the flask.After plating, check the cell density under the microscope, then culture the cells in a 5%carbon dioxide incubator for three to five days, making sure to change the hES media every two days. After 48 hours, the cells should grow as a monolayer. Generally after five days, cells achieve 80 to 90%confluency in a 75 centimeter flask.For siRNA transfection, begin by plating 800, 000 cells per well in a six well plate in triplicates for each condition, and incubate the cells in a 5%carbon dioxide incubator for two days to prepare for transfection. After two days, add 150 microliters of Opti-MEM serum free media in a five mL tube, followed by the addition of five microliters of transfection reagent. Mix well by inversion, and incubate for five minutes.Next add 100 microliters of Opti-MEM serum free media in a new five mL tube, followed by the addition of 16 nanomoles of SRC-2 siRNA. Then add 100 microliters of Opti-MEM serum free media in a five mL tube, followed by the addition of 16 nanomoles control siRNA. After incubation, combine the diluted transfection reagent with the SRC-2 siRNA and mix well by pipetting forward and reverse.Then combine the diluted transfection reagent with the control siRNA and mix well by pipetting forward and reverse. Then incubate the complexes for five minutes. During this incubation, aspirate out the hES media, and add one milliliter of change media to each well.After five minutes of incubation, centrifuge the complexes at 500x G for two minutes to collect the solution. Then add 250 microliters of the siRNA transfection reagent complexes to each well containing cells and media, and mix by gentle rocking, followed by incubation in a 5%carbon dioxide incubator for five hours at 37 degrees Celsius. After five hours, aspirate the media and change to hES media, and continue incubation for two days in preparation for an in-vitro decidualization assay.Post 48-hour transfection and prior to initiating the in vitro decidualization assay, collect the day zero control siRNA and SRC-2 siRNA transfected cells. First, aspirate the media and wash with one mL PBS per well. Then remove the PBS, and add 300 microliters of RNA lysis buffer per well and incubate for three minutes.After three minutes of incubation, collect the cells in RNA lysis buffer into a 1.5 ml tube for later downstream analysis. Then prepare the EPC media by aliquoting the appropriate volume of decidualization media, and add 10 nanomolar estradiol, one micromolar medroxyprogesterone acetate, and 50 micromolar cyclic adenosine monophosphate in a 50 milliliter tube. For remaining day six wells, add two milliliters of EPC media into each well, and incubate the cells in a 5%carbon dioxide incubator at 37 degrees Celsius for six days, changing the EPC media every 48 hours.After the sixth day, collect the day six control siRNA and day six SRC-2 siRNA transfected cell pellets in RNA lysis buffer for downstream analysis using real-time PCR. The induction of decidualization by estrogen medroxyprogesterone acetate and cyclic adenosine monophosphate, results in the cytoskeletal rearrangement of stromal cells from elongated fibroblastic cells to rounded epitheliod cells. Phalloidin staining of endometrial stromal cells further reveals the active fiber cytoskeleton reorganization characteristic of hES decidualization.In vehicle treated hES, cells remain fibroblastic in appearance, whereas in EPC treated cells, hES appeared epithelioid like. To confirm the efficacy of the in-vitro decidualization assay, mRNA transcript levels of decidualization markers prolactin and insulin-like growth factor binding protein-1 were assessed over a 6 day period. Upon decidualization of human endometrial stromal cells, prolactin and IGFBP1 levels increased in a time-dependent fashion.The time-dependent increase of secreted prolactin levels was also confirmed by examining the hES tissue culture media via ELISA method. Gene specific knockdown effects on the cellular and molecular changes of human endometrial stromal cell decidualization were assessed on the sixth day of culture post 48-hour siRNA transfection, with control or steroid receptor co-activator 2 siRNA. Control siRNA transfected hES demonstrated cellular and molecular changes, consistent with proper decidualization, including a cobblestone morphological change and increased transcript levels of decidualization markers PRL and IGFBP1.In SRC-2 siRNA knockdown, hES remained fibroblastic in appearance as compared to the control siRNA transfected hES, and showed significantly decreased PRL and IGFBP1 transcript levels. SRC-2 transcript levels were significantly decreased in SRC-2 siRNA transfected hES when compared to control siRNA, indicating the viability of this method for gene silencing. After watching this video, you should have a good understanding of how to isolate primary stromal cells from endometrial biopsies for the purpose of in-vitro decidualization and knockdown a specific gene of interest using siRNA transfection.

Research

JoVE Journal

Developmental Biology

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

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