Name: a primary human trophoblast model to study the effect of inflammation associated with maternal obesity on regulation of autophagy in the placenta
Uploaded: 2017-09-27T15:00:00
Description: Watch this Scientific Journal Video about A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta at JoVE.com

The authors thank women who donated their placentas for this study. We also thank the Labor and Delivery Department at OHSU and the Maternal and Fetal Research Team for coordinating the collection of placentas. We are grateful to Eric Wang, Ph.D., and Kelly Kuo, MD for support and help with experimental methods and optimization.
This work was funded by NIH HD076259A (AM) and AHA GRNT29960007 (AM).

Placentae were collected from the Labor and Delivery Unit at University Hospital under a protocol approved by the Institutional Review Board of Oregon Health and Science University in Portland, Oregon, with informed consent from the patients.
1. Collection of Placental Tissue
<ol>
	<li>Preparation 
	NOTE: All equipment that encounters tissue must be sterile.
	<ol>
		<li>Sterilize the dissecting equipment by autoclaving for 60 min at 121 &#176;C.</li>
		<li>Use personal ...

<ol>
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The placenta, responsible for regulating the growth of the fetus, exhibits compromised function in the obese environment6. Despite the high metabolic demands of trophoblasts, placentas with maternal obesity exhibit dysfunctional mitochondrial respiration6,19. Changes in placental metabolism may contribute to the increased incidence of complications and adverse fetal outcomes observed in obese pregnancies3,</sup...

Obesity is an inflammatory state characterized by chronic low-grade inflammation, stemming from excess adipose tissue and nutrient availability. In obesity, proinflammatory cytokines are elevated in metabolic tissues as well as systemically in circulation. A robust body of evidence has shown that TNF&#945; is significantly elevated in the setting of obesity with implications in insulin resistance and metabolic dysfunction1. Activation of TNF&#945; also contributes to disease pathogenesis in conditions such as cancer and autoimmunity, making it an attractive therapeutic target2.
Inflammation in obesity is compounded by pregnancy, also a proinflammatory state3,4. It has been previously shown that placental TNF&#945; content increases with maternal adiposity in pregnancies with female fetuses. Furthermore, TNF&#945; treatment inhibits mitochondrial respiration in female but not male trophoblast cells, suggesting that TNF&#945; is involved in regulating placental metabolism in a sexually dimorphic manner5. Maternal obesity is associated with the increased incidence of a variety of complications during pregnancy, including stillbirth, with male fetuses being the most susceptible3,6,7,8. Due to its key role at the maternal-fetal interface, changes in the functional capacity of the placenta in the obese intrauterine environment in response to inflammatory signaling may play an important role in mediating the outcomes of obese pregnancies.
Cytotrophoblasts and syncytiotrophoblasts in the villous tissue of the placenta are critical for endocrine signaling and nutrient and oxygen exchange between the mother and developing fetus9. Disruptions in the functional capacity of villous cytotrophoblasts (hereafter referred to as trophoblasts) may jeopardize fetal health and development. This protocol describes a method for sampling of villous tissue from the human term placenta by dissecting away the chorionic and basal plates along with an optimized procedure for the isolation of trophoblasts for primary cell culture. This protocol is derived from established methodologies involving enzymatic digestion of villous tissue to release cells from the extracellular matrix followed by differential density centrifugation to isolate trophoblasts10,11,12. This protocol details an approach in which primary trophoblasts from placentas from lean pregnancies are treated with culture media supplemented with TNF&#945; to simulate one component of the inflammatory milieu associated with maternal obesity. Finally, a simple procedure for harvesting total cell lysates from TNF&#945;-treated trophoblasts followed by Western blotting to detect changes in gene expression is described.
While this model does not recapitulate the obesogenic in utero environment in its entirety, it provides a controlled system that allows one to parse out the individual contribution of TNF&#945;-mediated inflammation in the trophoblasts&#39; response to maternal obesity. This model affords both the opportunity to discover or confirm molecular targets directly regulated by TNF&#945; signaling in trophoblasts as well as allows one to test if changes in gene expression patterns observed in vivo in placentas with maternal obesity may be a result of TNF&#945;-mediated inflammation.
The approach described here was implemented to test the effect of TNF&#945;-mediated inflammation on the regulation of autophagy in human trophoblasts. Trophoblasts from obese pregnancies with male fetuses exhibit disrupted autophagic turnover, or autophagosome maturation13. A protein called Rubicon (RUN domain protein Beclin1-interacting and cysteine-rich containing), which is localized to the lysosomes and late endosomes, has been recently described as a &#34;brake&#34; in the autophagic turnover process because it functions as a negative regulator of autophagosome maturation14,15. In fact, Rubicon is a rare example of a protein that restrains autophagy, which makes it a valuable therapeutic target. Very little information is available about the pathophysiological significance of Rubicon, except for its roles in the innate immune response to microbials16,17 and cardiomyocyte protection18. Using the protocol described here, it is found that Rubicon is upregulated in female primary trophoblasts in response to treatment with increasing concentrations of TNF&#945; up to 250 pg/mL. The regulation of Rubicon may play a role in how female fetuses fare better than males in pregnancies with maternal obesity. Recapitulating inflammation associated with maternal obesity ex vivo by exposing human trophoblasts to exogenous TNF&#945; provides a platform to study the impact of the obese intrauterine environment on the regulation of critical pathways in trophoblasts and by extension, placental function.

