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

The authors have nothing to disclose.

All human endometrium biopsies collected for this study were attained from the Washington University in St. Louis, Department of Obstetrics and Gynecology using an Institutional Review Board (IRB) approved written consent form.
1. Preparation
<ol>
	<li>Prepare 500 mL of 1x Hank&#39;s Balanced Salt Solution (HBSS) by adding 100 U/mL penicillin and 100 &#181;g/mL streptomycin to the media containing bottle (further referenced as HBSS+ medium).</li>
	<li>Prepare 500 mL of Dulbecco&#39;s Modifie...

<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>

The female reproductive menstrual cycle is characterized by a rise in progesterone levels throughout the luteal phase, thereby inducing the decidualization of ESC into round, epithelial-like secretory cells3,8. The initiation of decidualization is species dependent. In humans, decidualization occurs spontaneously upon the rise of progesterone concentration, whereas mice require blastocyst presence10,11. T...

Between the stages of menarche and menopause, women of reproductive age undergo monthly cycles of hormone-regulated endometrial proliferation, differentiation, and subsequent shedding in preparation for pregnancy in a process known as menstruation1,2. Such physical modifications of the human endometrium are necessary for proper embryo implantation into the uterine wall1. Alterations of the endometrium, including both morphological and biochemical adaptations, are mediated throughout the menstrual cycle via ovarian steroid hormones estrogen and progesterone (P4)3,4,5. Within the proliferative (or follicular) phase, preovulatory estrogen levels increase, initiating endometrium thickening. Following ovulation, the secretory (or luteal) phase promotes a significant rise in P4 concentrations, inducing the morphological transformation of endometrial stromal cells (ESC) from fibroblast-like appearance to rounded, epithelial-like decidual cells in a process known as decidualization4,6. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage4,7,8. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to the diagnosis and treatment of early pregnancy loss.
Currently, several methodologies are utilized to explore the underlying effects of decidualization on endometrial stromal cells. In vivo, the mouse uterus can be artificially induced for decidualization via mechanical stimulation (i.e.,, scratching) or oil injection in a hormonally primed uterus9. Distinct from humans, this synthetic stimulation promotes the differentiation of the uterine lumen by providing the appearance of blastocyst presence, a step that is required for the initiation of decidualization in rodents10,11. Accordingly, due to the translational complications associated with animal models and the ethical dilemmas surrounding in vivo based studies in humans, decidualization based models are most successfully studied in vitro.
In this study, subjects are recruited through the placement of advertisements in both local English and Spanish newspapers. Subjects identified as suitable candidates for this study are brought in to meet with the research coordinator, in which a full disclosure of potential risks are discussed. Upon confirmation of a complete understanding of potential risks involved, subjects&#39; consent is attained in both written and verbal forms. Subject consent includes permission to (1) undergo phlebotomy (2) long-term storage of their tissues for future research purposes and (3) agree to the creation of primary cultures from collected tissue specimens. Following consent, subjects are given a form to complete in which permitted self-identification of race/ethnicity and/or the right for nondisclosure. A subsequent visit is scheduled to attain the endometrium biopsy based on the subject&#39;s menstrual cycle. Volunteers recruited to this study reflect both the ethnic and racial demographics of the St. Louis metropolitan region as documented by the 2012 Census and did not involve the participation of any vulnerable population including pregnant women, fetuses, embryos, children under 18 years of age, or other vulnerable groups. Eligibility requirements for participation in the biopsy sample collection include (1) being between the ages of 18-45 years (2) having regular menstrual cycles (25-32 days) (3) having no current pregnancy or use of hormonal/intrauterine device contraceptives for 30 days prior to enrollment (4) having no current vaginal infection or sexually transmitted diseases (5) having no current antibiotic treatments, and (6) having no current abnormal Pap smear.
Within this study, human endometrial stromal cells (HESC) are cultured and artificially induced to undergo in vitro decidualization through the supplementation of hormones (estradiol (E2), medroxyprogesterone acetate (MPA), and cyclic adenosine monophosphate (cAMP)) to the medium. In this method, the degree of decidualization is altered based on the total number of days of hormonal treatment. In conjunction with cytoskeletal rearrangement, hormonal supplementation induces biochemical adaptations in which the decidual cells experience secretory-like qualities2,4. The expression of hallmark genes, such as prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1), can be utilized to confirm and quantify the degree of HESC decidualization5,12,13,14. Importantly, the viability of this protocol to conduct gene specific knockdown is also demonstrated.

<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

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Decidualization in HESC Culture
Following isolation, human endometrial stromal cells were cultured to a monolayer formation with 80-90% confluency and induced for in vitro decidualization by treating with 10 nM E2, 1 &#181;M MPA, and 50 &#181;M cAMP (EPC). Morphological shifts associated with in vitro decidualization were visualized in Figure 1A. Upon decidualizati...

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

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

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

Research

JoVE Journal

Developmental Biology

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

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

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In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

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In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...