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 Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) with phenol red, L-glutamine, and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) by adding 1% antibiotic-antimycotic solution (the final concentration is 100 U/mL Penicillin, 100 &#181;g/mL Streptomycin, and 0.25 &#181;g/mL Fungizone) to the media containing bottle (Further referenced as HESC Isolation Media).</li>
	<li>Prepare 500 mL of DMEM/F12 with phenol red, L-glutamine, and 15 mM HEPES by adding 10% Fetal Bovine Serum (FBS), 1x Na2HCO3, and 1% Penicillin (10,000 U/mL) and Streptomycin (10,000 &#181;g/mL) (Pen Strep) to the media containing bottle (Further referenced as HESC Media).</li>
	<li>Prepare 500 mL of Reduced Serum Medium with L-Glutamine, 2.4 g/L sodium bicarbonate, and HEPES with 2% Charcoal Stripped Fetal Bovine Serum (csFBS) and 1% Pen Strep to the media containing bottle (Further referenced as Decidualization Media).</li>
	<li>Prepare 500 mL of Phenol red free DMEM/F12 by adding 2% charcoal stripped fetal bovine serum (csFBS) in the media containing bottle (Further referenced as Change Media).</li>
	<li>Prepare 10 nM estradiol (E2), 1 &#181;M medroxyprogesterone acetate (MPA), and 50 &#181;M cyclic adenosine monophosphate (cAMP) in a 15 mL polypropylene conical tube, and add to a 2 mL per well aliquot of previously prepared decidualization media for in vitro decidualization assay (Further referenced as EPC Media).</li>
	<li>Prepare 50 mL of freezing media with 90% FBS and 10% Dimethyl sulfoxide (DMSO) in 50 mL polypropylene conical tube (Further referenced as Freezing Media).</li>
	<li>Pre-weigh 25 mg of Collagenase and 5 mg of DNase I in a 50 mL conical polypropylene tube and store at -20&#176;C.</li>
</ol>
2. Human Endometrium Biopsy Acquisition
<ol>
	<li>Obtain human endometrium biopsy samples collected in the proliferative phase (Day 9 - Day 12) of the menstrual cycle from the IRB approved clinic in HBSS.</li>
	<li>Wash biopsy samples with 1 mL of prewarmed 37 &#176;C HBSS+ medium and gently shake to remove excess blood and mucous.</li>
</ol>
3. Isolation of Human Endometrial Stromal Cells (HESC)
Note: This isolation procedure is performed in a sterile environment under a biological safety cabinet.
<ol>
	<li>Transfer the biopsy with sterilized forceps into a 50 mL conical polypropylene centrifuge tube containing 10 mL fresh HBSS.</li>
	<li>Centrifuge the biopsy at room temperature at 500 x g for 90 s.
	<ol>
		<li>If the cell pellet ruptures, re-spin the biopsy at 2,000 x g for 90 s.</li>
	</ol>
	</li>
	<li>Remove the media using a 1 mL pipet and wash with 10 mL of 37 &#176;C prewarmed HESC Isolation Media followed by centrifugation at 500 x g for 90 s.</li>
	<li>Remove the media using a 1 mL pipet, and vigorously add 3 mL of fresh HESC Isolation Media to dislodge the biopsy.</li>
	<li>Decant the biopsy into a Petri dish containing 5 mL of HESC Isolation Media.</li>
	<li>Mince the biopsy into as small pieces as possible using #5 forceps and fine straight stitch scissors for about 20 minutes.</li>
	<li>Prepare DNase I and Collagenase enzymes by adding 10 mL of HESC Isolation Media to pre-weighed DNase I (0.5 mg/mL) and Collagenase (2.5 mg/mL). Mix with pipet.</li>
	<li>Pipette cut tissue samples using a 1 mL pipet into a new 50 mL conical polypropylene tube. Wash with 7 mL of HESC Isolation Media and add to the polypropylene tube to acquire the full sample.</li>
	<li>Centrifuge the sample at room temperature at 800 x g for 2 min. Gently remove the supernatant by pipette.</li>
	<li>Filter the DNase I and Collagenase solution through 0.2 &#181;m filter attached to 10 mL syringe directly onto pellet.</li>
	<li>Vortex for 10 s to resuspend pellet.</li>
	<li>Digest in 37 &#176;C water bath for 1.5 h, vortexing for 10 s thoroughly every 10 min, until tissue is digested.</li>
	<li>Filter the sample through a 40 &#181;m cell strainer stacked over a 50 mL conical polypropylene tube to remove the epithelial cells and tissue debris. 
	NOTE: Stromal cells are in the 50 mL conical polypropylene tube.</li>
	<li>Centrifuge the 50 mL conical polypropylene tube at 800 x g for 2.5 min at room temperature. Gently remove the supernatant with a 1 mL pipette.</li>
	<li>Resuspend the pellet in 10 mL of HESC Isolation Media.</li>
	<li>Centrifuge at 800 x g for 2.5 min at room temperature. Aspirate the supernatant, and then resuspend cells in 6 mL of HESC Media and pipette forward and reverse to mix.</li>