<table>
	<tbody>
		<tr>
			<td>10X HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 (anhyd.)</td>
			<td>Sigma-Aldrich</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 (anhyd.)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease/Phosphatase inhibitors</td>
			<td>Thermofisher Scientific</td>
			<td>88668</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576-028</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Scientific</td>
			<td>BP166-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate.</td>
			<td>Fisher Scientific</td>
			<td>AAJ6228822</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDM)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum (NCS)</td>
			<td>Gibco</td>
			<td>26010-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Fisher Scientific</td>
			<td>23-427-098</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3177-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>70013-032</td>
			<td></td>
		</tr>
		<tr>
			<td>K2EDTA vacutainer blood collection tubes</td>
			<td>BD</td>
			<td>366450</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (Density Gradient Media, DGM)</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 2.0 mL, natural</td>
			<td>Fisher Scientific</td>
			<td>22363352</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25-900-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Mini-PROTEAN Tetra System</td>
			<td>Bio-Rad</td>
			<td>1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Mini Trans-Blot Cell</td>
			<td>Bio-Rad</td>
			<td>1658033</td>
			<td></td>
		</tr>
		<tr>
			<td>TGX FastCast Acrylamide Kit, 12%</td>
			<td>Bio-Rad</td>
			<td>1610175</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Protean 3 Multi-Casting Chamber</td>
			<td>Bio-Rad</td>
			<td>1654112</td>
			<td></td>
		</tr>
		<tr>
			<td>4X Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bio-Rad</td>
			<td>1610718</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubicon (D9F7) Rabbit mAb</td>
			<td>Cell Signalling Technology</td>
			<td>8465S</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-&#946;-Actin antibody produced in mouse</td>
			<td>Sigma-Aldrich</td>
			<td>A2228-100UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signalling Technology</td>
			<td>7074S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signalling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34578</td>
			<td></td>
		</tr>
	</tbody>
</table>

a primary human trophoblast model to study the effect of inflammation associated with maternal obesity on regulation of autophagy in the placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Term human placentas from lean (pre-pregnancy body mass index (BMI) &#60;25) mothers with uncomplicated pregnancies carrying female offspring were collected and sampled within 15 minutes of delivery by cesarean section (no labor). The placentas were examined for the absence of calcifications and typical development: weighing between 300-600 g with the umbilical cord and membranes removed, round in shape, between 15 - 25 cm in diameter, and umbilical cord inserted into the middle of the pl...

Watch this Scientific Journal Video about A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta at JoVE.com

