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 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 ...

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

Research

JoVE Journal

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 ...

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 various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

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 ...

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

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

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

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

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 rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

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 also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of 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 ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.