	<li>Slowly layer on 3 mL of density gradient media to the resuspended cells to separate out remaining blood cells from the stromal cells, being careful not to mix the layers.</li>
	<li>Centrifuge the 15 mL polypropylene tube at 400 x g for 30 min at room temperature.</li>
	<li>Collect 5 mL of the supernatant in a new 50 mL polypropylene tube and add an additional 5 mL HESC Media to resuspend the cells.</li>
	<li>Centrifuge at 800 x g for 2.5 min at room temperature, followed by removal of the supernatant and addition of 6 mL HESC Media. Vortex the sample for 10 s and mix by pipetting forward and reverse 5 times.</li>
	<li>Aliquot 10 &#181;L of re-suspended cells to a 1.5 mL microcentrifuge tube for cell count.</li>
	<li>Add 10 &#181;L of 0.4% Trypan Blue stain to cell aliquot and mix with pipet. Load 10 &#181;L of the aliquot and count the cells using a hemocytometer.</li>
	<li>Plate the cells in an appropriate flask dependent on total cell count in 15 mL HESC Media.
	<ol>
		<li>If the total number of cells is less than 1 million, plate all cells in 25-cm flask with 6 mL of HESC Media. If the total number of cells is more than 1 million, plate all cells in 75-cm flask with 15 mL of HESC Media.</li>
	</ol>
	</li>
	<li>Culture the cells in a 5% CO2 incubator at 37 &#176;C for a minimum of 6 h to ensure proper cell adhesion and change the media to HESC Media.</li>
	<li>Culture the isolated HESC in a 5% CO2 incubator at 37 &#176;C for a period of 3-5 days until the cells form a monolayer achieving 80 - 90% confluency in a plated flask.</li>
	<li>Change HESC Media every two days.
	<ol>
		<li>Warm HESC Media in a 37 &#176;C water bath for 15-20 min.</li>
		<li>Aspirate media from the flask and add fresh HESC Media to the flask (working volume: 8 mL or 16 mL per 25-cm or 75-cm flask respectively).</li>
		<li>Place flask back into the 5% CO2 incubator at 37 &#176;C until 80-90% confluency is achieved.</li>
	</ol>
	</li>
	<li>Once HESC become 80 - 90% confluent, transfer cells from 25-cm to 75-cm flask or from one 75-cm flask to three 75-cm flasks. 
	NOTE: HESC can be frozen and stored for later usage at this time for future experimentation.</li>
</ol>
4. Freezing and Thawing HESCs
<ol>
	<li>Aspirate media from the 75-cm flask and add 2 mL of trypsin-EDTA.</li>
	<li>Incubate in a 5% CO2 incubator for 2-3 min at 37 &#176;C until the cells dislodge from the flask.</li>
	<li>Add 7 mL of HESC Media and mix well by pipetting forward and reverse.</li>
	<li>Centrifuge at 800 x g for 2.5 min and aspirate the supernatant.</li>
	<li>Label freezing tubes with cell line, date, and passage number.</li>
	<li>Re-suspend the cell pellet in 5 mL of freezing media.</li>
	<li>Aliquot 1 mL of the re-suspended HESC to each tube and transfer tubes to -80 &#176;C freezer.</li>
	<li>Transfer tubes to liquid nitrogen within 24 hours. 
	NOTE: If planning to thaw cells in immediate future, store frozen cells in -80 &#176;C for one month.</li>
	<li>For thawing the HESCs preheat HESC Media for 20 min.</li>
	<li>Add 8 mL of preheated HESC Media&#160;to 25-cm flask.</li>
	<li>Retrieve the freezing tube from the liquid nitrogen tank or -80&#160;&#176;C and submerge the tube in 37 &#176;C water bath for 1-2 min to thaw the cells.</li>
	<li>Transfer thawed HESC to a 25-cm flask with HESC Media and incubate in a 5% CO2 incubator overnight at 37 &#176;C.</li>
	<li>Next day, change the HESC Media and wait for 2-3 days until HESC become confluent and transfer cells to 75-cm flask as detailed below in Protocol 5.
	<ol>
		<li>Warm HESC Media in a 37 &#176;C water bath for 15-20 min.</li>
		<li>Aspirate media from the flask and add fresh HESC Media to the flask (working volume: 8 mL or 16 mL per 25-cm or 75-cm flask respectively).</li>
		<li>Place flask back into the 5% CO2 incubator at 37 &#176;C until 80-90% confluency is achieved.</li>
	</ol>
	</li>
</ol>
5. Human Endometrial Stromal Cell (HESC) Culturing
Note: This Culturing procedure is performed in a sterile environment under a biological safety cabinet.
<ol>
	<li>Pre-heat HESC Media at 37&#176;C in a water bath for 20-30 min.</li>
	<li>Aspirate media from the flask and add 0.25% Trypsin ethylenediaminetetraacetic acid (EDTA) (1 mL for 25-cm flask or 2 mL for 75-cm flask).</li>
	<li>Incubate in a 5% CO2 incubator at 37 &#176;C for 2-3 min until the cells dislodge.</li>
	<li>Gently tap the flask walls to dislodge the remaining cells. Add 7 mL of HESC Media and mix well by pipetting forward and reverse.</li>
	<li>Pipet the cell mixture into a 50 mL conical polypropylene tube and centrifuge at 800 x g for 2.5 min at room temperature.</li>