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

The overall goal of this trophoblast cell culture model is to measure the effect of obesity-related inflammation on the regulation of critical pathways in trophoblasts. The main advantage of this control system is it establish a relationship between TNF alpha mediated inflammation and the gene regulation in placenta in the context of maternal obesity. The main advantage of this experimental system is it establishes relationships between TNF-alpha mediated inflammation and gene regulation in placenta in the context of maternal obesity.Demonstrating the procedure will be Bailey Simon and Matt Bucher, technicians from my lab. Villous tissue should be sampled from the placenta as soon as possible following delivery, preferably in 30 minutes or less. With the chorionic plate facing upwards, use forceps and scissors to excise, at random, two to three full-thickness sections, two to three centimeters in from the periphery of the placenta.Avoid parts of the placenta that appear abnormal. Trim away the chorionic and basal plates and any large blood vessels. Place the resulting villous tissue in warm, complete medium.Within 30 minutes of villous tissue sampling, begin trophoblast isolation. Place each piece of villous tissue in a 50 milliliter conical tube filled with room temperature PBS and gently agitate to release excess blood in the tissue. Decant the PBS and replace with fresh PBS.Repeat the PBS rinse until most of the excess blood is removed. The PBS rinse will be light red or pink when the tissue is thoroughly rinsed. Place the villous tissue in a sterile petri dish and remove as many blood vessels as possible by using a microscope slide to gently scrape off the soft villous tissue from the vessels.Using scissors, finely mince the resulting villous tissue. Transfer the minced villous tissue to a sterile bottle with digestion solution according to the calculated volumes based on the specific activity of trypsin and DNase. Incubate the digestion bottle in a 37 degree Celsius water bath with shaking at 70 rpm for 35 minutes.After 35 minutes, tilt the digestion bottle on its side and allow the undigested pieces of tissue to settle at the bottom of the bottle. Carefully draw up the supernatant with a serological pipette, avoiding the settled tissue. Dispense the supernatant through a 100 micron cell strainer equally between 50 milliliter conical tubes.Start the second digestion by adding digestion solution to the remaining settled tissue and incubating the bottle in the 37 degrees Celsius water bath. Gently layer three to five milliliters of newborn calf serum, or NCS, underneath the strained supernatant from the first digestion by slowly dispensing from a serological pipette at the bottom of the tube. A meniscus between the strained supernatant containing trophoblasts and NCS should be visible.Centrifuge the supernatants over NCS at 1, 250 times g at 20 degrees Celsius for 15 minutes. The resulting pellet will include red blood cells in the lowermost layer, followed by a white layer containing trophoblast cells. In the same way, strain the supernatant from the second and third digestion.Layer NCS underneath the strained supernatant and centrifuge at 1, 250 times g at 20 degrees Celsius for 15 minutes. Once all supernatants have been centrifuged and the NCS and supernatant layers removed, re-suspend each pellet in five milliliters of warm, complete medium and then pool the suspensions. Divide the cell suspension equally between two 50 milliliter conical tubes and centrifuge at 1, 250 times g at 20 degrees Celsius for 15 minutes.Gently remove the supernatant and re-suspend each of the cell pellets in six milliliters of warm, complete medium. To begin this procedure, divide the cell suspension equally between four density gradients by using a transfer pipette to slowly and carefully layer the cell suspension on top of the density gradients. Centrifuge the density gradients at 1, 250 times g at 20 degrees Celsius for 20 minutes with minimum acceleration and deceleration.This should produce distinguishable bands of sedimented cells. Slowly and carefully remove the top layer of density gradient media until the opaque band containing trophoblast cells is reached. Transfer the trophoblast bands into two 50 milliliter conical tubes and fill with warm, complete medium.Gently invert the tubes three to six times to mix and centrifuge at 1, 250 times g at 20 degrees Celsius for 15 minutes. Remove the supernatant and re-suspend each cell pellet in five milliliters of warm, complete medium. Combine the cell suspensions and count viable cells using a hemocytometer and trypan blue.The next step after cell counting is to plate the cells. Plate three million cells per well in each six well plate. Gently rock the plate back and forth and side to side to evenly distribute the cells.Leave the plated cells in the laminar flow vent for approximately 30 minutes to allow the cells to evenly distribute, settle, and begin to adhere to the bottom of the wells. After 30 minutes, place the plates in a 37 degree Celsius incubator with 5%carbon dioxide and 95%humidity. Examine the trophoblasts under a microscope after 24 hours of culture to confirm attachment and proper trophoblast morphology.The cells can be treated after 24 hours of culture. Aspirate the culture media from the cells and replace with two milliliters of TNF alpha supplemented medium per well. Return the plates to the 37 degree Celsius incubator.After 24 hours of TNF alpha exposure, replace the TNF alpha supplemented medium with complete medium. Incubate for another 24 hours. 24 hours after removal of TNF alpha, aspirate media and gently rinse the cells with room temperature PBS.Add directly to each well 80 microliters of ice cold radioimmunoprecipitation assay buffer containing freshly added protease and phosphatase inhibitors. Remove the cells from the plate with a cell scraper. Transfer the cells to a 1.5 milliliter microcentrifuge tube, pooling wells within treatment groups.Ice the cells by vortexing the tubes on high for at least three intervals of 15 seconds. Incubate the cells at four degrees Celsius with rocking for 15 minutes. Vortex the tubes again on high.Centrifuge at 10, 000 times g for five minutes at four degrees Celsius to pellet cellular debris. Transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. Store the cellular protein samples at 80 degrees Celsius until analysis by Western blotting for the protein of interest.After 72 hours of culture, immunocytochemical analysis of cytokeratin 7, shown in red, in primary trophoblasts confirm that the cells underwent syncytialization evidenced by the presence of multi-nucleated cell masses. Furthermore, immunocytochemical analysis of vimentin, also in red, revealed very few contaminating fibroblasts. Nuclear counter-staining is shown in blue in both images.Treatment with TNF alpha up to 250 picograms per milliliter up-regulated rubicon expression. At TNF alpha concentrations greater than 250 picograms per milliliter, rubicon expression levels decreased back toward baseline and even below. Likewise, rubicon is significantly up-regulated in flash frozen villous tissue biopsies from placentas from obese pregnancies with female fetuses compared to lean controls.Once mastered, isolation of trophoblasts from villous tissue can be done in six to seven hours if it is performed properly. While attempting this procedure, it's important to prepare materials and reagents ahead of time so you can work deliberately and efficiently. Following this procedure, other assays, such as those that measure cellular bioenergetics, can be performed to answer additional questions, like does inflammation in the obese intrauterine environment affect the metabolic capacity of trophoblasts?After this development method paved the way for placental researcher to establish the effect of maternal environment on trophoblast function. After watching this video you will have a good understanding of how to isolate trophoblasts from villous tissue for your cell culture experiment and how to determine the effect of TNF alpha mediated inflammation in regulation of critical pathways in the trophoblasts. Don't forget that working with human tissue, cultured cells, DMSO, acrylamide and reducing reagents can be extremely hazardous and precautions, such as wearing proper PPE and using aseptic technique should always be taken while performing this procedure.

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