	<li>Aspirate the supernatant and re-suspend the cell pellet in HESC Media.
	<ol>
		<li>Add 15 mL for 25-cm flask or 45mL for 75-cm flask (15 mL each).</li>
	</ol>
	</li>
	<li>Add 5 mL or 15 mL cell suspension to each 25-cm or 75-cm flask respectively.</li>
	<li>Culture the isolated HESC in a 5% CO2 incubator at 37 &#176;C for a period of 3-5 days until the cells form a monolayer achieving 80 - 90% confluency in a plated flask.</li>
	<li>Change HESC Media every two days.
	<ol>
		<li>Warm HESC Media in a 37 &#176;C water bath for 15-20 min.</li>
		<li>Aspirate media from the flask and add fresh HESC Media to the flask (working volume: 8 mL or 16 mL per 25-cm or 75-cm flask respectively).</li>
		<li>Place flask back into the 5% CO2 incubator at 37 &#176;C until 80-90% confluency is achieved.</li>
	</ol>
	</li>
</ol>
6. Human Endometrial Stromal Cell (HESC) Plating and siRNA Transfection
Note: This transfection procedure is performed in a sterile environment under a biological safety cabinet.
<ol>
	<li>Aspirate HESC Media from the 75-cm flask and add 2 mL of 0.25% trypsin-EDTA solution.</li>
	<li>Incubate in 5% CO2 incubator at 37 &#176;C for 2-3 min until the cells dislodge.</li>
	<li>Once cells are dislodged from the flask, add 7 mL of HESC Media and mix gently by pipetting forward and reverse 5 times.</li>
	<li>Pipet the cell mixture into a 50 mL conical polypropylene tube and centrifuge at 800 x g for 2.5 min at room temperature. Aspirate the supernatant and re-suspend the cell pellet in 10 mL of HESC Media. 
	NOTE: If plating cells from more than one flask, simply increase the HESC Media volume.</li>
	<li>Count cells using a Hemocytometer.
	<ol>
		<li>Aliquot 10 &#181;L of re-suspended cells into a 1.5 mL microcentrifuge tube.</li>
		<li>Add 10 &#181;L of 0.4% Trypan Blue stain to cell aliquot and mix by pipetting forward and reverse.</li>
	</ol>
	</li>
	<li>Plate 0.8 x 105 cells per well in 6-well plates. After two days, cells should be 60-70% confluent for optimal transfection.</li>
	<li>For each siRNA transfection sample, prepare complexes using siRNA (60 nanomoles) to transfection reagent.</li>
	<li>Dilute 5 &#181;L of transfection reagent in 150 &#181;L of reduced serum free media and incubate for 5 min.</li>
	<li>Dilute 60 nanomoles of siRNA in 100 &#181;L of reduced serum free media and incubate for 5 min.</li>
	<li>After 5 minutes, combine diluted siRNA with diluted transfection reagent solution and mix gently by pipetting forward and reverse 5 times. 
	NOTE: If transfecting one or more siRNA in multiple wells, prepare master mix in 10 mL polystyrene tubes.</li>
	<li>Incubate siRNA-transfection reagent complexes for 5 min at room temperature.</li>
	<li>During this incubation, add 1 mL of Change Media to each well.</li>
	<li>After 5 min of incubation, centrifuge tubes at 500 x g for 2 min to collect solution and add the 250 &#181;L of siRNA-transfection reagent complexes to each well containing cells and media.</li>
	<li>Mix by gently rocking and incubate the cells at 37&#176;C in a 5% CO2 incubator for 5 h.</li>
	<li>After 5 h, change the media to HESC Media.</li>
	<li>Incubate the cells at 37 &#176;C in a 5% CO2 incubator for 2 days in preparation for an in vitro decidualization assay.</li>
</ol>
7.&#160;In Vitro Decidualization Assay
Note: This assay is performed in a sterile environment under a biological safety cabinet.
<ol>
	<li>Prepare the EPC media prior to initiating the in vitro decidualization treatment as described in Camden et al.3 (See 1.6).</li>
	<li>Aspirate the media from the post-48 hour transfected line of HESC incubated from step 6.16.</li>
	<li>Add 2 mL of EPC Media into each well. 
	Note: As a control, leave one set of cells untreated with EPC Media. Instead, add 2 mL of HESC Media to the control wells.</li>
	<li>Incubate the cells in 5% CO2 incubator at 37 &#176;C for 6 days.
	<ol>
		<li>Change the EPC Media every 48 hours.
		<ol>
			<li>Warm and prepare EPC Media in a 37 &#176;C water bath for 15-20 min.</li>
			<li>Aspirate media from the well and add fresh EPC Media.</li>
			<li>Place the plate back into the 5% CO2 incubator at 37 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After the sixth day, analyze the extent of stromal cell decidualization via ELISA and qRT-PCR (as described below).</li>
</ol>
8. Validation of In Vitro Decidualization utilizing Quantitative Real Time PCR (qRT-PCR)
Note: qRT-PCR is completed as previously described in Kommagani et al10.
<ol>
	<li>Isolate the total RNA from HESC utilizing the protocol from a commercially available RNA isolation kit.
	<ol>
		<li>Analyze the RNA quantity and purity (A260/A280) using a NanoDrop or similar methodology as described in Garcia-Elias et al15.</li>
	</ol>
	</li>
	<li>Synthesize cDNA from 1 &#181;g of total RNA through a commercially available reverse transcription kit protocol.</li>
	<li>Run a qRT-PCR reaction as described in a commercially available qRT-PCR kit protocol.
	<ol>
		<li>Perform the reaction using a commercially available Real-Time PCR Master Mix and gene specific probes along with internal system controls (18s, PRL, and IGFBP1).</li>
	</ol>
	</li>
</ol>
9. Validation of In Vitro Decidualization utilizing ELISA
<ol>
	<li>Quantify the secreted Prolactin levels from the tissue culture media utilizing a commercially available Prolactin ELISA kit.</li>
</ol>
10. Validation of In Vitro Decidualization utilizing Phalloidin staining.
<ol>
	<li>Insert sterile coverslips into each well of a 12 well plate.</li>
	<li>Repeat steps 6.1 - 6.5.</li>
	<li>Plate 0.8 x 105 cells per well of a 12 well plate.</li>
	<li>Incubate the cells in 5% CO2 incubator at 37 &#176;C for 2 days.</li>
	<li>Aspirate the media.</li>
	<li>Repeat steps 7.3 - 7.4.</li>
	<li>Fix the cells in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at room temperature for 30 min.</li>
	<li>Aspirate the fixation solution and wash 3 times with 1 mL of PBS for 5 min each.</li>
	<li>Add 0.1% non-ionic surfactant Polyethylene glycol tert-octyl phenyl ether in PBS for 5 min to the fixed cells to increase permeability.</li>
	<li>Wash 3 times with 1 mL of PBS for 5 min each.</li>
	<li>Add 100 &#181;L of phalloidin solution per coverslip and incubate at room temperature for 90 min.
	<ol>
		<li>Prepare 1:100 dilution in PBS from using a phalloidin stock solution in 1 unit per 5 &#181;L.</li>
	</ol>
	</li>
	<li>Wash 3 times with 1 mL of PBS for 5 min each.</li>
	<li>Mount the slides with mounting medium containing 4&#39;,6-Diamidino-2-Phenylindole (DAPI).</li>
	<li>Observe the cells utilizing a confocal microscope.</li>
</ol>

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This work is supported by funding from National Institutes of Health (NIH)/National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K. We would like to thank the Washington University Fertility Clinic for providing the endometrial biopsy samples.

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><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

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...

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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 ...

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Universidad de Cartagena UDEC

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Developmental Biology

10.1K Views.  Washington University School of Medicine. 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.

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.

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.

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.

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.

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.

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;

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.

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.

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.

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

3D-Printed Scaffold Model to Investigate Embryo Implantation Mechanisms

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.

Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

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.

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

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Summary

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Chapters in this video

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

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

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

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

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

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

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

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

Establishing 3D Endometrial Organoids from the Mouse Uterus

Co-culture Model Using Two Types of Adherent Cell Lines